MxB inhibits long interspersed element type 1 retrotransposition.

Long interspersed element type 1 (LINE-1, also L1 for short) is the only autonomously transposable element in the human genome. Its insertion into a new genomic site may disrupt the function of genes, potentially causing genetic diseases. Cells have thus evolved a battery of mechanisms to tightly control LINE-1 activity. Here, we report that a cellular antiviral protein, myxovirus resistance protein B (MxB), restricts the mobilization of LINE-1. This function of MxB requires the nuclear localization signal located at its N-terminus, its GTPase activity and its ability to form oligomers. We further found that MxB associates with LINE-1 protein ORF1p and promotes sequestration of ORF1p to G3BP1-containing cytoplasmic granules. Since knockdown of stress granule marker proteins G3BP1 or TIA1 abolishes MxB inhibition of LINE-1, we conclude that MxB engages stress granule components to effectively sequester LINE-1 proteins within the cytoplasmic granules, thus hindering LINE-1 from accessing the nucleus to complete retrotransposition. Thus, MxB protein provides one mechanism for cells to control the mobility of retroelements.

Introduction

Non-long terminal repeat (LTR) retrotransposons include long interspersed element 1 (LINE-1), Alu and SINE-VNTR-Alu (SVA). They have proliferated over the past 80 million years in primates, and together account for approximately 45% of the human genome with LINE-1 alone taking 17% [1-3]. Among ~500,000 LINE-1 copies in human genome, about 80 to 100 LINE-1 elements are still capable of retrotransposition [1]. LINE-1 encodes two proteins called ORF1p and ORF2p. ORF1p is an RNA-binding protein and associates with LINE-1 RNA [4-7]. ORF2p has endonuclease and reverse transcriptase activities [8,9]. ORF1p, ORF2p and LINE-1 RNA together form an RNP complex that enters the nucleus where LINE-1 RNA is reverse transcribed and integrated into cellular DNA [10,11]. LINE-1 reverse transcription and integration is one continuous process, named target primed reverse transcription (TPRT) [12,13]. LINE-1 proteins also assist SVA and Alu mobilization [14,15]. Inevitably, retrotransposition creates various deleterious effects on the structure and function of the human genome [16]. Not surprisingly, more than 100 single-gene genetic diseases have been reported due to LINE-1 and Alu insertion [17,18].

Given the mutagenic nature of LINE-1 retrotransposition, cells have developed various defense mechanisms to restrict LINE-1 mobilization [19]. One arsenal of mechanisms are provided by innate immune factors that have been shown to inhibit virus infections. These include APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide 3) [20,21], TREX1 (Three Prime Repair Exonuclease 1) [22], MOV10 (Moloney leukemia virus type 10 protein) [23,24], SAMHD1 (SAM domain and HD domain containing protein 1) [25,26], RNase L (Ribonuclease L) [27], and ZAP (zinc-finger antiviral protein) [28,29]. These host factors operate by targeting LINE-1 RNP, and either directly degrading LINE-1 RNA by RNase L, mutating newly synthesized LINE-1 DNA by APOBEC3 proteins, degrading ORF1p by TREX-1, or sequestering LINE-1 RNP in the cytoplasmic granules by MOV10, SAMHD1 or ZAP. In 2015, Goodier and colleagues screened a panel of ISGs and restriction factors with known antiviral activities for effects of their expression on L1 retrotransposition. They demonstrated that BST2, ISG20, MAVS, MX2, and ZAP showed strong L1 inhibition [28]. However, the mechanism of MxB inhibiting LINE-1 has not been completely elucidated.

Mx proteins are dynamin-like large GTPases. They have been reported to inhibit distinct groups of viruses by different mechanisms. MxA is cytoplasmic protein, inhibits a plethora of viruses from diverse families, including influenza A virus, vesicular stomatitis virus, LaCrosse virus, hepatitis B virus and others [30]. The antiviral activity of MxA relies on the GTP binding and hydrolysis domain, oligomerization of the stalk domain and the intact BSE (bundle signaling element). In contrast, much fewer viruses have been reported to be restricted by MxB. These viruses include HIV-1 [31-33], HBV [34], HCV [35] and herpesviruses [36,37]. MxB is mainly observed at the nuclear envelope, inhibits HIV-1 and herpesviruses through targeting viral capsid and blocking nuclear import of viral DNA [38]. The N-terminal domain (NTD) and the stalk domain are crucial for MxB to intercept HIV-1. Our data demonstrate that MxB restricts LINE-1 retrotransposition by engaging the stress granule pathway.

Results

MxB inhibits LINE-1 retrotransposition

We used the CMV-L1-neoRT reporter to measure LINE-1 retrotransposition. This construct has a neomycin resistance gene inserted into the 3’ untranslated region of LINE-1 in such a way that this gene can only be expressed from the reverse transcribed LINE-1 DNA, leading to cell resistance to G418 [39,40]. To test whether Mx proteins affect LINE-1 retrotransposition, we co-transfected HeLa cells with human Mx plasmid DNA and the CMV-L1-NeoRT reporter DNA (S1A Fig), followed by G418 selection. The results of colony assays showed that the ectopically expressed MxB diminished LINE-1 retrotransposition by 4.7-fold (p<0.01), whereas MxA exerted no effect (S1B Fig). We generated a defective L1 control which has the ORF1 RR261/262AA mutations inserted in the CMV-L1-neoRT DNA [40]. As a control for the specificity of MxB inhibition, the pEGFP-N1 vector DNA was tested with MxB overexpression, and no effect on the formation of G418 resistance cell colonies was observed (S1B Fig). A dose-dependent inhibition of LINE-1 by MxB was observed, with the lowest amount (200 ng) of MxB DNA causing significant loss of LINE-1 activity (S1C Fig). Cell viability was not affected by MxB overexpression (S1D Fig). Next, we performed quantitative PCR to measure levels of reverse transcribed LINE-1 DNA in cells transfected with CMV-L1-neoRT [41]. In agreement with the data of colony assay, MxB reduced LINE-1 DNA level by more than 2-fold (S1E Fig).

Although MxB is an interferon (IFN) stimulated gene, MxB has been shown to express at measurable levels in several human tissues such as lymph node, liver, and less expressed in brain and reproductive system (S2A Fig). When we used RT-qPCR and Western blot to measure endogenous MxB RNA and protein levels in HeLa cells, MxB expression in the absence of IFN treatment was detected as previously reported (S2C and S2D Fig) [42,43]. We then generated endogenous MxB knockout HeLa cell line using CRISPR/Cas9, and confirmed MxB knockout by sequencing (S2B Fig) and western blot (Fig 1D). We observed that LINE-1 activity increased by 1.9-fold (p<0.01) in the MxB knockout HeLa cells (Fig 1A). In addition, we measured the viability of MxB knockout cells and found no significant difference between MxB knockout and control cell lines (S2E Fig). The same observations were made with cells stably expressing MxB (S2F Fig). We further tested CMV-L1-neoRT transposition in HeLa cells stably expressing MxB, and observed 2.9-fold (p<0.01) decrease compared to that in the control cells (Fig 1B). Together, these data demonstrate an inhibition of LINE-1 restrotransposition by MxB, in agreement with previous report [28].

Fig 1

MxB restricts L1 retrotransposition.

(A) MxB knockout (KO) HeLa cells were transfected with 250 ng CMV-L1-neoRT DNA. Neomycin-resistant cell colonies were scored, and the results of three independent experiments are presented in the bar graph (mean ± SEM; paired t-test) (B) MxB stably expressing HeLa cells were transfected with CMV-L1-neoRT DNA (250 ng). G418-resistant cell colonies were scored, and the results of three independent experiments are presented in the bar graph (mean ± SEM; paired t-test). (C) Western blots to measure the expression of ORF1p and MxB in HeLa cells stably expressing ectopic MxB. (D) Levels of ORF1p in MxB knockout (KO) HeLa cells. (E) Location of the amplified regions in the LINE-1 genome. Red, 5’UTR; brown, ORF1p; green, ORF2p. (F, G) Levels of LINE-1 RNA in HeLa cells stably expressing MxB or with MxB knockout, as determined by real-time RT-PCR. Data are the average from three independent experiments (mean ± SEM; paired t-test). *, P<0.05; **, P< 0.01; ***, P<0.001.

IFN has been shown to inhibit LINE-1 replication [44]. Since MxB is an IFN stimulated gene, we investigated the role of MxB in IFN-mediated LINE-1 inhibition. Activity of CMV-L1-neoRT in the control and MxB knockout cells was measured with and without IFN. The results of colony assay showed that LINE-1 retrotransposition was strongly inhibited by IFN in the control cell line, but this inhibition was significantly lost in MxB knock-out cell line (S2G Fig), which indicates that IFN-induced MxB contributes to IFN inhibition of LINE-1.

In support of MxB inhibition of LINE-1 retrotranspotion, the expression level of LINE-1 ORF1p diminished in HeLa cells stably expressing ectopic MxB (Fig 1C), and increased when MxB was knocked out (Fig 1D). We also investigated the effect of MxB on the activity of endogenous LINE-1 by performing quantitative RT-PCR to measure the level of endogenous LINE-1 RNA, using three pairs of primers that specifically amplify regions in the 5’UTR, ORF1 or ORF2 (Fig 1E) [45]. In MxB knockout HeLa cells, levels of endogenous LINE-1 RNA, as measured by all three primer pairs, significant increased, as opposed to the marked decrease seen in HeLa cells stably expressing MxB (Fig 1F and 1G). In summary, these results suggest that MxB is an inhibitor of LINE-1 retrotransposition.

MxB promotes sequestration of LINE-1 ORF1p to cytoplasmic bodies

We next investigated the molecular mechanisms by which MxB inhibits LINE-1 activity. In HeLa cells stably expressing MxB-EGFP, when the CMV-L1-neoRT reporter DNA was transfected, the ORF1p protein was seen to form cytoplasmic granules and co-localize with MxB (Fig 2A), which was also observed when only ORF1p protein was expressed in the absence of ORF2p (Fig 2B). These data were reproduced with transiently expressed MxB (S3A and S3B Fig).

Fig 2

MxB interacts with ORF1p and sequesters LINE-1 RNP within the cytoplasm.

(A) Immunofluorescence microscopy of MxB and ORF1p in HeLa cells which stably expressed MxB-EGFP and were transfected with CMV-L1-neoRT DNA. The white arrows indicate nuclear ORF1p. (B) Co-localization of stably expressed MxB-EGFP with ORF1p in HeLa cells that were transfected with ORF1p-Flag DNA. The white arrows show nuclear ORF1p. (C) Immunofluorescence microscopy of MxB and L1-ms2x6/MS2-GFP plasmids. The transcribed LINE-1 RNA contains six copies of MS2-binding site that are bound with MS2-GFP. The cytoplasmic punctate green fluorescence indicates localization of LINE-1 RNA. (D, E, F) Co-immunoprecipitation of transiently expressed MxB-Flag and ORF1p which was expressed either from the ORF1p-Myc DNA (D), the CMV-L1-neoRT DNA (E), or the endogenous LINE-1 in HEK293T cells (F). (G) Effect of RNase on co-immunoprecipitation of MxB and ORF1p. 293T cell were co-transfected with CMV-L1-neoRT vector and MxB-Flag. Co-IP was performed with or without RNase A. (H, I) Nucleocytoplasmic fractionation of HeLa cells transfected with the MxB DNA (48 hours). Presence of ORF1p, either ectopically expressed from the CMV-L1-neoRT DNA (H) or the endogenous form (I), in the nuclear (N) and cytoplasmic (C) fractions was detected by Western blots. In the Western blots, the nuclear fraction samples were from cells 5 times more than the cells of the cytoplasmic fraction samples. The intensities of protein bands were quantified and the results are presented in the bar graphs. To calculate the ratio of nuclear/cytoplasmic MxB, the amount of nuclear ORF1p normalized with the nuclear LAMN protein level, the amount of cytoplasmic ORF1p with tubulin, then nucleus/cytoplasm ORF1p ratio was calculated by dividing nuclear ORF1p value by the cytoplasm ORF1p value. At the end, the nucleus/cytoplasm ORF1p ratio of the vector control is arbitrarily set at “1”. N, nucleus; C, cytoplasm (mean ± SEM; paired t-test) (J) The Operetta High-Content Screen system (PerkinElmer) was utilized to detect the ORF1p fluorescence signals within the nuclei and the cytoplasm in HeLa cells which were transfected with MxB-EGFP and ORF1p-Flag. Ratios of the nuclear and cytoplasmic ORF1p signals were calculated. Results from the control group, which was transfected with the pEGFP-N1 vector, were arbitrarily set as 1. The results are summarized in the bar graph (mean ± SEM; paired t-test). **, P< 0.01; ***, P<0.001.

We also investigated the relationship of subcellular location between MxB and LINE-1 RNP which were measured by the LINE-1-ms2x6/MS2-GFP system. As described previously, the expressed LINE-1 RNA contains 6 copies of MS2-binding sites which is bound by MS2-GFP for visualization of LINE-1 RNA [46]. When LINE-1-ms2x6 and MS2-GFP DNA were co-expressed with MxB-Flag, LINE-1 RNA was seen to co-localize with MxB in the cytoplasm (Fig 2C). The association of MxB with LINE-1 RNP was also supported by the data of co-immunoprecipiation experiments performed with ectopic ORF1p (Fig 2D and 2E) or endogenous ORF1p (Fig 2F). The association of LINE-1 ORF1p and MxB was reduced when RNase was added during co-immunoprecipiation to remove RNA (Fig 2G). These results suggest that MxB associates with ORF1p in an RNA-dependent manner.

MxB has been shown to inhibit the nuclear import of HIV-1 DNA [43,47,48]. We asked whether MxB also impairs the nuclear import of LINE-1 RNP. To answer this question, we performed nucleocytoplasmic fractionation experiments to measure the levels of ORF1p in the cytoplasm and the nucleus. Indeed, MxB reduced the nuclear ORF1p which was either ectopically expressed or endogenous (Fig 2H and 2I). This observation was supported by the data of high-content imaging analysis (Fig 2J), which calculates the ratios of ORF1p signals in the nucleus and cytoplasm in HeLa cells. We could also observe nuclear ORF1p in control cells, which disappeared with MxB overexpression (Fig 2A and 2B, indicated by white arrows).

Once in the nucleus, LINE-1 ORF2p cleaves cellular DNA by its endonuclease activity to initiate reverse transcription. This DNA cleavage event causes DNA damage response, shown by the increase of γH2AX foci. If MxB prevents the nuclear import of LINE-1 RNP, we expected to detect fewer LINE-1-induced γH2AX foci with MxB expression. When we immunostained γH2AX, we detected a great number of γH2AX foci in cells transfected with the CMV-L1-neoRT DNA. As expected, MxB stable expression dramatically reduced the number of these foci (Fig 3A, 3B and 3C). In agreement with the MxB overexpression data, greater numbers of γH2AX foci were scored in MxB knockout HeLa cells with CMV-L1-neoRT transfection than in the control cells, concurrent with increased LINE-1 ORF1 expression (Fig 3D, 3E and 3F). When LINE-1 DNA was transfected into MxB knockout cell lines, γH2AX level and foci number increased compared with control cells (S4A, S4B and S4C Fig). Ectopic MxB decreased the γH2AX level induced by CMV-L1-neoRT (Fig 3G). The defective L1-ORF1 (RR261/262AA) did not increase γH2AX level. We also observed that MxB overexpression alone diminished the number γH2AX foci, and MxB knockout itself led to an increase in γH2AX foci, which is likely a result of the suppression of endogenous LINE-1 by MxB. These data further suggest that MxB reduces the formation of double-strand DNA breaks associated with LINE-1 retrotransposition.

Fig 3

MxB decreases the nuclear γH2AX foci induced by L1 retrotransposition.

(A) Western blots to show the levels of γH2AX in HeLa cells which stably express MxB and were transfected with the CMV-L1-neoRT DNA for 48 hours. (B) Detection of γH2AX foci in HeLa cells which stably express MxB and were transfected with the CMV-L1-neoRT DNA. Immunofluorescence was performed 24 hours post transfection. (C) The γH2AX foci were scored in more than 50 cells for each treatment. The average number of γH2AX foci per cell is presented in the bar graph (mean ± SEM; paired t-test). (D) Western blots to detect γH2AX in the control or MxB knockout HeLa cells without transfection of LINE-1 DNA. (E, F) Detection of γH2AX foci in the control and MxB knockout cells without LINE-1 DNA transfection. The γH2AX foci were scored in 50 cells, the results are presented in (F) (mean ± SEM; paired t-test). ** indicates P<0.01; ***, P<0.001. (G) Western blot was performed to detect γH2AX in cells transfected with CMV-L1-neoRT or inactive L1-ORF1 (RR261/262AA), and MxB-Flag.

Knockdown of stress granule marker proteins abrogates MxB inhibition of LINE-1

LINE-1 ORF1p has been reported to associate with stress granules [49-51]. Several LINE-1 inhibitors have also been reported to co-localize with LINE-1 ORF1p in stress granules, including SAMHD1 [26], ZAP [28], APOBEC [52,53] and MOV10 [51,53]. We therefore asked whether the MxB- and ORF1p-containing cytoplasmic foci also bear stress granule markers. To this end, we performed immunofluorescence staining experiment to detect stress granule marker G3BP1 or TIA1. Indeed, the MxB/ORF1p foci contained G3BP1 and TIA1 in HeLa cells that stably expressed MxB and were transfected with CMV-L1-neoRT reporter or ORF1p plasmid (Figs 4A, 4B, S5A and S5B). We have scored the number of ORF1p-containing stress granules with and without MxB expression, observed no significant difference between the stable MxB-expressing cell line and control cell line (Figs 4A, 4B, S5A and S5B). However, we observed the enlarged ORF1p-containing foci under MxB-EGFP overexpression by visually inspecting more than 100 cells. The co-localization was also observed in HeLa cells that were transiently transfected with MxB plasmid DNA (S6A, S6B, S6D and S6E Fig). Moreover, the LINE1-ms2x6 RNA was also detected in the MxB/G3BP1 (Fig 4C) or TIA1 (S5C Fig) cytoplasm foci. In support of the localization of MxB, ORF1p with G3BP1 and TIA1, these four proteins were co-immunoprecipitated with each other, which was not observed with MxA (Fig 4D).

Fig 4

Inhibition of LINE-1 by MxB depends on the stress granule pathway.

(A, B) Co-localization of stably expressed MxB-EGFP with ORF1p and G3BP1 in HeLa cells. ORF1p was either expressed from the transfected CMV-L1-neoRT DNA (A) or ORF1p vector DNA (B). White arrows indicates nuclear ORF1p. ORF1p/G3BP1-containing SGs were scored in more than 50 cells for each treatment. The average number of ORF1p-containing SGs per cell is presented in the bar graph (mean ± SEM; paired t-test). (C) Co-localization of LINE-1 RNA, MxB-Flag and G3BP1 in cells transfected with MxB-Flag (500 ng), LINE-1-ms2x6 (750 ng) and MS2-GFP (250 ng) DNA. The LINE-1 RNA bears 6 copies of MS2-binding sites which are bound by MS2-GFP and detected as cytoplasmic puncta. (D) Co-immunoprecipitation to detect the association of MxB with ORF1p in HEK293T cells which were co-transfected with CMV-L1-neoRT and MxA-Flag or MxB-Flag DNA. (E, F) Effects of G3BP1 (E) or TIA1 (F) knockdown on MxB inhibition of LINE-1 in HeLa cells which were co-transfected with MxB and CMV-L1-neoRT DNA. The number of G418-resistant cell colonies was determined for each condition. The results are presented in the bar graphs (mean ± SEM; paired t-test). ns, not significant. ** denotes P<0.01; ***, P<0.001.

We next tested whether MxB inhibition of LINE-1 depends on stress granule formation. We thus depleted endogenous G3BP1 or TIA1, and examined whether MxB still inhibits LINE-1. Results of LINE-1 reporter assays showed that MxB lost inhibition of LINE-1 when either G3BP1or TIA1 was knocked down (Fig 4E and 4F). We also observed 50% increase in LINE-1 retrotransposition with knockdown of either G3BP1 or TIA1 in the absence of MxB expression (Fig 4E and 4F) [26]. In agreement with these functional data, knockdown of endogenous G3BP1 or TIA-1 led to the loss of ORF1p/MxB-EGFP granules in the cytoplasm (S6C and S6F Fig), further supporting the stress-granule nature of the ORF1p/MxB foci.

Arsenite (AS) is commonly used to induce canonical stress granules. Interestingly, MxB did not localize to stress granules that were induced with arsenite, neither the endogenous MxB induced by IFN-α in HeLa cell (S7A Fig) nor ectopically expressed MxB (S7B Fig), which suggests that MxB itself is not recruited to arsenite-induced stress granules, but rather is located to G3BP1- and TIA1-bearing foci through interaction with LINE-1 ORF1p. Together, these data demonstrate that MxB associates with LINE-1 RNP in the stress granules, and enhances cytoplasmic sequestration of LINE-1 RNP. We treated both the control and MxB knockout cell lines with AS. The number of SGs increased significantly with arsenite treatment, yet there was no difference between MxB knockout and control cell lines (S7C Fig). When the CMV-L1-neoRT plasmid was transfected, the number of ORF1p-containing SGs did not differ between MxB knockout and control cell lines with or without arsenite induction (S7D Fig). These results suggest that arsenite -induced ORF1p-containing SGs is not affected by endogenous MxB expression.

The inhibition of HIV-1 and HCV by MxB is dependent on cyclophilin A (CypA) [35,54]. To answer whether CypA affects the MxB inhibition of LINE-1, we used shRNA of CypA to knock down endogenous CypA in HeLa cells and performed colony assay of cells transfected with MxB and CMV-L1 neoRT reporter DNA. The results showed that LINE-1 retrotransposition was inhibited by MxB as much in CypA knockdown cells as in the control cells (S8A and S8B Fig), which indicates that MxB does not require CypA to restrict LINE-1 retrotransposition. Fewer G418-resistant cell colonies were scored with CypA-knockdown, which indicates a positive role CypA in LINE-1 retrotransposition.

MxB requires its NLS, GTPase activity and oligomerization to inhibit LINE-1

MxB has a GTPase globular domain and a stalk domain which mediates MxB oligomerization [55-61]. In addition, the N-terminal sequence of MxB has a nuclear localization signal (NLS) [31,62]. To understand the contribution of the different MxB domain to LINE-1 inhibition, we generated a series of MxB mutants (Fig 5A). We first deleted the N-terminal 25 amino acids which contain the NLS, and generated mutant MxBdel25. We also replaced this 25-amino acid sequence with the NLS from the SV40 large T antigen, this mutant is named MxBdel25+NLS. Lastly, we replaced the first 42 amino acids of MxA with the first 25 amino acids of MxB, to generate the MxA+B25 chimeric protein (Fig 5A). The subcellular localization of these Mx proteins were examined with confocal microscopy (Fig 5B). MxA was detected in the cytoplasm, while MxB was localized at the nuclear envelope as previously reported [43,63]. When the NLS in N-terminal 25 amino acids of MxB was deleted, the MxBdel25 mutant changed subcellular location to the cytoplasm, the distribution was the same as the natural short isoform of MxB [64]. Results of LINE-1 reporter assays showed that MxBdel25 lost the ability of inhibiting LINE-1 (Fig 5C). MxBdel25+NLS inhibited LINE-1 as strongly as the wild type MxB, and the MxBdel25+NLS mutant was detected at the peri-nuclear region, a distribution pattern not exactly the same as MxB itself (Fig 5B and 5C). Interestingly, the MxA+B25 protein, which has its first 42 amino acids changed to the first 25 amino acids of MxB, was localized to the peri-nuclear region, and did not inhibit LINE-1 (Fig 5B and 5C). These data suggest that the NLS is essential but not sufficient for Mx proteins to inhibit LINE-1.

Fig 5

Effect of MxB mutations on the inhibition of LINE-1 retrotransposition.

(A) Illustration of wild-type (WT) and mutated MxB proteins. NLS, nuclear localization signal; G domain, GTPase domain; B, bundle signaling element. (B) Subcellular localization of MxB and its mutants. (C) HeLa cells were co-transfected with CMV-L1-neoRT (250ng) and wild type MxB (250 ng) or its mutants, followed by G418 selection for resistant cell colonies. G418-resistant cell colonies were scored. The results of three independent experiments are presented in the bar graph (mean ± SEM; paired t-test). Expression of MxB and its mutants was determined by Western blot. (D) HeLa cells were co-transfected with CMV-L1-neoRT or pEGFP-N1 DNA and wild type MxB or the indicated MxB mutants. G418-resistant cell colonies were scored, the results of three independent experiments are presented in the bar graph (mean ± SEM; paired t-test). Expression of MxB and its mutants was determined by Western blot. **, P< 0.01; ***, P<0.001. (E) MxB (500 ng DNA) or its mutants were transfected into HeLa cell. Their subcellular localization was detected by immunofluorescence staining. Bars represents 10 μm.

It was reported that MxB is localized at cytoplasmic face of nuclear core complex (NPC) and interact with NPC protein NUP214, as well as transport receptor transportin-1 (TNPO-1) [47,65]. We thus investigated whether NUP214 or TNPO-1 are required for LINE-1 inhibition by MxB. We observed that TNPO-1, but not NUP214, was localized to the MxB/ORF1p cytoplasm foci (S8C and S8D Fig). When we knocked down endogenous NUP214 or TNPO-1, we found that NUP214 knockdown led to moderate decrease of LINE-1 retrotransposition, whereas TNPO-1 knockdown moderately increased LINE-1 retrotransposition (S8E and S8F Fig). Knockdown of neither NUP214 nor TNPO-1 changed MxB inhibition of LINE-1. These data suggest a modest role of NUP214 and TNPO-1 in LINE-1 retrotransposition, but neither of these two proteins affect MxB inhibition of LINE-1.

We next generated more MxB mutants, including K131A which is defective in GTP binding ability [43], M574D/Y651D which disrupt MxB dimerization, as well as mutation R449D/F495D that prevent MxB oligomerization [59,60]. When these MxB mutants were tested in LINE-1 reporter assays, none of them affected LINE-1 activity (Fig 5D), in agreement with the decreased association of these MxB mutants with G3BP1 or ORF1p (S9A and S9B Fig), and none of these mutants affected the nuclear import of LINE-1 RNP (S9C and S9D Fig). These MxB mutants exhibited dispersed cytoplasmic localization (Fig 5E). Together, these results suggest that both the GTPase activity and oligomerization are essential for MxB to inhibit LINE-1.

Inhibition of LINE-1 by Mx2-like proteins

We next tested whether MxB homologs of different species can also inhibit LINE-1. We first established a phylogenetic tree of Mx proteins from mammals (S10 Fig). The results showed that Mx proteins from most mammals segregate into two lineages, MxA/Mx1 and MxB/Mx2, with the exception of the mouse Mx proteins, mouse Mx1 and Mx2 are orthologous with human MxA. Human MxB ortholog in mouse was lost in evolution. We then tested the anti-LINE-1 activity of several Mx proteins from the MxA-like and MxB-like groups. The results showed that MxB protein from Macaca mulatta (Mac) and African green monkey (AGM) strongly inhibited LINE-1 (Fig 6A), whereas murine Mx1 and Mx2 did not affect LINE-1 activity (Fig 6B). The Equus caballus Mx2 also inhibited LINE-1 retrotransposition, the Ovis aries Mx2 exhibited week inhibition of LINE-1 (Fig 6B). To understand the variable effect of MxB-like proteins on LINE-1, we examined their subcellular localization. MacMx2 and AGMMx2 showed similar localization to the nuclear envelope as human MxB (Fig 6C). In the case of Equus and Ovis MxB proteins, their N-terminal sequences significantly differ from human MxB NLS. Nonetheless, Equus MxB was detected at the nuclear envelope as opposed to the Ovis MxB that was diffused in the cytoplasm (Fig 6C). These data suggest a correlation of localization to the nuclear envelope and the inhibition of LINE-1 by MxB-like proteins.

Fig 6

Inhibition of LINE-1 by Mx2-like proteins.

(A) CMV-L1-neoRT reporter assay to measure the effect of non-human primate MxB on LINE-1 retrotransposition. Expression of MxB was determined by Western blot. (B) Effect of MxA-like and MxB-like proteins on LINE-1 activity. hu: human, m: mouse, Mac: Macaca mulatta, AGM: African green monkey, E: Equus caballus, O: Ovis aries. (C) Subcellular localization of MxB-like proteins in HeLa cells, detected with immunoflourescence microscopy. (D) Effect of stably expressed human MxB-Flag on CMV-L1-neoRT in the BALB/3T3 clone A31 cell line, as determined by colony assay. (E) Effect of stably expressed human MxB protein on mouse endogenous retroelements in the BALB/3T3 clone A31 cell line, as determined by RT-qPCR. Vector control was shown as grey dot, MxB data are shown as yellow square. (F) Effect of human MxB on mouse endogenous retroviruses reporter, IAP-neoTNF or MusD-neoTNF, in BALB/3T3 clone A31 cells. G418 was added 48 hours post transfection. G418-resistant cell colonies were scored, and the results of three independent experiments are presented in the bar graph. Vector control was shown as gray dot, MxB data as yellow square. All data were plotted as mean values, with variation as SEM. Statistical significance was calculated by Student’s two-tailed t test. *, P<0.05; **, P< 0.01; ***, P<0.001.

Finally, we tested whether human MxB can inhibit LINE-1 in non-human cells such as in murine BALB/3T3 clone A31 cell line. We stably expressed human MxB in the BALB/3T3 clone A31 cell line, and then transfected with human LINE-1 reporter CMV-L1-neoRT DNA. A strong inhibition of human LINE-1 retrotransposition was observed (Fig 6D). Levels of endogenous mouse LINE-1 subfamilies (L1A, L1Gf and L1Tf) and SINE RNA were measured by RT-qPCR with promoter specific primers in BALB/3T3 clone A31 cells that stably express human MxB. Marked inhibition of these mouse LINE-1 subfamilies (L1A, L1Gf and L1Tf) and SINE was observed (Fig 6E). IAP (Intracisternal A-type Particle elements) and MusD are active mouse ERVs (endogenous retroviruses) (also named LTR (long terminal repeat) retrotransposons) [66]. When we transfected IAP-neoTNF and MusD-neoTNF reporter DNA into human MxB-expressing BALB/3T3 clone A31 cells, the retrotranspostion of mouse IAP and MusD was strongly inhibited (Fig 6F). These results suggest that Mx2-like (orthologous to human MxB) proteins inhibit a wide range of retrotransposons in different species, thus play an important role in safeguarding the genome.

Discussion

LINE-1 has approximately 100 full-length copies in human genome. Because of the mutagenesis nature of its retrotransposition into new loci, LINE-1 activity is tightly regulated by the host. Among the multi-layered mechanisms humans have evolved to control LINE-1 retrotransposition, it is not surprising that the innate antiviral factors, which defend against exogenous viruses, also have the ability of inhibiting LINE-1. This concept is strongly supported by a study from Goodier and colleagues who systematically tested a group of known interferon-induced antiviral proteins and found several of these factors with anti-LINE-1 function, including BST2, ISG20, MAVS, ZAP, and MxB (also called Mx2) [28]. In this study, we confirmed LINE-1 inhibition by MxB and further showed that MxB does so by sequestering LINE-1 RNP within cytoplasmic bodies containing stress granule marker proteins G3BP1 and TIA1. We further showed that the LINE-1 inhibition ability of MxB depends on its nuclear envelope subcellular location, the GTPase activity and oligomerization.

MxB has been shown to inhibit several important pathogenic viruses, including HIV-1 [31-33], herpesviruses [36,67], hepatitis B virus [34], hepatitis C virus, Japanese encephalitis virus, and Dengue virus [35]. MxB exerts its antiviral function by targeting specific viral proteins. The capsids of HIV-1 [54,61,68-70] and herpesvirues [36,67] are the target of MxB. MxB inhibits HCV through binding to viral NS5A protein [35]. Our data showed that MxB is able to associate with LINE-1 protein ORF1p, enhancing the sequestration of LINE-1 RNP in G3BP1-containing cytoplasmic granules. In analogy to MxB inhibition of the nuclear import of HIV-1 and herpesvirus DNA, MxB sequesters LINE-1 RNP within the cytoplasm, thus prevents LINE-1 RNP from accessing the nucleus to complete reverse transcription. Therefore, MxB manages to use similar strategies to suppress virus infection and LINE-1 retrotransposition.

Clearly, there are differences in the detailed mechanisms by which MxB inhibits different viruses and LINE-1. For example, the requirement for the GTPase activity differs. The GTPase defective MxB still inhibits HIV-1 [31,32,54] but not herpesviruses [36,67]. Our data showed that MxB needs its GTPase function to suppress LINE-1. Although MxB depends on the cellular peptidylprolyl isomerase, cyclophilin A (CypA), to inhibit HIV-1 [33,54] and HCV [35], MxB inhibition of LINE-1 is CypA-independent (S8A and S8B Fig). These different requirements may reflect the different natures of the viral proteins which MxB targets, as well as the different replication strategies that the inhibited viruses employ. Regardless of these mechanistic variations, MxB strongly depends on its oligomerization and N-terminal sequence to inhibit its target viruses and LINE-1. It has been reported that MxB N-terminal sequence participates in the recognition of HIV-1 capsid structure and this activity is subject to regulation by phosphorylation [71,72]. It is possible that MxB interaction with LINE-1 ORF1p also requires its N-terminal sequence. We also noted that the N-terminal sequence was also reported to be dispensable for MxB to inhibit LINE-1 [28]. This discrepancy with our observation may be a result of the different experimental systems used in different studies including cell lines and protein tags. More studies are warranted to resolve this discrepancy.

Our data showed that MxB inhibition of LINE-1 depends on stress granule marker proteins G3BP1 and TIA1 (Fig 4E and 4F). LINE-1 proteins ORF1p and ORF2p as well as LINE-1 RNA have been reported to associate with cytoplasmic granules containing stress granule markers and p body markers [49-51]. This suggests that sequestering RNA to stress granules and p bodies serves as an intrinsic cellular strategy to control the activity of LINE-1, which operates through sequestering LINE-1 RNP within cytoplasmic bodies and therefore preventing LINE-1 from accessing the nucleus and completing retrotransposition. However, this strategy by itself may not be quite effective, as knockdown of G3BP1 or TIA1 only marginally increased LINE-1 activity (Fig 4E and 4F). MxB appears to enhance this intrinsic anti-LINE-1 mechanism, since we observed much greater LINE-1 ORF1p localization to G3BP1 or TIA1/MxB-positive cytoplasmic granules compared to the relatively more dispersed cytoplasmic distribution of ORF1p in the absence of MxB expression (Figs 4A, 4B, S5A and S5B). MxB is not the only factor stimulating sequestration of LINE-1 RNP in cytoplasmic granules, we and others have reported that ZAP [29] and SAMHD1 [26] employ a similar mechanism to assist cellular control of LINE-1 retrotransposition. It appears that cells have evolved means to regulate the stress granule-based anti-LINE-1 mechanism. One possible scenario is that high-level LINE-1 activity may stimulate the expression of MxB and other anti-LINE-1 factors that in turn suppress LINE-1 retrotransposition through engaging the stress granule pathway. In retrospective, sequestration of MxB to stress granules as a result of interaction with ORF1p alters subcellular localization of MxB, and may potentially influence its other cellular functions.

Stress granules are a typical membraneless organelles (MLOs) which are formed by liquid-liquid phase-separated in cytoplasm. MLOs regulate diverse cellular function, such as protein turnover, mitosis mRNA storage and translation, virus replication, and antiviral activity. Murine Mx1 and Mx2 were observed in nuclear bodies and granular cytoplasmic structures [73]. Coincidentally, exogenously expressed as well as IFN induced human MxA protein also forms as membrane-less condensates in the cytoplasm, and interacts with and sequesters viral nucleocapsid proteins. Endogenous human MxB is localized to the cytoplasmic face of nuclear pore which also has phase-separated nature. Therefore, we speculate that Mx family proteins all have the propensity of forming MLO, which contributes to their antiviral function. MxB is not an integral component of stress granules, since we did not observe MxB localization with stress granules that were induced with arsenite treatment (S7A and S7B Fig). Expression of MxB itself did not cause stress granule formation either. Although there are MxB-containing cytoplasmic foci with MxB overexpression as seen in S3A Fig, these likely result from MxB aggregation. We suspect that MxB association with ORF1p stimulates G3BP1/TIA1-dependent aggregation of ORF1p, either through the GTPase activity of MxB and/or the ability of MxB to oligomerize. This mechanism allows MxB to inhibit LINE-1, also sends MxB to the ORF1p/G3BP1/TIA1 positive cytoplasmic granules.

Our data showed that the GTPase mutation K131A prevents MxB localization to the nuclear envelope and impairs its interaction with ORF1p. Furthermore, Betancor and colleagues reported that the GTPase domain cooperates with the N-terminal domain in Mx2 binding to the HIV-1 capsid [61]. We speculate that the GTPase activity may influence MxB conformation which is involved in interaction with LINE-1 ORF1p (S9C Fig) and inhibiting LINE-1 retrotransposition (Fig 5D).

We further investigated Mx proteins from different species for their ability to inhibit LINE-1. Mx2 proteins from non-human primates, including Macaca mulatta (Mac) and African green monkey (AGM), which are homologous to human MxB, also effectively restrict LINE-1 (Fig 6A), whereas Mx1 and Mx2 proteins from rodents, both of which share homology with human MxA, exhibit no effect (Fig 6B). Equus caballus Mx2 strongly inhibited LINE-1, whereas the Ovis aries Mx2 exhibited modest inhibitory effect (Figs 6B and S10). We also observed that anti-LINE-1 Mx2 or MxB proteins are located to nuclear envelope. Furthermore, classical nuclear localization signal of the SV40 large T antigen enables the MxBdel25 mutant to inhibit LINE-1 (Fig 5C). These results suggest a correlation of MxB-like protein localization to nuclear envelope and their anti-LINE-1 function.

Furthermore, the retrotranspostion of mouse SINE, IAP and MusD was strongly inhibited by stably expressed human MxB in BALB/3T3 clone A31 cells. This suggests that MxB regulates a wide spectrum of retroelements. SINE does not encode proteins, is unable to retrotransposition autonomously, its activity depends on LINE-1 ORF1p and ORF2p [14,15]. MxB is thus expected to inhibit SINE through sequestering LINE-1 ORF1p as we observed in this study. IAP and MusD are endogenous retroviruses, they encode Gag protein as HIV-1 does [74]. It is likely MxB targets Gag and inhibits IAP and MusD, similar to MxB inhibiting HIV-1 by targeting viral capsid. Since MxB also inhibits retrotransposons in murine cells, it can be envisioned that MxB either exerts its inhibition by itself or is assisted by factors shared by human and murine cells

In conclusion, our data support MxB as an anti-LINE-1 factor which functions by sequestering LINE-1 RNPs within the cytoplasmic granules through engaging the stress granule marker proteins G3BP1 and TIA1. This finding further substantiates the importance of the stress granule pathway in controlling LINE-1 and potentially other transposable elements. It will be interesting to investigate whether MxB uses similar mechanisms to restrict the infection of particular viruses.

Materials and methods

Plasmids and antibodies

CMV-L1-neoRT reporter DNA contains the complete human LINE-1 DNA and a neomycin resistance gene as a reporter of LINE-1 retrotransposition [26]. The ORF1-Myc DNA was cloned into the pCMV-Tag 3B expression vector. The L1-ORF1 (RR261/262AA) plasmid was generated by inserting ORF1 261/262RR to AA mutations into the CMV-L1-neoRT plasmid, as reported in [40]. IAP-neoTNF and MusD-neoTNF reporter plasmids contain mouse LTR retrotransposon and a neomycin resistance cassette in reverse orientation [23]. The L1-ms2x6 contains six tandem MS2 CP binding sites near the 3’ end of L1-RP in 99-PUR L1-RP vector. MS2-GFP plasmid was generated by inserting MS2 sequence in pEGFP-N1 vector. The MxB-Flag DNA was inserted into the pQC-XIP vector. The MxB mutations del25, K131A, R449D/F495D and M574D/Y651D were created using PCR-based mutagenesis method. The wild type and mutated MxB-EGFP were generated by fusing EGFP sequence to C-terminal end of MxB. The MxB sequence was inserted to the N-terminus of EGFP in pEGFP-N1 vector between the cleavage sites of restriction enzymes BamH1 and Age1, to generate MxB-EGFP fusion protein. Mouse Mx1 and Mx2, Equus caballus Mx2, Ovis aries Mx2, Mac and AGM Mx2 sequences were synthesized by Beijing Ruibio BioTech Co. Ltd company. These Mx cDNA sequences were cloned into the pQC-XIP vector.

Mouse anti-Actin antibody (66009-1-Ig), mouse anti-Myc antibody (67447-1-Ig), rabbit anti-Tubulin antibody (10094-1-AP), rabbit anti-G3BP1 antibody (13057-2-AP), rabbit anti-TIA1 antibody (12133-2-AP), and rabbit anti-TNPO-1 antibody (20679-2-AP) were purchased from Proteintech. Mouse anti-Flag (F1365) antibody, rabbit anti-Myc (C3956) antibody, rabbit anti-GFP antibody (G1544) and rabbit anti-LAMN1 antibody (L1293) were purchased from Sigma. Mouse anti-G3BP1 antibody (05–1938), mouse anti-ORF1p antibody clone 4H1 (MABC1152), mouse anti-γH2AX antibody (05–636) were purchased from MILLIPORE. Alexa fluor 555-labled donkey anti-rabbit antibody (A-21428) and Alexa fluor 647-labled donkey anti-mouse antibody (A-21236) were purchased from Thermo Fisher Scientific. The anti-MxB antibody was generated by immunizing rabbits with recombinant MxB protein [33]. Rabbit anti-ORF1p antibody was generated by immunizing rabbits with recombinant ORF1p [26]. Anti-Flag M2 affinity gel (A2220) was purchased from Sigma.

Cell lines and cell culture

Human embryonic kidney cell line HEK293T expressing the SV40 T-antigen (ATCC, CRL-3216), human cervical carcinoma cell line HeLa (ATCC, CCL-2), and murine BALB/3T3 clone A31 (ATCC, CCL-163) cell line were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin.

MxB KO cells were generated using the CRISPR-Cas9 system. Cells were transfected with lentiCRISPRv2 (52961, Addgene) [75] carrying single guide RNAs (sgRNAs) that target MxB. The gRNA sequence was listed in S1 Table. Following selection with puromycin (0.8 μg/ml), the resistant cells were serially diluted in 96-well plates to obtain single cell clones.

HeLa and BALB/3T3 clone A31 cells stably expressing MxB were generated using the retroviral vector system pQC-XIP (Clontech) which expresses MxB.

Cell viability assay

Cell viability was measured using the cell-counting kit-8 (96992, Sigma) according to the protocol from the manufacturer. MxB/EGFP stably expressing cell lines or MxB knockout cell lines were cultured in 96-well plates with 2×103 cells per well. The MxB DNA was transfected into HeLa cells using PEI before cell viability was assessed. 10 μl cell-counting kit-8 was added into each well. After 3 hours, OD450 was measured with a microplate reader (Multiskan FC, ThermorFisher).

Cell colony assay

The LINE-1 retrotransposition assay was performed as described previously [23,26]. In brief, 1.5 × 105 cells/well were seeded in 6-well plates. After 18 hours, cells were transfected with the CMV-L1-neoRT reporter DNA with or without MxB DNA, using PEI (sigma). 48 hours post-transfection, G418 (0.75 mg/ml) was added to select for resistant cells because of LINE-1 retrotransposition. Twelve days after selection, the G418-resistant colonies were fixing with 4% paraformaldehyde, stained with 0.5% crystal violet, and scored. IAP-neoTNF or MusD-neoTNF reporter assays were similarly performed as the CMV-L1-neoRT reporter assay.

Quantification of LINE-1 cDNA by PCR

HeLa cells were transfected with MxB or empty vector and CMV-L1-neoRT reporter DNA.72 hours after transfection, total cellular DNA was extracted using the QIAamp DNA Mini kit (QIAGEN). The same amounts of DNA (250 ng) were subjected to PCR with primers L1cDNAF/L1cDNAR to amplify the reverse transcribed LINE-1 cDNA [46]. The forward primer L1cDNAR crosses neomycin resistance gene junction, thus only amplifies the spliced and reverse transcribed DNA. Levels of β-globin DNA were determined in PCR using primers β-globinF/ β-globinR. The results were used to normalize the levels of LINE-1 DNA [76]. The PCR products were separated in 1% agarose gels, and stained with Ethidium Bromide. All primer sequences are listed in S1 Table.

Quantification of LINE-1 RNA by RT-qPCR

HeLa cells were transfected with CMV-L1-neoRT reporter DNA, with or without MxB DNA. 36 hours later, cellular total RNA was extracted using Trizol reagent (Invitrogen). The same amount of total cellular RNA was treated with DNase. The RNA was dissolved in RNase free water and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instruction with the oligo dT primer. Endogenous LINE-1 RNA levels were determined by qPCR using the Luna Universal qPCR Master Mix (NEB, M3003), and analyzed with the ΔΔCT method. The primers were named 5’UTRF/5’UTRR, ORF1pF/ORF1pR, ORF2pF/ ORF2pR [45]. The GAPDH RNA was measured as the internal control. RNA levels of endogenous retrotransposition elements of mouse LINE-1 and SINE in BALB/3T3 clone A31 cells was measured by RT-qPCR with primers: mL1_TfF/mL1_TfR, mL1_GfF/mL1_GfR, mL1_AF/mL1_AR, mSineF/mSineR. The internal control is mRrm2 [77]. All primer sequences are listed in S1 Table.

Gene silencing

To knock down target gene by siRNA, HeLa cells was transfected with siRNA oligos (30 nM) by Lipofectamine RNAiMAX (Invitrogen) before plasmid transfection in 6-well plates. The knockdown efficiency was examined by Western blot. The siRNAs used were listed in S1 Table. The expression of CypA was silenced by shRNA targeting CypA (TRCN0000049228; Sigma).

Immunofluorescence microscopy

Cells were grown on coverslips before transfection with the indicated plasmid DNA. 24 hours after transfection, cells were washed with phosphate buffered saline (PBS) (pH7.2), fixed with 4% paraformaldehyde for 10 min at room temperature, followed by a 10 min permeabilization with 0.3% TX-100 at room temperature. Cells were then blocked with 5% BSA (bovine serum albumin) in PBS, and further incubated for 2 hours with primary antibodies (anti-ORF1p (1:1000 dilution), anti-γH2AX (1:1000 dilution) or anti-G3BP1 (1:1000)) at room temperature. Alexa fluor 555-conjugated donkey anti-rabbit antibody (A-21428) and Alexa fluor 647-conjugated donkey anti-mouse antibody (A-21236) were used as secondary antibodies. Confocal images were recorded with a Leica TCS SP5 (Leica Microsystems) mounted on an inverted microscope (DMI6000; Leica Microsystems), with an oil immersion 63x/NA1.4 objective len (HCX PL APO CS; Leica Microsystems).

Immunoprecipitation

Transfected HEK293T Cells were harvested and lysed in the RIPA buffer (0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 10 mM Tris [pH 7.5], 1 mM EDTA). The cell lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4°C. One milligram of the cell lysates was incubated with 50 μl of anti-Flag M2 affinity gel (A2220 Sigma) for overnight at 4°C. The beads were then washed three times with the RIPA buffer, followed by incubation with the 2X loading buffer at 95°C for 8 minutes to elute the bound proteins. The eluted materials were separated in SDS-12% PAGE, and further analyzed in Western blotting.

Operetta high-content screening

To quantify the distribution of LINE-1 RNP complex in the nucleus and the cytoplasm, cells were visualized and analyzed on an Operetta High-Content Screen system (PerkinElmer). HeLa cells were transfected with ORF1-Flag and pMxB-EGFP. 48 hours after transfection, cells were seeded in a Cell- Carrier-96 plate (6005550, PerkinElmer) in triplicates at 20000 per well. 24 hours later, cells were fixed and permeablilized, and then incubated with DAPI to stain the nuclear DNA for 10 min at room temperature. After washing with PBS, plates were scanned using the Opertta HTS imaging system (PerkinElmer) to collect images which were further analyzed using the harmony software to quantify the percentage of ORF1p in the nucleus the cytoplasm in each cell [26].

Cytoplasmic and nuclear fractionation

Subcellular fractionation was performed as described previously [78] Briefly, cells were harvested and lysed in buffer A (20 mM Tris, pH 7.6, 0.1 mM EDTA, 2 mM MgCl2, 0.5 mM NaF, 0.5 mM Na3VO4 supplemented with protease inhibitors (Sigma-Aldrich, S8830)) for 2 min at room temperature and for another 10 min on ice. Nonidet P-40 (NP-40) was then added at a final concentration of 1% (vol/vol). The lysates were homogenized by gently vortexing or inverting the tube. Cytoplasmic fraction was collected by centrifugation in a pre-chilled centrifuge at 500xg for 3 min at 4°C. The pelleted nuclei were washed three times in buffer A containing 1% NP-40, then suspended in one to two pellet volumes of the extraction buffer B (20 mM HEPES (pH 7.9), 400 mM NaCl, 25% (vol/vol) glycerol, 1 mM EDTA, 0.5 mM NaF, 0.5 mM Na3VO4 and 0.5 mM DTT), with vigorous vortex. The mixture was snap-frozen twice in liquid nitrogen and incubated for 20 min on ice. Lastly, the soluble nuclear extracts were collected by centrifugation at 20,000xg for 20 min at 4°C. To visualize nuclear ORF1p, we examined in the Western blots nuclear fraction samples from cells 5 times more than the cytoplasmic fraction samples.

To calculate the ratio of nuclear/cytoplasmic MxB, we first normalized the amount of nuclear ORF1p with the nuclear LAMN protein level, the amount of cytoplasmic ORF1p with tubulin, then nucleus/cytoplasm ORF1p ratio was calculated by dividing nuclear ORF1p value by the cytoplasm ORF1p value. At the end, the nucleus/cytoplasm ORF1p ratio of the vector control is arbitrarily set at “1”.

Western blotting

Cells were lysed in the RIPA buffer containing 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 10 mM Tris (pH 7.5), and 1 mM EDTA. The lysates were separated in SDS-polyacrylamide gel (12%) (WB1103, Beijing Biotides Biotechnology Co. Ltd.). Proteins were transferred onto nitrocellulose membranes (Whatman). The membranes were probed with the indicated antibodies, followed by incubation with IRDye secondary antibodies (1:20000; LI-COR Biotechnology). Protein bands were visualized on a LI-COR Odyssey instrument (LI-COR Biotechnology). Intensities of protein bands were determined with ImageJ (National Institutes of Health) [79].

Phylogenetic analysis

Orthologous Mx2 sequences were downloaded from Genbank. The protein sequences were aligned using Clustal Omega and converted to a codon alignment using MEGA 7. The Neighbor-Joining tree was calculated in MEGA 7 with 1,000 bootstrap replicates. The following Mx2 sequences were used: Homo sapiens MxB (NM_002463.1), Macaca mulatta (NM_001079696.1), Loxodonta Africana Mx2 (XM_023559110.1), Equus caballus Mx2 (XM_005606159.2), Sus scrofa Mx2 (NM_001097416.1), African green monkey Mx2 (XM_037984842.1), Myotis davidii Mx2 (XM_015569000.1), Bos taurus Mx2 (NM_173941.2), Ovis aries Mx2 (NM_001078652.1), Canis lupus familiaris Mx2 (XM_038443558.1), Vulpes vulpes Mx2 (XM_025983361.1), Mustela putorius furo Mx2 (XM_013061286.1), Ursus maritinus Mx2 (XM_008710207.2), Canis lupus familiaris Mx1 (NM_001003134.1), Eumetopias jubatus Mx1 (XM_028126469.1), Myotis davidii Mx1 (XM_006754326.2), Homo sapiens MxA (NM_001178046.3), Macaca mulatta Mx1 (NM_001079693.2), Sus scrofa Mx1 (NM_214061.2), Bos taurus Mx1 (NM_173940.2), Ovis aries Mx1 (NM_001009753.1), Mus musculus Mx1 (NM_010846.1), Rattus norvegicus Mx1 (NM_001271058.1), Mus musculus Mx2 (NM_013606.1); Rattus norvegicus Mx2 (NM_134350.2).

Statistics

All experiments were performed three or more times independently under similar conditions. All data were plotted as mean values, with variation as SEM. Statistical significance was calculated by Student’s two-tailed t test. P values of statistical significance are represented as *** P<0.001; **P <0.01; *P<0.05.

MxB diminishes L1 activity.

(A) Illustration of the L1-neoRT reporter cassette. CMV-L1-neoRT reporter DNA contains the complete human LINE-1 DNA and a neomycin resistance gene as a reporter of LINE-1 retrotransposition and a CMV promoter before 5’UTR. (B) HeLa cells were transfected with CMV-L1-neoRT, defective L1-ORF1 (RR261/262AA) or pEGFP-N1 (carrying neomycin resistant gene) DNA together with MxA or MxB DNA. G418-resistant cell colonies were scored and results of three independent experiments are presented in the bar graph. Images of representative colony assays are shown. Ectopic expression of MxA-Flag or MxB-Flag was examined by Western blot. (C) HeLa cells were transfected with CMV-L1-neoRT or pEGFP-N1 DNA together with increasing doses of MxB DNA. Results of three independent experiments are presented in the bar graphs. (D) Cell viability of HeLa cells transfected with different doses of MxB-Flag plasmid DNA for 48h. (E) HEK293T cells were co-transfected with the CMV-L1-neoRT DNA and MxB DNA. Levels of the newly synthesized LINE-1 DNA were determined by semi-quantitative PCR. Levels of β-globin DNA were measured as internal controls. Intensities of DNA bands were quantified, the results are summarized in the bar graph (mean ± SEM; paired t-test). *, P<0.05; **, P< 0.01; ***, P<0.001.

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MxB knockout with CRISPR-Cas9.

(A) MxB RNA levels in human tissues. Consensus Normalized eXpression (NX) levels for 55 tissue types and 6 blood cell types, created by combining the data from the three transcriptomics datasets (HPA, GTEx and FANTOM5), reference from www.proteinatlas.org. (B) The two guide RNA sequences are shown in red letters. The protospacer adjacent motifs (PAMs) are shown in orange letters. The mutated MxB sequences at the gRNA target sites are presented for each cell clone which was selected and used in this study. (C, D) Endogenous MxB protein and its mRNA level were determined with Western blot and RT-qPCR after stimulation with IFNα1 (25 ng/mL) for 16 hours. (E, F) Cell viability and cell growth rate of MxB knockout cell lines (E) and stable MxB-expressing cell line (F) were detected by cell-counting kit-8. (G) Colony assay was performed with MxB knockout or control cell lines which were transfected with CMV-L1-neoRT plasmid (500 ng) for 24 hours and treated with IFNα1 (2.5 ng/mL) for 24 hours. The results was presented in the bar graph (mean ± SEM; paired t-test). ns, not significant. *, P<0.1; **; P<0.01; ***, P<0.001.

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Co-localization of MxB with L1 ORF1p.

(A, B) HeLa cells were co-transfected with MxB-EGFP and CMV-L1-neoRT reporter (A) or ORF1p-Flag DNA (B). Distribution of MxB and ORF1p was examined by immunofluorescence microscopy. The white arrows showed nuclear ORF1p. Fluorescence intensity analysis was performed at the position which indicated by white lines in merge panel to quantify the co-localization of EGFP or MxB-EGFP with ORF1p (A). Co-localization analyzed with fluorescence intensity used software from LAS AF (Leica). Scale bar, 10 μm.

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CMV-L1-neoRT induces γH2AX in MxB knockout cells.

(A) Western blot to detect γH2AX in the control or MxB knockout HeLa cells which were transfected with CMV-L1-neoRT plasmid. (B, C) Detection of γH2AX foci in the control and MxB knockout cells transfected with CMV-L1-neoRT plasmid. The γH2AX foci were scored in 50 cells, the results are presented in (C) (mean ± SEM; paired t-test). ** indicates P<0.01; ***, P<0.001. Scale bar, 10 μm.

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Co-localization of stably expressed MxB-EGFP with LINE-1 RNP and TIA1 in HeLa cells.

(A, B) Co-localization of stably expressed MxB-EGFP with ORF1p and TIA1 in HeLa cells. ORF1p was either expressed from the transfected CMV-L1-neoRT DNA (500ng) (A) or ORF1p vector DNA (500ng) (B) for 24 hours. White arrows indicate nuclear ORF1p. ORF1p/TIA1-containing SGs were scored in more than 50 cells for each treatment. The average number of ORF1p-containing SGs per cell is presented in the bar graph (mean ± SEM; paired t-test). ns, no significant. (C) HeLa cells were co-transfection with MxB-Flag (500 ng), LINE-1-ms2x6 (750 ng) and MS2-GFP (250 ng) plasmid DNA. Subcellular location of LINE-1 RNA was indicated by the binding of MS2-GFP to the 6 MS2-binding sites in the LINE-1 RNA. Scale bar, 10 μm.

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Co-localization of MxB, ORF1p and G3BP1/TIA1 with transiently transfected MxB.

(A, B) Immunofluorescence microscopy to detect ORF1p, G3BP1 and MxB in HeLa cells co-transfected with MxB-EGFP (500ng) and CMV-L1-neoRT DNA (500ng) (A) or the ORF1p-Flag DNA (500ng) (B) for 24 hours. The white arrows indicate nuclear ORF1p. (C) The MxB-EGFP stably expressing HeLa cell lines were transfected with siRNAs targeting G3BP1 and then transfected with CMV-L1-neoRT DNA (500ng). Immunofluorescence was performed to detect ORF1p, G3BP1 and MxB 24 hours after transfection. Expression of G3BP1 was examined by Western blot. Scale bar, 10 μm. (D, E) Immunofluorescence was performed to detect ORF1p, TIA1 and MxB in HeLa cells co-transfected with MxB and CMV-L1-neoRT DNA (500ng) (C) or the ORF1p-Flag DNA (500ng) (D). The white arrows indicate nuclear ORF1p. (F) MxB-EGFP stably expressing HeLa cells were transfected with siRNAs targeting TIA1 and then transfected with CMV-L1-neoRT DNA. Immunofluorescence was performed to detect ORF1p, TIA1 and MxB 24 hours post transfection. Expression of TIA1 was examined by Western blot. ORF1p-containing SGs were scored in more than 50 cells for each treatment. The average number of ORF1p-containing SGs per cell is presented in the bar graph (mean ± SEM; paired t-test) in (C and F). ***, P<0.001. Scale bar, 10 μm.

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MxB does not associate with stress granules induced by arsenite.

(A) HeLa cells were treated with IFN-α (25 ng/mL) for 24 hours, followed by exposure to arsenite (500 μM) for 30 min. G3BP1 and MxB were detected by immunostaining and fluorescence microscopy. (B) HeLa cells which stably express MxB-EGFP were treated with arsenite (500 μM) for 30 min. G3BP1 and MxB were detected by immunofluorescence microscopy. (C, D) MxB knockout cells were transfected CMV-L1-neoRT DNA (D) or vector control (C) for 24 hours, and then treated with arsenite (500 μM) for 30 min. SGs number was scored in more than 50 cells for each treatment, the results of three independent experiments are presented in the bar graph. The average number of SGs per cell is presented in the bar graph (mean ± SEM; paired t-test) in (C and D). ns, not significant. ***, P<0.001. Scale bar, 10 μm.

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MxB inhibits LINE-1 independent of NUP214, TNPO1 and CypA.

(A, B) CypA was knocked down before MxB and CMV-L1-neoRT were co-transfected into HeLa cells. Western blot was performed 48 hours post transfection to measure CypA and MxB levels (A). Meanwhile, G418 was added at 48 hours post transfection. G418 resistant colonies were scored and results of three independent experiments are presented in the bar graph (B). (C, D) Endogenous NUP214 (C), TNPO1 (D), CMV-L1-neoRT ORF1p and stably expressed MxB-EGPF were detected by immunofluorescence. (E, F) Effect of knocking down endogenous NUP214 (E) or TNPO1 (F) on MxB inhibition of CMV-L1-neoRT in HeLa cells. Results of three independent experiments are presented in the bar graph (mean ± SEM; paired t-test). Images of representative colony assays are shown. Expression of NUP214 and TNPO1 was examined by Western blot. ns, not significant. ** indicates P<0.01; ***, P<0.001. Scale bar, 10 μm.

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Effect of MxB mutations on association and subcellular localization with LINE-1 RNP.

(A) Immunofluorescence microscopy analysis of ORF1p, G3BP1 and MxB in HeLa cells co-transfected with CMV-L1-neoRT and MxB or its mutants. Scale bar, 10 μm. (B) 293T cells were co-transfected with CMV-L1-neoRT DNA and MxB-Flag or its mutants. Immunoprecipitation was performed with anti-Flag antibody 48 hours post transfection. Presence of ORF1p in the precipitated materials was detected by Western blot. (C) HeLa cells were co-transfected with CMV-L1-neoRT and MxB or its mutant DNA. Nuclear and cytoplasmic fractions were prepared and further examined in Western blot to determine the levels of ORF1p. The bar graph at the S9C right was the quantification of the immunoblot. (D) The Operetta High-Content Screen system (PerkinElmer) was utilized to determine the ratios of the nuclear and cytoplasmic ORF1p. The results are summarized in the bar graph (mean ± SEM; paired t-test). ns, not significant. **, P<0.01; ***, P<0.001.

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Click here for additional data file.

Phylogenetic analysis of of Mx genes from different species.

Neighbor-Joining method was used for the evolution analysis. Orthologous Mx2 sequences were acquired from Genbank. The protein sequences were aligned using Clustal Omega and converted to a codon alignment using MEGA 7. The Neighbor-Joining tree was calculated in MEGA 7 with 1,000 bootstrap replicates. Bootstrap values (>70) were also tested. Mx genes were selected from MxA-like and MxB-like major clades. “*” indicates the Mx genes that were investigated in this study for inhibiting LINE-1 retrotransposition.

(TIF)

Click here for additional data file.

Primers and RNAs used in this study.

(DOCX)

Click here for additional data file.

10 Sep 2021

Dear Dr Guo,

Thank you very much for submitting your Research Article entitled 'MxB inhibits Long interspersed element 1 retrotransposition' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by four independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. In particular, the reviewers found that the claims of relocalization or sequestration of ORF1p by MxB were not well supported by the immunofluorescence data as presented. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

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Reviewer's Responses to Questions

Comments to the Authors:

Reviewer #1: In this work Huang and colleagues investigate the inhibitory activity of the interferon stimulated protein MXB on the transposable element LINE-1. Authors describe how ectopically expressed MXB inhibits integration of LINE-1 in HeLa cells. Authors present evidence for the interaction and colocalization of MXB and ORF1P, in a complex that also colocalize with stress granule marker G3BP1. For inhibition to happen, MXB requires its N-terminal nuclear envelope localization signal, its GTPase and oligomerization activities, as well as the presence of stress granules components G3BP1 and TIA1. In a final set of experiments, they broaden the spectrum of MXB inhibition to other transposable elements such as IAP and Mus D.

This paper is interesting because it further widens the functions of MXB (MX2) as an "antiviral" factor, addressing its activity against retrotransposon elements. The notion that MXB itself has a potent inhibitory activity on restricting LINE-1 nuclear transport could be specially interesting for tissues with a “high” basal levels of MXB (such as granulocytes). In addition, it provides further insight on the tight regulation of transposable elements.

However, I think the paper has a series of weaknesses.

1. MXB is an Interferon (IFN) stimulated gene and in most cells, is virtually absent in the absent of IFN stimulation. On the other hand, LINE-1 has been linked to type 1 IFN induction before. However, the authors fail to make this connection. Is LINE-1 inducing an IFN response and then upregulating MXB? Or is this mechanism (LINE-1 inhibition) only valid for cells/tissues with some basal level of MXB? An easy experiment to link MXB to the inhibition of LINE-1 would be adding IFN to a CRISPR control cell line and to the MXB KO lines and quantify the extent of the IFN inhibition, where it should be reduced for the MXB KO lines.

2. Authors claim that nuclear envelope localization of MXB is an important determinant for the LINE-1 inhibitory mechanism. In the case of HIV-1, several articles have linked the importance of nuclear pore proteins to the MXB antiviral activity. I am missing some evidence on this regard. Are any nuclear pore proteins involved in LINE-1 inhibition by MXB? Are any nuclear pore proteins located to the MXB-containing stress granules?

3. Authors claim that MXB and ORF1P accumulates to stress granules and that transient depletion of G3BP1 or TIA1 depletes MXB of its inhibitory activity. I would like to see further confirmation of this. Specifically, will depletion of G3BP1 and/or TIA1 affect the accumulation of ORF1P and MXB to cytoplasmic bodies? I don’t think figures 4e and 4f really show the formation of stress granules.

4. I am not convinced by the microscopy throughout the paper, specifically:

a. Fig 2: MXB distribution should be the same in the EGFP panel for the MXB L1 NEO condition of fig 2a and the MXB-EGFP + L1 NEO panel from S3a, and in the Flag panel for the MXB-Flag condition in fig 2c. However, there is no MXB located to the nuclear envelope in fig 2c, while this is clearly noticeable in fig 2a and S3a.

b. Fig 6C: again, I am not convinced by this figure. None of the nuclear envelope-located proteins seem to be locating there. Staining with MXB and/or nucleoporins creates a tight ring around the nucleus (see for example Kane et al., 2018), while in here there is some space between the DAPI signal and the Flag signal, as if between the nucleus and the nuclear envelope was some “empty” space. In the case of human MXB there is not even such a ring, but just aggregation of protein in the proximity of the nucleus.

c. Lines 196-197: “in agreement with the inability of these MxB mutants to interact with G3BP1 or ORF1p, (Fig. S6A and B)”. This is not what I understand from S6a and S6b. Firstly, if I am not mistaken, in S6a colocalization of MXB, G3BP1 and L1 is indicated by the light-pink colour on the merge image. While this might be absent in the case of del25 and the M574D/Y651D mutant, I can see it in the K131A and R449D/F495D mutants.

5. Figure legends are too succinct. Some of the figures are virtually impossible to understand looking only at the legend, in others some graphics are not explained (as is the case of the arrows in microscopy images) and in most of them further explanation is needed to understand what they are showing.

In addition, I have other comments:

1. Author summary needs some correction, number of grammatical mistakes and apparent lack of text, such as:

a. Line 30: “Retrotransposons have been existed as ancient components”.

b. Line 32: “and inserts in genome by its endonuclease, which staggers”.

c. Line 35: “Among the risk of LINE-1 out of control, host develops”.

d. RNP is not defined

2. Line 70: HBV, a reference is missing.

3. Fig S1: It is not clear to me how the colony assay is quantified. It is not specified in M&M (where it probably should be), and in the legend of fig S1 it reads “Number of neomycin-resistant colonies with the control vector is arbitrarily set as 1”. I assume authors mean set as 100. Nevertheless, in figures depicting this assay (such as S1b and 1a &1b), the average of the vector control is not 100 (in S1b for example is clearly above it). Taking again S1b as an example, it seems that only one of the dots has a value of exactly 100, while the other 2 are clearly higher. Does this mean that one of the vector control experiments was set as a 100, and then all the other results (for vector and MX proteins) normalized to this value, rather than on each experiment setting the vector control to 100 and then normalizing the MX proteins to it?

4. Fig S1d doesn’t have a legend. In addition, -globin is misspelled as, -glubin.

5. Fig S2: The order of the different panels should be modified: S2e is presented in the text before any other panel from S2.

6. S2b: the level of MXB in both lines seems the same to me. One of the ref given on the text for previously showing presence of MXB in HeLa cells in the absence of IFN doesn’t show that, on the contrary, there is a total absence of MXB in the no IFN lane (see fig 1g from ref 36).

7. S2d: immunblot lacks enough quality to draw any conclusions from it.

8. Line 123: a “was” is missing.

9. Line 129: experiments instead of experiment.

10. Fig 2H: wouldn’t be expected an increased signal for L1ORF1 in the cytoplasm as the nuclear accumulation is reduced under MXB expression?

11. Fig 2I: I fail to see any signal for L1ORF1 in the nucleus.

12. Fig 2j: why is there less ORF1 p positive cells when expressing EGFP than when expressing MXB-EGFP?

13. Fig 3a: shouldn’t the level of L1ORF1 been reduced under MXB expression? This was shown in fig 1c.

14. Fig 3b: the intensity of the H2AX staining needs to be increased. It is quite difficult to see it as it is now.

15. Fig 3e: needs more intensity.

16. Fig 3g: I am not convinced by these results. First, what is mock here? No explanation in the figure legend. Second, why is there a marked increase of H2AX from mock to EN- if the level of ORF1P is the same? Third, I do see a small reduction of the H2AX signal in the EN- under MXB expression, is this statistically different from the reduction seen in the EN+ condition?

17. Fig 4a and 4b: the arrows shown are not mentioned in the figure legend, what are they signalling?

18. Fig 4b: why is the distribution of ORF1P more diffuse in the absence of MXB (also seem in S4a)? This is not the case in Fig 4a.

19. Fig 4c: Why is MXB not localize to the nuclear envelope here?

20. Lines 152-153: it reads “Indeed, the MxB/ORF1p foci had G3BP1 and TIA1 in HeLa cells that stably expressed MxB and were transfected with CMV-L1-neoRT reporter (Fig. 4A)”. However, TIA1 staining is not shown.

21. S4a: figure labelling contains a MXBE instead of MXB.

22. Fig 4d: it would enrich the paper seeing also the interaction with TIA1.

23. Line 185: MXB-B25 should be MXA-B25 or even MXA+B25, to keep consistency with line 181.

24. Fig 5a: there is no explanation for the MXB domains labelling.

25. Fig 5b: why is MXA aggregating in the cytoplasm? Previously it has been reported as a diffuse cytoplasmic protein (see for example Goujon et al., 2014 or Betancor et al., 2019) and it behaves this way in fig 6c. Also, none of the proteins with a nuclear envelope localization signal (MXB and MXA+B25) or an NLS (MXBdel25 + NLS) seems to accumulate at the nuclear envelope or at the nucleus.

26. In previous works, experiments using MXA chimaeras bearing the N-terminal domain of MXB and showing transfer of the antiviral activity have used MXA proteins where the whole N-terminal domain of MXA has been substituted with the whole N-terminal domain of MXB (see for example Goujon et al., 2014). I would recommend doing this experiment rather than attaching the first 25 amino acids of MXB to MXA, since this could potentially interfere with the function of the N-terminal domain.

27. Line 194: should say mutant M574D/Y651D instead of M574D, Y651D

28. Lines 194-195: since the authors are describing mutants, they should state they are using the R449D/F495D mutant, instead of saying they also mutate those 2 positions, since in the current format the text is not clear on whether they created 2 independent mutants or a double mutant (as they did).

29. Fig S6: I found confusing the arrangement of the figure. It seems like both bar charts at the bottom belong to S6d. The lack of any mention to the quantification of the immunoblots from S6c makes it more confusing. A further explanation on the figure legend is required.

30. The IP showed in S6b doesn’t show the lack of interaction of MXB mutants with L1ORF1: there is a clear signal for the latter in all the lanes and in the case of the vector control. Better experiments are needed to be able to make this claim.

31. Line 225: what are IAP and MusD?

32. Fig 6e: colour code description of the bars is missing in the figure.

33. Lines 257-258: “MxB inhibition of LINE-1 is CypA-independent (data not shown)”. While ideally such a claim should be accompanied by the data showing this, authors could further explained how they found this (CRISPR KO?, siRNA? Use of cyclosporine?, etc).

34. Line 288: remove “in” at the end of the line.

35. Line 289: a space is missing before “(65)”. Also see point 45.

36. Line 303: “Equus caballus Mx2, but not Ovis aries Mx2, inhibits LINE-1”. This claim is not supported by data in Fig 6B, since there are less colonies in cells expressing Ovis MX2 than in cells expressing MXA (with statistical significance).

37. Line 320: a “the” is missing in “D205A point mutation in endonuclease”.

38. Lines 321-322: the sentence “IAP-neoTNF, MusD-neoTNF reporter plasmids which contain mouse LTR retrotransposon and reverse direction neomycin resistance cassette(19)” is missing something.

39. In general, the “plasmids and antibodies” part of M&M needs rewriting since there are several grammatical mistakes.

40. Line 325: the mutation is M574 not N574.

41. Line 326: how is the MXB-EGFP generated? Is EGFP at the N or C-terminal end of MXB?

42. Line 333: is missing “antibody” at the end.

43. Line 335: is also missing antibody after “(05-1938)”.

44. Line 336: is Millipore

45. Throughout the text there are many spaces missing between the text and the reference call.

46. In “cell colony assay” an explanation on how the results are normalized is missing.

47. The way the number of hours is depicted is not consistent throughout the text. For example, in line 358 it says “18 hours”, while in line 367 it says “seventy-two hours”

48. Line 401: the “1,000” is the only time authors used a “,” all the other 1000 are without it. Also, some other numbers throughout the text are depicted with or without “,”. Consistency is needed.

49. Line 412: “were” should be “was”.

50. The M&M are in general too summarized. In most cases the amount of DNA transfected or the time after transfection is not indicated.

51. Line 432: “Na3VO4” should be written as Na3VO4.

52. Line 439: Should say “by centrifugation” instead of “with centrifugation”.

53. Line 443: What viruses?

Reviewer #2: In their manuscript “MxB inhibits Long Interspersed Element 1 retrotransposition”, Huang et al. demonstrate that the cellular antiviral protein MxB blocks L1 retrotransposition in cultured cells, and then investigate the mechanism by which this inhibition occurs using cultured cell retrotransposition assays, immunofluorescence imagining, and biochemistry approaches. Over all, the manuscript presents results that will be interesting to the L1 and virology fields. However, some claims are over-stated particularly with regards to the mechanism of inhibition, and some of the experiments could be presented more clearly particularly in the figures and legends.

Major points:

1. MxB was demonstrated to restrict L1 retrotransposition by Goodier and colleagues in 2015 (PMID 26001115). This publication is mentioned in the discussion of the present manuscript, but should be mentioned in the abstract or introduction. Otherwise the manuscript gives the initial impression that L1 inhibition by MxB is a novel finding. In addition, Doucet et al. (PMID: 20949108) used epitope-tagging strategies to investigate the subcellular localization of L1 proteins and RNA. This work should be cited.

2 While the results as a whole demonstrate that MxB mediates the localization of L1 ORF1p to stress granules, the conclusion that MxB inhibits L1 specifically by preventing L1 RNPs from entering the nucleus is not sufficiently supported. Figure 2 is titled with this claim, but the immunofluorescence images in A-C do not convincingly or quantitatively show ORF1p localizing to the nucleus in the absence of MxB, and previous studies have shown that wild-type ORF1p does not normally localize to the nucleus (PMID 29309036, PMID 20949108, PMID 20147320). Figure J claims to show this quantitatively using IF, but YFP (26.2 kDa) is a very bulky tag and is likely to affect cellular localization of ORF1p (40 kDa). This experiment would be much stronger if it utilized a smaller epitope tag already demonstrated to minimally impact L1 retrotransposition (eg, T7--PMID 20949108, PMID 16183655)

3 The fractionation experiment in panels H and I is a bit confusing: in H, the amount of ORF1p found in the nucleus with vector-only is dramatically more than in the cytoplasm, but this pattern is not supported by the immunofluorescence experiments in panel A. The quantitation shown in panels H and I should be better explained—the actual ratio of ORF1p in nucleus/cytoplasm for vector control, for example, is clearly not 1. How was this normalized? In addition, in panel I the detection of endogenous ORF1p in HeLa cells is not supported by previous studies, which describe HeLa cells to express very little or no ORF1p (PMID 20686575). Over all, the claim that MxB prevents nuclear import of L1 RNPs should be softened.

4 In the paragraph beginning with line 100, the authors say that the reduced detection of L1 RNA and ORF1p is consistent with MxB inhibiting retrotransposition. The rationale for this statement should be clarified—if the authors are proposing a sequestration model (rather than an inhibition of L1 expression), one would not necessarily expect the L1 RNA and protein levels to be affected.

5 Throughout the manuscript, the figure legends and labels, particularly on panels showing western blots, could be clearer and more informative. For example, many IF images have white arrows but it is never stated what these indicate. In figure 3A, the gel segment labeled “EGFP” shows detection in the MxB-EGFP transfection but not in the EGFP alone transfection. Presumably this is because the molecular weights of MxB-EGFP and EGFP alone are different, but this should be clarified. The figure legend for supplemental figure 1D is missing entirely.

6 Throughout the paper, sequestration of ORF1p by MxB is only shown by IF for exogenously expressed ORF1p. Given that the authors appear to have an antibody that apparently can detect endogenous ORF1p by western blot, it would greatly strengthen the paper to demonstrate that MxB sequesters endogenous ORF1p via immunofluorescence.

Minor comments

1. Line 54: cite a reference for the number of human genetic disease cases attributed to L1-mediated mutagenesis.

2. Line 69: Would be informative to discuss in more detail the mechanism(s) by which MxB and MxA inhibit viruses.

3. Line 86: state quantitatively what the “lowest amount” of plasmid was.

4. Figure 6D: which mouse L1 retrotransposition constructs were used? No papers cited and not shown in methods.

Reviewer #3: In this manuscript, Huang et al. invested the mechanism of MxB-mediated LINE-1 suppression. They started by confirming this reported phenomenon in a neomycin-resistance-based LINE-1 retrotransposition assay. Then IF and IP experiments were conducted, results of which suggested that MxB and LINE-1 ORF1p co-localizes in some dot-like organelles, which were later determined as stress granules. The authors further demonstrated the MxB suppresses LINE-1 replication through stress granule pathway, because shutting down expression of SG-associated proteins such as G3BP1 and TIA1 significantly compromises MxB’s efficiency in LINE-1 inhibition. Tests with mutated MxB indicated that nuclear membrane localization, oligomerization, and GTPase activity are all essential for MxB to suppress LINE-1. At last, the authors presented evidence suggesting that only some members of the Mx protein family are effective in LINE-1 regulation, and MxB can suppress other retrotransposons such as murine LINE-1, IAP, and MusD.

Taken together, this study suggested that changing the subcellular localization of ORF1p in host cells is the key mechanism for MxB-mediated LINE-1 suppression. And combining with previous reports, restricting ORF1p onto SG and/or p-bodies might be a common mechanism shared by various LINE-1 suppressors. Such information is both interesting and important for the LINE-1 community, and might shed light on future research in the Genetics and Genomics field. However, I am not fully convinced because some conclusions in this manuscript were not fully supported, while some phenomena were not fully investigated.

Major issues:

I first found it impressive that the authors tested MxB through three different systems: knockout, stable expression, and transient expression. And then I found myself confused because at most times it was difficult to find out which system the authors used for a statement in the text or a panel in a figure. For instance, the authors performed IP experiments in Fig. 2D, E, and F, but there was no description whether the experiments where performed in MxB stably expressed or transiently expressed HeLa cells. It would be nice if the authors clearly state which system was used when describing data in the text.

Some of the statements were not logical. In Line 121, the authors stated, “The association of MxB with LINE-1 RNA…”, while no LINE-1 RNA detection was performed with MxB interactants. The interaction between ORF1p and MxB cannot guarantee the binding between MxB and LINE-1 RNA. Because it is possible that, by binding ORF1p, MxB can sabotage the interaction between ORF1p and LINE-1 RNA. Besides, Fig. 2G needs improvement, as the ORF1p bands in the IP samples are hard to see, esp. when comparing to previous results in Fig. 2D, E, and F.

The use of Fig. S7 is also puzzling. Though it separates different Mx proteins from different species, the result had no impact on the selection of Mx proteins for subsequent LINE-1 retrotransposition assay. My best guess is that the authors chose some Mx proteins from each branch for subsequent tests. If true, the authors should elaborate the reason(s) in more details. Also, the procedures for the reconstruction of the phylogenetic tree was not mentioned. In addition, the tree should be bootstrap tested, with bootstrap values (>70) shown on the nodes of the tree to indicate the credibility. There is only one panel in Fig. S7, so the panel letter “A” is not necessary.

Some of the experiments were poorly designed and/or presented. For example, the authors should at least show one negative result for neomycin selection in Fig. S1B where the Neo-based LINE-1 retrotransposition assay is first presented. Such a negative result can be generated by transfecting HeLa cells with any retrotransposition-incompetent vectors (such as pJM111) reported by Moran et al. (Cell 1996), and can help to confirm LINE-1 retrotransposition events shown in Fig. S1B.

Why no elevation of ORF1p expression could be detected in cells transfected with LINE-1-EN- or LINE-1-EN+ in Fig. 3G?

Lines 149-154: The authors mentioned TIA1 here but no TIA1 images were shown in Fig. 4A and B.

Which experiment was conducted to generate Fig. 6E? And why some of the “relative mRNA levels” were not set as 1 for vector (control) groups?

Fig. S6B is not acceptable. The ORF1p band detected in the IP eluant of the negative control (i.e., the vector group) significantly compromises the interpretation of the whole IP results. The authors should repeat this experiment to achieve a better image as they did in Fig. 2D-F.

And the investigation for observed phenomena was not thorough in some cases. In Lines 100-108, the authors suggested that by suppressing LINE-1 retrotransposition, MxB has an impact on the expression of ORF1p. However, the authors completely neglected the possibility that MxB might induce the depletion of ORF1p, even when they later found that MxB can interact with ORF1p. Such an idea can be verified simply through Western blotting tests on cells transfected with ORF1p and MxB expressing vectors.

In Lines 165-171, the authors concluded that “MxB is located to G3BP1- and TIA1-bearing foci through interaction with ORF1p.” This also means the presence of ORF1p also altered the subcellular distribution of MxB which normally associates with nuclear membrane. The authors should test it to confirm, as such a phenomenon might interrupt other functions of MxB.

MxB suppressing murine SINE, IAP, and MusD is another example. Although it is impressive to known that MxB has a wider spectrum in regulating retroelements, these data introduce confusion to the study. To suppress LINE-1, MxB alters the subcellular localization of ORF1p. If ORF1p is an important component for the replication of murine SINE, IAP, and MusD, the authors should state it clearly, with proper references. If not, then the authors should investigate or at least speculate (with reasonable facts) whether MxB suppresses these retroelements through similar mechanisms.

In Discussion, the authors should provide possible reason(s) why the GTPase activity is essential for MxB-mediated LINE-1 suppression, or ORF1p re-localization.

Minor issues:

Text:

Full name for LINE-1 should be corrected to "long interspersed element type 1". No "nuclear" in the name. This convention dates back to early eighties (see Singer MF, Cell 1982). “L1” is also ok, which was used in the manuscript (e.g., L1-neo) but the authors failed to explain what “L1” strand for.

The authors used both “ORF1” and “ORF1p” to refer the open reading frame encoding the 40kDa protein. Be consistent. And please notice that “ORF1p” with the small p in most LINE-1-related literatures indicates the protein but not the ORF.

In Line 46, the authors stated, “LINE-1 is still active”, which can be misleading. Among ~500,000 LINE-1 copies in one single cell, only a very small fraction of them are competent in retrotransposition (this has been mentioned in many LINE-1 research papers and reviews). In addition, the word “active” is confusing, as many LINE-1 copies, though incompetent in replication, are active in transcription.

In Line 63, the authors stated, “digesting LINE-1 DNA by TREX-1”, while Li et al. reported that the exonuclease activity is not involved in TREX1-mediated LINE-1 suppression; instead, TREX1 inhibits LINE-1 by inducing the proteasome-mediated proteolysis of ORF1p (NAR 2017).

What did the authors mean by “LINE-1 DNA production” in Line 88?

Line 149: I believe the word “APOBC” here should be “APOBEC”.

The following statements need to be referenced:

Line 53: Not surprisingly,…and Alu insertion.

Line 70: HBV(ref) (I believe the authors meant to insert references here but forgot).

Figure:

Some of the figure legends are sloppy and confusing. For example, the description for Fig. 3G focused on tested HeLa cells, while the panel actually shows Western blotting results. The authors should correct such errors.

Fig. 2A, B, and C: Transfected vectors should be shown clearly by each set of images. Also, “LINE-1-MS2” in Fig. 2C is not a proper description, as green fluorescence can only indicate the localization of MS2-GFP, but not its status for LINE-1 RNA binding.

Fig. 2H and I: explain what the letters “C” and “N” stand for. And I suggest that the space between panel H and I should be increased. In current form, it is hard to determine whether the label “L1-Neo” belongs to H or I.

Bar charts in Fig. 2H, I, and J, and Fig. S6: the titles of y-axes should be changed, as it is not difficult to determine which protein has been targeted for nucleocytoplasm distribution.

Fig. 4A and B: was it MxB or MxB-EGFP used in these tests?

Fig. 6D: what is “huMaB” that was shown in the bar chart?

Explain those arrows used in Fig. 2A, 2B, 4A, 4B, S3, and S4.

In CMV-LINE-1-neoRT (the authors needs to confirm whether it is the pJM101 plasmid used in Moran et al. Cell 1996), there is a CMV promoter next to the 5’ side of LINE-1 5’-UTR. The authors missed it in Fig. S1A.

In the figure legend for S1B, it was stated that, “Number of neomycin-resistant colonies with the control vector is arbitrarily set as 1”. However, the y axes of bar charts in S1B (and other LINE-1 assay results) are showing numbers of colonies. And it should not be labeled as “colony assay” in the bar chart, which is a test but not a result. A proper title for the y-axes in bar charts showing neomycin-resistance-based L1 assay results can be found in Herrmann et al. Mol DNA 2018.

There is no description in the figure legend for Fig. S1D.

Fig. S4A: What is “MxBE”?

Explain the difference between Fig. S6D and the bar chart next to it (on the right)

Reviewer #4: The present study from Haung et al. investigates the anti-viral restriction factor MxB as also an inhibitor of LINE-1 retrotransposons. Some data reported here duplicates that of a previous report by Goodier et al. (2015, PLoS Genetics) including that MxB (but not MxA) decreases L1 retrotransposition, that MxB associates with L1 ORF1p in an RNA-dependent manner, and that the K131 MxB mutation which affects GTP binding restores retrotransposition. (These confirmatory data should be noted as such).

New data include demonstrating increase in L1 retrotransposition and endogenous L1 RNA and protein in an MxB KO cell line, decrease in L1 RNA and protein in a MxB knock-in cell line, and that MxB associates with the L1 RNP in cytoplasmic stress granules. Furthermore, the authors report that MxB reduces the amount of nuclear vs cytoplasmic ORF1p with a consequent reduction in H2AX-detected DNA damage, and that inhibition of stress granule components limits MxB inhibition of the L1. Thus, this study expands considerably on the previous limited work and proposes a mechanism for the effect of MxB on LINE-1s. As such it is worthy of consideration for publication in PLoS Genetics. However, while detailed supporting data has already been presented, I believe some additional controls and experiments are required to more strongly support the interesting claims made in this paper.

MAJOR:

There are no obvious specifically stated toxicity and cell growth rate assays for potential effects of MxB in transfected and knock-out cell lines. These should be shown. The N1-EGFP transfection control (p. 4, line 83) might be considered as an toxicity assay.

p. 4, line 111 (also abstract, p. 11, line 240, p. 12, line 248). The section title and conclusion, "MxB sequesters LINE-1 ORF1p in cytoplasmic bodies" is not necessarily correct. If this is claimed, the numbers of cells with ORF1p in SGs in the presence or absence of overexpressed MxB should be counted. It is stated on p. 13, line 272 that "we observed much greater LINE-1 ORF1p localization to G3BP1/MxB-positive cytoplasmic granules compared to the relatively more dispersed cytoplasmic distribution of ORF1p in the absence of MxB expression ((Fig. 4A, B and S4A, B)." If so this needs to be backed up by cell count quantification. In our experience overexpressed EGFP-ORF1p forms many stress granules in numbers of unstressed cell lines, including HeLa cells.

Furthermore, on p. 8, line 165 (also p. 14, line 291) it is noted that ectopic or endogenous MxB does not form arsenite-induced SGs in the absence of transfected ORF1p. That would suggest that overexpressed ORF1p sequesters MxB in SGs rather than the other way around. However, Fig. S3A, second row shows MxB-EGFP alone forming many SGs; furthermore, in the fourth row there are a significant number of ORF1p SGs that do not contain MxB protein -- so this is confusing regarding the authors' conclusion.

The definitive experiment is to see in the MxB KO cell line vs its wild-type control if overexpressed and endogenous ORF1p forms fewer stress granules/in fewer cells (both unstressed and arsenite-stressed). If the above claim is to be made, these experiments should be done. The monoclonal 4H1 anti-ORF1p antibody from Millipore is a good one; I believe it is available in China.

Fig. 3, F. This is unclear to me: are the controls and KO cell lines transfected with the L1? If not, is there any data for transfected cells?

Also, Fig. 3G, and p. 7, line 142. It is stated in the text that, "As a control, the endonuclease inactivated LINE-1 (EN-) did not increase γH2AX foci, and MxB overexpression had no effect (Fig. 3G)." No cell counts are presented in this figure to show the effect of the EN- control on γH2AX foci production: these should be shown. Also it is stated, "As a control, the endonuclease inactivated LINE-1 (EN-) did not increase γH2AX foci". However, there is a significant increase in γH2AX protein in both EN- and EN-transfected cells, which seems contradictory. Quantification of foci numbers could help in clarification.

p. 8, line 169. Also, it is concluded "Together, these data demonstrate that MxB associates with LINE-1 RNP in the stress granules, thus prevents nuclear import of LINE-1 proteins and diminishes LINE-1 retrotransposition." The authors are implying that MxB sequesters ORF1p in SGs to prevent further function. However, as noted above, to me the converse appears true, that ORF1p sequesters MxB.

Fig. 4D. The IP of G3BP1 by MxB is not entirely convincing due to background presence of MxB in the other two lanes. Also, there seems to be somewhat more G3BP1 in the MxB lane compared with vector. Can a better blot be shown? Quantification of band intensities might also help.

p. 9, line 186. There is a discrepancy with previous findings. In the present study, MxBdel25 lost the ability to inhibit retrotransposition while Goodier et al. reported that deleting the first 25 aa of MxB had no effect on retrotransposition (although their K131A mutation did, in agreement with the present study, a fact that should be also mentioned). This discrepancy should be noted and discussed. Perhaps epitope tags are having some effect.

MINOR.

p. 4, line 87. I point out that possible artifacts of quantitative PCR to measure levels of LINE-1 integrated cDNA in cells have been proposed in the literature, including generation of non-integrated ectopic cDNA by L1 RT which would then be amplified by PCR and misinterpreted as increased genomic insertions. I would suggest removing the word from '...to measure levels of LINE-1 integrated cDNA in cells".

Please note what arrows in all figures indicate. Some appear to show foci overlapping nuclei which is confusing.

Fig. S2D. The quality of the Western showing loss of MxB protein in KO cells is poor. Can a better one be presented?

Fig. 4E,F. Is the apparent increase in retrotransposition seen by knock-down of TIA-1 or G3BP1 significant? If so, this would appear worthy of comment in the text.

p. 7, line 144. It is noted, "These data further suggest that MxB inhibits the nuclear

import of LINE-1 RNP composed of LINE-1 RNA, ORF1p and ORF2p." While it is often assumed, I believe it has never been proven that ORF1p enters the nucleus bound to the L1 RNA. This caveat could perhaps be noted.

Fig. 6E, p. 11, line 223. It needs to be made clearer in the text that these mouse elements were detected by RT-PCR. Where were the primers located in the L1s?

p. 12, line 257. It is noted, "MxB inhibition of LINE-1 is CypA-independent (data not shown)". Show the data please if you wish to state this.

There are a number of quite minor English grammar errors scattered through the manuscript, a majority involving missing articles.

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes: Gilberto Betancor Quintana

Reviewer #2: Yes: Sandra Richardson

Reviewer #3: No

Reviewer #4: No

22 Nov 2021

Submitted filename: responses to reviwers PGENETICS-D-21-01057 R1.docx

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14 Dec 2021

Dear Dr Guo,

Thank you very much for submitting your Research Article entitled 'MxB inhibits long interspersed element type 1 retrotransposition' to PLOS Genetics.