The emerging roles of retromer and sorting nexins in the life cycle of viruses.

Retromer and sorting nexins (SNXs) transport cargoes from endosomes to the trans-Golgi network or plasma membrane. Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses, including members of Coronaviridae, Flaviviridae and Retroviridae. Key components of retromer/SNXs, such as Vps35, Vps26, SNX5 and SNX27, can affect multiple steps of the viral life cycle, including facilitating the entry of viruses into cells, participating in viral replication, and promoting the assembly of virions. Here we present a comprehensive updated review on the interplay between retromer/SNXs and virus, which will shed mechanistic insights into controlling virus infection.

Introduction

Retrograde transport is a highly regulated and selective process, which is widely described as the flow of proteins and lipids from the plasma membrane to the Golgi, from endosomes to the Golgi, or from Golgi to the endoplasmic reticulum (ER) (Bonifacino and Rojas, 2006; Johannes and Popoff, 2008). The endocytic recycling pathway is responsible for sending the endocytic cargoes back to the plasma membrane, maintaining the stability of the plasma membrane composition and ensuring the normal progress of a variety of cellular biological processes.

An important protein complex for vesicle transport is the retromer complex (Bujny et al., 2007). Retromer is an endosomal membrane-binding protein complex and its main function is to select the cargoes for retrograde transport from the early endosome to the Golgi (Seaman, 2012; Burd and Cullen, 2014). In eukaryotic cells, retromer transports cargoes from mature endosomes to newly synthesized endosomal tubules through recognizing and sorting, and then transports back to the Golgi (Arighi et al., 2004; Seaman, 2004, 2012; Bonifacino and Hurley, 2008). There are growing evidences that retromer is also involved in transport processes from endosome to plasma membrane (Temkin et al., 2011; Choy et al., 2014). Transport pathways mediated by retromer were outlined in Fig. 1.

Fig. 1

Transport pathways mediated by retromer and SNXs in the absence of virus. Retromer and SNXs mediate two transport pathways including endocytic recycling from the recycling endosome to the plasma membrane and retrograde transport from the endosome to TGN. Retromer also bridges the endosome and autophagosome. SNX, sorting nexin; TGN, trans-Golgi network; Vps, vacuolar protein sorting; WASH, Wiskott-Aldrich syndrome protein and SCAR homologue; CCC, COMMD/CCDC22/CCDC93; TBC1D5, Tre2-Bub2-Cdc16 (TBC) Domain Family Member 5.

In mammalian cells, retromer is comprised of Vps35, Vps26 and Vps29 (Seaman et al., 1998; Seaman, 2005; Wassmer et al., 2007; Attar and Cullen, 2010). The cargo-selective trimer Vps26-Vps29-Vps35 forms a stable tertiary structure, which participates in the binding of cargoes (Haft et al., 2000; Arighi et al., 2004; Kerr et al., 2005; Bugarcic et al., 2011). Retromer-mediated trafficking in cargoes works by directly binding to Vps35 or Vps26 (Arighi et al., 2004; Shi et al., 2006; Seaman, 2007; Belenkaya et al., 2008; Tabuchi et al., 2010; Fjorback et al., 2012).

Sorting nexins (SNXs) are consisted of a series of proteins that are involved in the identification and sorting of cargoes during retrograde transport from endosome to the Golgi or plasma membrane (Cullen and Korswagen, 2011). At present, 33 kinds of SNXs are known in mammals (Cullen, 2008), some of which can bind to retromer (Arighi et al., 2004). SNX1, SNX2, SNX5, SNX6 and SNX32 can associate with retromer to deform membrane (Carlton et al., 2004; Griffin et al., 2005; Wassmer et al., 2007). Many studies have shown that mammalian SNXs are able to function in recognizing and trafficking cargoes, independently of retromer (Kvainickas et al., 2017; Simonetti et al., 2017; Yong et al., 2020).

Many protein cargoes reach the cell surface through endocytic recycling by retromer, which requires the participation of COMMD/CCDC22/CCDC93 (CCC), Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex and SNX27 (Li et al., 2015; Phillips-Krawczak et al., 2015; Bartuzi et al., 2016; Lee et al., 2016). WASH complex is fundamental for endosomal cargo sorting, which is composed of five proteins, in which FAM21 is located on the endosome near retromer by binding to Vps35 (Harbour et al., 2012). The WASH complex is able to promote the formation of actin fiber network in the part of the endosome, thus boosting the sorting of cargoes into retromer, while FAM21 can direct SNX27/retromer cargoes to the plasma membrane (Phillips-Krawczak et al., 2015; Bartuzi et al., 2016; Lee et al., 2016).

Rab protein is a small guanosine triphosphatase (GTPase) that regulates membrane transport events by cycling between the membrane-related GTP-binding and GDP-binding types (Guerra and Bucci, 2016; Stroupe, 2018). Tre2-Bub2-Cdc16 (TBC) Domain Family Member 5 (TBC1D5) is a Rab7-specific GTPase activating protein, which binds closely to retromer in vitro and is a pivotal component of the retromer complex (Seaman et al., 2009). TBC1D5 not only bridges the endosome and autophagosome via its C-terminal LIR motif (Popovic et al., 2012), but also associates with ATG9 and the active ULK1 complex during autophagy (Popovic and Dikic, 2014). ATG9A is recycled to the trans-Golgi network (TGN) in a SNX4/retromer-dependent manner (Ravussin et al., 2021). The involvement of retromer complex in autophagy is shown in Fig. 1.

Retrograde transport and endocytic recycling pathways have been used by various viral proteins to mediate cytotoxicity and pathogenic mechanisms. Accumulating studies have revealed the emerging roles for retromer/SNXs in the life cycle of viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human Papillomavirus (HPV). Retromer/SNXs components may participate in multiple steps of the viral life cycle directly or indirectly. In this review, we will summarize the interplay between retromer/SNXs and virus sorted by virus families.

The roles for retromer in the life cycle of virus

Coronaviridae

The coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, is a huge threat to public health and global economy. Several protein-protein interaction studies and functional genome-wide screening studies have shown that the involvement of retromer in the life cycle SARS-CoV-2. Daniloski et al. found a consistent theme among the enriched complexes for SARS-CoV-2 life cycle was endosome function, of which Vps26A, Vps29, Vps35 and SNX27 were identified (Daniloski et al., 2021).

The SARS-CoV-2 entry is mediated by membrane fusion at the plasma membrane, while the deletion of RRAR furin cleavage site in spike (S) protein drives virus entry to endosomal pathway. Genome-wide CRISPR screening with RRAR deletion mutant identified a series of SARS-CoV-2 host factors related to intracellular transport, such as Vps35, Vps29, SNX27, CCC, WASH, Arp2/3 (Zhu et al., 2021). Knockout of key components of retromer, such as SNX27 and CCC, reduced surface level of angiotensin-converting enzyme 2 (ACE2), indicating the key role for SNX27/retromer in SARS-CoV-2 entry (Zhu et al., 2021). SNX27 PDZ domain recognizes SNX27/retromer complex cargoes and mediates the recycling of transmembrane cargoes from the endosome to the plasma membrane (Ghai et al., 2013; Gallon et al., 2014; Kliche et al., 2021). Interestingly, C-terminal cytoplasmic tail of ACE2 possesses a type I PDZ binding motif (PBM) and associates with SNX27 PDZ domain (Kliche et al., 2021), raising the possibility that ACE2 is a SNX27/retromer cargo. Thus SNX27 may promote SARS-CoV-2 entry by regulating the endocytic recycling of ACE2 (Fig. 2A). Indeed, silencing SNX27 or knockout of SNX27 reduced the surface level of ACE2 (Yang et al., 2022).

Fig. 2

The schematic models that demonstrate the mechanistic view of the function of retromer and SNXs for typical viruses. A SARS-CoV-2 receptor ACE2 and S protein are transported to cell surface by SNX27-retromer. B Retromer and its cargo CIMPR are required for HCV replication. C Retromer affects HIV through trafficking Env. D VACV uncoating requires WASH component FAM21. E Retromer facilitates AAV to enter the cell. SNX, sorting nexin; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; ACE2, angiotensin-converting enzyme 2; S, spike; NSP, non-structural protein; TGN, trans-Golgi network; HCV, hepatitis C virus; CIMPR, cation-independent mannose-6-phosphate receptor; PI4P, phosphatidylinositol-4-phosphate; Env, envelope glycoprotein envelope; HIV, human immunodeficiency virus; VACV, vaccinia virus; MV, mature virion; WASH, Wiskott-Aldrich syndrome protein and SCAR homologue; AAV, adeno-associated virus; GARP, Golgi associated retrograde protein complex.

In addition to ACE2, S protein of SARS-CoV-2 could interact with SNX27 (Cattin-Ortolá et al., 2021; Zhao et al., 2021). PDZ domain of SNX27 associated with the “MTSC” motif of S protein (Cattin-Ortolá et al., 2021; Zhao et al., 2021). One study showed that S mutant deficient for SNX27 binding did not alter its accumulation on the plasma membrane (Cattin-Ortolá et al., 2021). However, in another study, S mutant losing SNX27 binding ability reduced surface level of S protein (Zhao et al., 2021). Moreover, knockout of SNX27 decreased S protein cell surface expression and reduced S-based pseudovirus production. Therefore, SNX27 could mediate the endocytic recycling pathway of S (Fig. 2A).

Flaviviridae

Hepatitis C virus (HCV) infection is an important cause of liver disease. More than 170 million people worldwide are chronically infected with HCV (Shepard et al., 2005). Chronic HCV usually develops into liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (Yamane et al., 2013), which seriously affects human health. The completion of the life cycle of HCV requires the participation of many host factors. Among host factors of HCV, phosphatidylinositol-4-phosphate (PI4P) is a specific inositol phosphate rich in the HCV replication region, which plays a vital role in the replication of HCV (Hsu et al., 2010; Reiss et al., 2011; Tai and Salloum, 2011; Bianco et al., 2012; Hong et al., 2014; Li et al., 2014). The effectors of PI4P are involved in many steps of the life cycle of HCV. PI4P could promote the dissociation of retromer and its cargoes on the TGN (Niu et al., 2013), while retromer and other PI4P effectors OSBP, FAPP2, GOLPH3 and CERT jointly promote the process of virus life cycle (Amako et al., 2009; Lim and Hwang, 2011; Khan et al., 2014; Wang et al., 2014; Yin et al., 2016). The domain I of NS5A protein interacts with Vps35, the core component of retromer. Moreover, PI4P induced by HCV can elevate the unloading of a typical retromer cargo cation-independent mannose-6-phosphate receptor (CIMPR) in the virus replication region (Yin et al., 2016) (Fig. 2B). Knockdown of retromer and its cargo CIMPR inhibited HCV replication. Given that the CIMPR is necessary for trafficking lysosomal hydrolases to lysosomes (via endosomes) and that retromer regulates the endosome-to-Golgi retrieval of the CIMPR, it seems likely that loss of retromer is impacting on CIMPR localization, altering lysosome function which is then affecting virus replication. In the process of HCV replication, retromers located in autophagosomes, indicating that retromers may also regulate the replication of virus through autophagy (Yin et al., 2017).

Retroviridae

Human T cell leukemia virus type 1 (HTLV-1) was the first discovered human retrovirus (Poiesz et al., 1980). The HTLV-1 endemic areas are spread all over the world, such as Japan, the Caribbean Islands, Central America, South America, and Africa. HTLV-1 is the pathogen of adult T cell leukemia (ATL), and associated myelopathy/tropic spastic paraparesis (Yoshida et al., 1982; Goncalves et al., 2010).

The regulatory gene Tax-1 is required for efficient virus replication, as it drives transcription of viral gene products. PBM at the carboxyl terminus of Tax-1 is significant for HTLV-1 induced cellular transformation. SNX27 is an interacting partner of Tax-1, and their interaction is mediated by the Tax-1 PBM and SNX27 PDZ domain. SNX27 can promote the plasma membrane localization of glucose transport 1 (GLUT1). Tax-1 alters GLUT1 localization via its interaction with SNX27. Through their observations, Al-Saleem et al. demonstrated that overexpression of Tax-1 in cells reduced the level of GLUT1 on the plasma membrane, while knockdown of SNX27 increased virion release and decreased HTLV-1 infectivity (Al-Saleem et al., 2019).

The envelope glycoprotein envelope (Env) of human immunodeficiency virus (HIV) is not only the key determinant of virus infection, but also the main target of humoral immunity. Env is composed of 856 amino acids and synthesizes a 160 kDa leader chain (gp160) in the ER, which is then split into two subunits (Checkley et al., 2011): the receptor-bound surface subunit gp120 and transmembrane subunit gp41. Subunit gp41 has cytoplasmic binding domain and can regulate the intracellular transport of Env. In addition, the long cytoplasmic tail of Env has a potential effect on the assembly of infectious virions and prevents Env from being exposed to the immune system. Under normal circumstances, most of the envelope glycoproteins are located on the Golgi and traverse the whole pathway from the Golgi to the plasma membrane. The envelope glycoproteins that are not packaged into the virus will be endocytosed back to the Golgi (Berlioz-Torrent et al., 1999; Wyss et al., 2001; Byland et al., 2007). Nevertheless, little is known about the detailed mechanism of this process.

Experiments have shown that retromer regulates the intracellular transport of Env and promotes the retrograde transport from endosome to Golgi (Fig. 2C). In virus-infected cells, HIV-1 envelope glycoprotein Env can co-localize with retromer (Groppelli et al., 2014). Knockdown of the retromer component Vps26 or Vps35 increases Env expression on the plasma membrane. These functions depend on the direct interaction between C-terminal 100 amino acids of Env gp41 and retromer complex (Groppelli et al., 2014). Interfering with the function of retromer could inhibit the transport of envelope glycoproteins, resulting in the inability of endogenous envelope glycoproteins to return to the Golgi, changing the location of Env, and ultimately affecting the process of Env incorporation into newly synthesized virions (Groppelli et al., 2014).

Using high-throughput small interference RNA (siRNA) screening, Bayliss et al. identified the trafficking proteins, including Rab7L1 also known as Rab29, are required for HIV virological synapses (VS) formation (Bayliss et al., 2020). Rab7L1/Rab29 functions in intracellular transport and endosomal sorting of lysosome-bound membrane proteins and participates in retromer activity (Kuwahara et al., 2016). By using the retromer pathway, internalized HIV virions can be delivered onto the plasma membrane, promoting the formation of VS, and enabling trans-infection between monocyte derived Dendritic cell (MDDC) and T cell (Bayliss et al., 2020). Retromer affects the late step of HIV-1 replication and regulates virus assembly, which is a new discovery of its function. It also provides a new direction for the future research on the relationship between virus and retromer.

Papillomaviridae

HPV is a non-enveloped DNA virus that mainly infects epithelial cells of the skin and mucous membranes (Griffin et al., 2014). More than 200 types of human papillomavirus have been identified, among which HPV16 and HPV18 subtypes are the most common high risk strains. Genome-wide siRNA screening confirmed that retromer was a crucial protein for HPV to enter cells (Lipovsky et al., 2013). The capsid protein of HPV penetrates the plasma membrane and employs retromer to transport to TGN. Its genome is transmitted to TGN during infection. Studies have demonstrated that inhibition of TGN function significantly reduces virus infectivity. After endocytosis, the incoming HPV virions remain in the membrane vesicles until they reach the nucleus, where viral DNA replication occurs (Schelhaas et al., 2012; Day et al., 2013, 2019; Lipovsky et al., 2013; DiGiuseppe et al., 2016; Aydin et al., 2017; Siddiqa et al., 2018).

The entry of HPV16 into cells was mediated by retromer (Pim et al., 2021). Upon knocking down the core components of retromer, Vps26, Vps29 or Vps35, respectively, HPV16 infection is restricted. Moreover, other types of Papillomaviridae such as HPV18 also require retromer to enter the host cells (Ganti et al., 2016a). The internalized HPV exists in the endosome cavity. Although the L2 capsid protein is not a transmembrane protein, it is still a cargo transported by retromer. Part of the viral L2 capsid protein extends into the cytoplasm through the membrane, where it can bind to retromer and several other L2 binding partners (such as SNX17) (Bergant Marusic et al., 2012; Bronnimann et al., 2013; Lipovsky et al., 2013; Popa et al., 2015). Retromer directly binds to a highly conserved sequence motif at the C-terminal of L2 protein (Zhang et al., 2018), which is considered as a retromer binding motif mimic. The knockdown or mutation of this motif will interfere with the ability of L2 to bind to retromer, resulting in the accumulation of L2 protein in vivo and inhibiting the transport of virus from early endosome to TGN (Popa et al., 2015; Lipovsky et al., 2017). This defect was remedied by replacing the main binding site of retromer on L2 with retromer sorting signal from intracellular proteins. In vitro experiments illustrated that this site on L2 could directly bind to retromer (Popa et al., 2015). L2 protein also contains a cationic cell-penetrating peptide (CPP) sequence, which triggers retromer-mediated HPV transport during virus entry (Zhang et al., 2018, 2020). CPP from L2 mediates transient insertion of L2 into the endosome membrane, which is stabilized by retromer-L2 binding. Those results demonstrated that retromer plays a key role in CPP-mediated membrane insertion (Xie et al., 2021).

SNX17 and SNX27 play an important role in the process of HPV infection. SNX17 can interact with L2, which is related to the transport of L2 from late endosomes. A PDZ domain in SNX27 is critical for the interaction between HPV16 L2 and SNX27, which modulates virion trafficking (Pim et al., 2015). Knockdown of SNX17 decreased the level of L2 in late endosomes and weakened pseudoviral infection (Bergant and Banks, 2013). SNX27 exists in the early endosomes and binds to cargo through its carboxyl terminal amino acid residues (Cai et al., 2011). Knocking down SNX27 limits pseudoviral particles infection. When both SNX17 and SNX27 were knocked down, the viral infection efficiency were further decreased. However, overexpression of these two proteins led to different localization of L2 protein. SNX27 overexpression did not change the nuclear localization of L2, while overexpression of SNX17 caused L2 to localize only in the cytoplasm, suggesting that SNX17 may participate in the late stage of virus entry (Zhang et al., 2016).

GTPase cycle of Rab7b is critical for HPV trafficking (Day et al., 2013; Lipovsky et al., 2013; Young et al., 2019). During HPV infection, binding of the retromer to L2 recruits Rab7-specific GTPase activating protein TBC1D5 to retromer, inducing the disassembly of retromer-HPV complex from the membrane (Seaman et al., 2009; Jia et al., 2016; Borg Distefano et al., 2018; Jimenez-Orgaz et al., 2018). Cargo binding plays a central role in membrane recruitment and assembly of active retromer complexes (Harrison et al., 2014; Lucas et al., 2016). TBC1D5 stimulates the hydrolysis of Rab7-GTP, driving the disintegration of the retromer-HPV complex, which in turn causes dissociation of the retromer from the endosomal membrane, and vesicles containing cargoes are transported to TGN, where the cargoes are delivered through membrane fusion to coordinate retrograde transport (Rojas et al., 2008; Seaman et al., 2009; Liu et al., 2012; Harrison et al., 2016; Lucas et al., 2016; Jimenez-Orgaz et al., 2018).

Oncoprotein E6 from multiple high-risk HPV types associates with SNX27 by a classical PBM-PDZ interaction. E6, SNX27 and the retromer form a complex, which facilitates the SNX27 cargoes, such as GLUT1, recycling back to the cell surface. Thus, E6 plays an important role to maintaining high levels of glucose uptake by regulating GLUT1 transport (Ganti et al., 2016b).

Stannin is an important regulator of HPV entry. It is located in the lysosomal compartment and does not affect the binding of HPV16 to cells, virus absorption or virus uncoating, whereas can selectively inhibit the entry of several types of HPV. It is expressed in human keratinocytes and interacts with L1 major capsid proteins. Stannin could weaken the interaction between L2 capsid protein and retromer, inhibit HPV16 transport into TGN in the cell, and stimulate HPV degradation, thus leading to the low infectivity of HPV. Although endogenous level of Stannin inhibits HPV16 infection, its role in natural infection remains unclear (Lipovsky et al., 2017).

Herpesviridae

Herpesvirus saimiri (HVS) tyrosine kinase-interacting protein (Tip) is needed for the immortalization of T lymphocytes in vitro. Tip interacts with Vps35 through a conserved glutamate-rich N-terminal motif. CIMPR is a receptor for lysosomal hydrolases and other endosomal proteins. The expression of Tip diminishes the level of CIMPR, leading to lysosomal degradation and reducing the redistribution of Vps35 into lysosomes. Conversely, the existence of Tip does not affect the stability of the retromer subunit. Tip inhibition of retromer activity is related to down-regulation of CD4 expression on cell surface and the transformation of human initial T cells in vitro (Kingston et al., 2011).

Human cytomegalovirus (HCMV) belongs to the beta herpesvirus subfamily of herpesviruses (Davison et al., 2009). It causes asymptomatic infection in healthy people and poses a serious threat to patients with low immune function. HCMV induces the formation of cytoplasmic viral assembly center (cVAC), where mature virus particles are formed by a variety of viral proteins. HCMV UL35 gene encodes the envelope protein ppUL35, which participates in the assembly of cytomegalovirus. Maschkowitz et al. found that SNX5 interacted with UL35 protein. The expression of wild-type protein rather than SNX5 binding deficient mutant leads to the redistribution of intracellular CIMPR, indicating that UL35 protein binds to SNX5 and negatively regulates the cellular transport pathway. In addition, the binding of UL35 proteins to SNX5 is necessary for efficient viral replication and transport of the most abundant HCMV glycoprotein B (gB, gpUL55) to cVAC (Maschkowitz et al., 2018). cVAC consists of circular vesicles from the Golgi and endosomal network (Das et al., 2007; Das and Pellett, 2011). In the late stages of HCMV infection, the viral capsid is transported to cVAC, where it is surrounded by a membrane protein layer and a viral membrane.

Viruses could induce the degradation of key components of immune system by selective autophagy of aggregates (aggrephagy). Murine cytomegalovirus (MCMV) M45 and herpes simplex virus type 1 (HSV-1) ICP6 recruit Vps26B and TBC1D5 to facilitate the degradation of receptor-interacting protein kinase 1 (RIPK1) by aggrephagy, which represents a novel viral immune-evasion mechanism (Muscolino et al., 2020).

Poxviridae

Vaccinia virus (VACV) belongs to Orthopoxvirus genus of Poxviridae family. It is a large double-stranded DNA virus, which is capable of infecting a wide range of cell lines and animals (Van Vliet et al., 2009). VACV mature virion (MV) enters the host cells through macropincytosis (Mercer and Helenius, 2008). In the stage of intracellular cargo transport, Rab5 exists in the early endosome, while Rab7 is located at the late endosome and lysosome (Hsiao et al., 2015). Studies have shown that before membrane fusion and virus decapitation, MV is transported to early endosomes, where Rab5 is active and further binds to recycling endsomes marked by Rab11 and Rab22. Rab11 and Rab22 work together to regulate VACV entry into cells.

In early endosome, FAM21-retromer protein complex is required for MV sorting (Hsiao et al., 2015) (Fig. 2D). The invasion of vaccinia virus MV into HeLa cells depends on FAM21 (Huang et al., 2008). FAM21 is a key component of WASH protein, which is necessary for endosomal sorting of vesicles containing MV. The sorting of endosome containing MV also requires the interaction of FAM21 with retromer complex components Vps26, Vps29 and Vps35 (Harbour et al., 2012; Jia et al., 2012). WASH and retromer cause membrane fusion and VACV core uncoating in the cytoplasm. The discovery of FAM21-retromer complex regulating the intracellular transport of VACV has enlightened us on the relationship between VACV and early endosomal MV sorting.

Western Reserve (WR) strain of VACV open reading frame (ORF) WR039 (K7R) encodes a non-essential VACV protein K7 (Schroder et al., 2008; Benfield et al., 2013). Li et al. found that K7 preferentially bound to host proteins related to vesicle transport, including COPI, retromer and class C Homologues in Endosome-Vesicle Interaction complex (CHEVI) (Li et al., 2017). Experiments uncovered that K7 interaced with Vps35, Vps26A and Vps26B, and the C-terminal leucine in K7 was required for retromer binding (Li et al., 2017). Therefore, K7 may be involved in the intracellular transport of the virus and combine with FAM21-retromer complex, thereby promoting the uncoating of vaccinia virus. Another possibility is that K7 blocks the cargo binding site of retromer by interacting with retromer (Li et al., 2017).

Parvoviridae

Recombinant adeno-associated virus (AAV) has low immunogenicity which infects a variety of tissues in vivo and provides long-term transgene expression in tissues after mitotic (Grimm and Kay, 2003). Thus it is considered to be one of the most powerful tools for gene therapy, which is a promising way to treat life-threatening diseases that cannot be cured by any other form of medicine. AAV vectors are currently being evaluated for the treatment of Duchenne muscular dystrophy, hemophilia, heart failure, Parkinson's disease and other diseases.

AAV capsids accumulate in TGN and Golgi (Bantel-Schaal et al., 2002; Pajusola et al., 2002; Johnson et al., 2011; Nonnenmacher and Weber, 2011), which indicates that AAV exploits the retrograde transport mechanism from endosome to TGN, involving retromer complex (Vps29 and Vps35) and Golgi associated retrograde protein complex (GARP) (Vps51, Vps52, Vps53, and Vps54) (Fig. 2E). Using recently developed Golgi targeting drugs and siRNA-mediated knockdown of a specific transporter protein, Nonnenmacher et al. have proved that transport to the Golgi is necessary for AAV transduction. SNX5-mediated retrograde transport to the Golgi is a widely conserved feature of AAV transport, which seems to have nothing to do with the identity of the receptor used for virus attachment. They also observed that virions escaped the endosomal system directly from the TGN/Golgi compartment (Nonnenmacher et al., 2015). Different AAV serotypes may vary in endocytosis rate and transport mechanism (Keiser et al., 2011; Weinberg et al., 2014). Unexpectedly, all the capsids of AAV will converge into a common retrograde transport route to TGN. These results suggest that retromer/SNXs and retrograde transport mechanism play a vital role in the transportation of AAV.

Orthomyxoviridae

For centuries, influenza viruses have been the cause of recurrent epidemics (Taubenberger and Morens, 2010). Although a variety of new drugs and vaccines have been developed, drug resistance has emerged because influenza A virus (IAV) has the strong ability to mutate and evolve. IAV has a segmented single-stranded negative sense RNA genome (Cheung and Poon, 2007), and matrix protein 2 (M2) is the spliced product of the seventh segment of IAV genome. It is a type III transmembrane protein that forms a homotetramer and belongs to ion channel proteins. Although a small amount of M2 was incorporated in virions, a large number of M2 existed in the endosomal chamber of infected cells (Henkel et al., 1998). Bhowmick et al. detected that M2 was retrogradely transported to the cytoplasm through endosome and Golgi network (Bhowmick et al., 2017). It prevents the fusion of endosome and lysosome when moving from endosome to TGN. M2 colocalized with SNX1 and SNX4 proteins and was considered as a retromer cargo. In addition, the exit of M2 to the cytoplasm from ER required the interaction with wild-type p97ATPase. Accordingly, the M2 is transported from the endoplasmic reticulum to the cytoplasm bypassing the cytoplasmic proteasome, and then transported to TGN to escape degradation (Bhowmick et al., 2017).

Pneumoviridae

Human respiratory syncytial virus (HRSV) is one of the most common viruses causing respiratory tract infections every year (Nair et al., 2011). HRSV belongs to the genus Orthopneumoviruses of the family Pneumoviridae, and its negative-stranded RNA genome encodes 11 kinds of proteins. Among those viral proteins, nucleoprotein (N) protein associates with viral RNA and plays the key role in inclusion bodies formation induced by virus, whereas the matrix protein (M) protein is involved in virus assembly and budding on the cell surface.

HRSV N and M proteins interact with retromer component SNX2 evidenced by proximity ligation assay and co-immunoprecipitation experiment (Cardoso et al., 2020). Another retromer component Vps26 also exists in inclusion bodies and filamentous structures. Silencing SNX1 and SNX2 causes the decrease of viral protein, the size of HRSV inclusion body, the formation of syncytia, and the generation of progeny viruses (Cardoso et al., 2020). In summary, SNX2 plays a significant role in the transport of HRSV structural proteins to assembly sites, indicating the role for retromer/SNXs in the biogenesis of HRSV particles (Cardoso et al., 2020).

Togaviridae

Alphaviruses belong to a group of viruses vectored in nature by blood-feeding insects. Sindbis virus (SINV), chikungunya virus (CHIKV) and Mayaro virus (MAYV) are pathogenic alphaviruses which pose great challenges to public health in the world. However, there are few specific drugs or vaccines available against these viruses (Chattopadhyay et al., 2013).

One host protein SNX5 was shown to be critical for the replication of these pathogenic alphaviruses. Schuchman et al. constructed a stable HEK293 SNX5 gene knockout cell line (Schuchman et al., 2018). Compared with wild-type HEK293 ​cells, they found that the production of SINV was significantly inhibited, and similar results were revealed in MAYV and CHIKV (Schuchman et al., 2018). Thus, it is speculated that SNX5 is essential for the growth of these three arboviruses viruses.

Conclusions

During infection, viruses hijack many host transport pathways for their own benefit. Here, we review the roles of the host protein trafficking complex retromer/SNXs in viral infection. What is striking is the large diversity of viruses that require retromer/SNXs or retromer-associated proteins. With the extensive study of the interplay between virus and retromer/SNXs, scientists found that numerous viruses utilized retromer/SNXs, most often for cell entry (Fig. 3). Retromer/SNXs associates with a variety of virus proteins to affect virus entry and replication (Table 1). For some viruses, the role of retromer/SNXs is fairly well understood, and in others the data just hint at a role. In several cases, SNX proteins interact with virus proteins. It is possible that retromer/SNXs is also involved in the life cycle of those viruses.

Fig. 3

Transport pathways mediated by retromer and SNXs in the presence of virus. HCV, SINV, CHIKV, MAYV, HIV, HPV, AAV, IAV, HRSV and HCMV use the retrograde transport pathway of retromer to promote infection. SARS-CoV-2, VACV, HVS and HTLV hijack the recycling pathway of retromer and SNXs. The detailed interaction between virus proteins and retromer components was illustrated in the right. HCV, hepatitis C virus; SINV, Sindbis virus; CHIKV, chikungunya virus; MAYV, Mayaro virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; AAV, adeno-associated virus; IAV, influenza A virus; HRSV, human respiratory syncytial virus; HCMV, human cytomegalovirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VACV, vaccinia virus; HVS, Herpes saimiri virus; HTLV, human T-cell leukemia virus; SNX, sorting nexin; Vps, vacuolar protein sorting.

Table 1

Selected examples of the interplay between virus and retromer/SNXs.

Virus	Viral protein	Retromer/SNXs component	Interplay between virus and retromer/SNXs	Reference
SARS-CoV-2	S,NSP2,NSP7,NSP12,NSP16	WASH,CCDC22,SNX1/2, Vps26A, Vps29, Vps35, SNX27	Retromer promotes the endocytic recycling of ACE2.	(Daniloski et al., 2021; Kliche et al., 2021; Papa et al.,. 2021; Zhu et al., 2021)
HCV	NS5A	Vps35	Retromer participates in the replication of HCV.	Yin et al., (2016)
HIV-1	Env	Vps35, Vps26	Retromer participates in the assembly of HIV-1 virions.	Groppelli et al., (2014)
HPV	L2, E6	SNX17, SNX27, Vps complex, TBC1D5	Retromer facilitates HPV to enter the cell.	(Bergant and Banks, 2013; Lipovsky et al., 2013; Pim et al., 2015; Popa et al., 2015; Xie et al., 2020)
VACV	–	WASH, FAM21	Retromer facilitates VACV to enter the cell.	Hsiao et al., (2015)
HVS	Tip	Vps35	Tip inhibits the activity of retromer, which in turn downregulates the level of CD4 on the cell surface.	Kingston et al., (2011)
AAV	–	Vps29, Vps35, SNX5	Retromer facilitates AAV to enter the cell.	Nonnenmacher et al., (2015)
IAV	M2	SNX complex	M2 is transported to TGN via retromer and escapes degradation.	Bhowmick et al., (2017)
HRSV	M, N, F	SNX2, Vps26	Retromer facilitates HRSV to enter the cell.	Cardoso et al., (2020)
HCMV	UL35	SNX5	Retromer participates in the assembly of HCMV virions.	Maschkowitz et al., (2018)
SINV, CHIKV, MAYV	nsP2	SNX5	SNX5 affects the replication pathway of SINV, CHIKV and MAYV.	Schuchman et al., (2018)
HTLV	TAX-1	SNX27	The interaction between SNX27 and TAX-1 alters GLUT1 localization.	Al-Saleem et al., (2019)

SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; S, spike; NSP, non-structural protein; WASH, Wiskott-Aldrich syndrome protein and SCAR homologue; SNX, sorting nexin; Vps, vacuolar protein sorting; ACE2, angiotensin-converting enzyme 2; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; VACV, vaccinia virus; HVS, Herpesvirus saimiri; Tip, tyrosine kinase-interacting protein; AAV, adeno-associated virus; IAV, influenza A virus; TGN, trans-Golgi network; HRSV, human respiratory syncytial virus; HCMV, human cytomegalovirus; SINV, Sindbis virus; CHIKV, chikungunya virus; MAYV, Mayaro virus; HTLV, human T-cell leukemia virus; GLUT1, human glucose transporter 1.

There are two transport routes regulated by retromer/SNXs: (1) retrograde transport, in which retromer/SNXs transports protein cargo from the endosome to TGN (Fig. 1); (2) endocytic recycling pathway, in which retromer/SNXs transports proteins from the recycling endosome to the cell surface (Fig. 1). Endocytic pathway is widely hijacked by viruses beneficial for their infection. Key components of host cellular machineries that function at endosomes including retromer components will likely be involved in viral infection. HCV, SINV, CHIKV, MAYV, HIV, HPV, AAV, IAV, HRSV and HCMV use the retrograde transport pathway of retromer/SNXs to promote infection (Fig. 3). In addition, retromer and SNXs regulate the recycling of many cell surface proteins, some of which are virus receptors. For instance, SARS-CoV-2 receptor ACE2 is a SNX27-retromer cargo. Therefore, retromer and SNXs may regulate virus infection by controlling the cell surface levels of virus receptors. SARS-CoV-2, VACV, HVS and HTLV proteins manipulate the recycling pathway of retromer (Fig. 3). Thus retromer and SNXs could regulate viral infection directly or indirectly. Additional, retromer and SNXs play important roles in other pathogens such as bacteria and parasites. Thus, retromer and SNXs could be potential drug targets to combat infectious diseases.

Conflict of interest

The authors declare that there are no conflicts of interest.