Virus infection-induced host mRNA degradation and potential application of live cell imaging.

Viruses exist wherever there is life. They can cause allergy, immune response, inflammation, and even fatal diseases directly or indirectly. Accumulating evidence shows that host RNA undergoes rapid degradation during virus infection. Herein, we focus on several possible mechanisms of infection-induced host RNA turnover, which seems to be a common strategy for both prokaryotic and eukaryotic viruses during the very early stage of infection and a potential application of live cell imaging on its visualization.

Introduction

When a virus/bacteriophage infects a host cell, it utilizes its host's molecular machinery to replicate, efficiently generating more than hundredfold of its progeny. In particular, the virus relies on the ribosomes in the host cell to translate viral messenger RNA (mRNA) into polypeptides. Many viruses also impair the translation of cellular mRNA [1], [2], [3], one of the mechanisms during the shift of gene expression from host to virus, a process termed “host shutoff”, in order to prevent the production of anti-viral, host protecting proteins [4].

Both eukaryotic and prokaryotic viruses are reported to have numerous mechanisms during ‘host shutoff’ that are either essential or can facilitate their propagation under changing environment, such as host interferon antagonist, host DNA damage, host transcriptional and translational apparatuses modification [5], [6], [7]. Among them, virus infection-induced host RNA degradation has not been paid enough attention to, which might be the very early step after viral infection in most cases. Strict control of their gene expression regulates the timing of host factor suppression to maximize their replication.

Dominant host mRNA decay pathways

mRNA decay occurs in various pathways and is highly regulated in both eukaryotes and prokaryotes. Here we will mainly summarize the common pathways.

In mammalian cells, a majority of mRNAs are capped by a 5′ 7-methyl-guanosine (m7G) and tailed by poly adenine (A) at 3′ end. These features physically protect the mRNA ends from exonucleolytic decay that remove nucleotides from 5′-end (Xrn1 mainly in the cytoplasm and Xrn2 in the nucleus) or 3′-end (the exosome complex) [8]. These structures of mRNA also serve to recruit translation initiation machinery [9]. Cellular deadenylase enzyme complexes, CCR4-NOT, PAN2-PAN3, and PARN are in charge of the deadenylation, which is the first and often the rate-limiting step of mRNA decay, resulting in the shortening of the poly (A) tail. Decapping enzymes are required in some cases for their mRNA turnover such as Dcp1, Dcp2, etc. [10].

In bacterial cells, the 3′-end and 5′-end of mRNA are likewise protected from exonucleolytic decay. mRNAs are 5′-end triphosphated instead of a 5′ cap and transcriptionally terminated at 3′-end with stem-loop [11], which is sometimes followed by a poly (U) or poly (A) cluster. Endonucleolytic cleavage via endoribonuclease is one of the basal pathways for bacterial mRNAs such as that in Escherichia coli (E. coli) endoribonuclease E plays an essential role in its mRNA turnover process [12]. Another common route for bacteria mRNA decay is that a removal of two of the triphosphates via RNA 5′ Pyrophosphohydrolase (RppH) [13] leaving a 5′ monophosphate, causes the message to be destroyed by the exonuclease RNase J, which degrades 5′ to 3′, such as the mRNA turnover in Bacillus subtilis [11].

Virus/bacteriophage induces host RNA degradation

Promoting global mRNA degradation is reported to be one of the host shutoff mechanisms to block host gene expression among at least three different viral subfamilies, alphaherpesviruses, gammaherpesviruses and betacoronaviruses [4], [5]. Moreover, Gaglia et al.’s work showed that viral encoded proteins trigger host mRNA degradation by a primary endonucleolytic cleavage causing shutoff of host gene expression and a host exonuclease such as Xrn1, an important 5′ to 3′ exonuclease in human cells, were required in subsequent completion of host mRNA turnover [5]. As shown in Fig. 1 A and B, nearly all viruses known drive widespread mRNA degradation triggered by either internal endonucleolytic cleavage or removal of 5′ cap structure of the mRNA.

Fig. 1

Possible host mRNA degradation mechanisms induced by virus/bacteriophage infection [5], [10], [34].

One typical example of alphaherpesviruses is herpes simplex virus 1 (HSV-1). Virion host shutoff protein (Vhs, previously UL41) having an RNase activity [14], [15], is packaged in virions and released after infection [16], [17]. Vhs is directed to mRNAs through interactions with the translation initiation factors eIF4H and eIF4AI/II [18] and thus induces host mRNA decline.

Several examples of gammaherpesviruses were also characterized: SOX in Kaposi's sarcoma-associated herpesvirus (KSHV) [19] is the homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome [20] as an endonuclease. During KSHV infection, SOX initiates host mRNA degradation by its endonucleolytic cleavage followed by rapid exonucleolytic degradation of host Xrn1 [21]. Recently, Mendez et al. demonstrated that SOX cleaves its target mRNA without other factors by in vitro cleavage assay [22]. Moreover, Muller et al. revealed that some mRNAs that ‘overrided’ SOX were also degraded, suggesting multiple viral endonucleases' existence [23].

BGLF5 in Epstein–Barr virus (EBV) plays a role in processing non-linear or branched viral DNA intermediates in order to promote the production of mature packaged unit-length linear progeny viral DNA molecules [24], serves as an endoribonuclease of host mRNA after infection [25]. Similar to BGLF5, muSOX in murine herpesvirus 68 (MHV68), which is also alkaline exonuclease homolog, a member of the PD(D/E)XK restriction endonuclease superfamily have been demonstrated to induce host mRNA decay after infection via endonucleolytic attack [26].

Nsp1, a protein with no known similarity to cellular or viral nucleases from severe acute respiratory syndrome (SARS) coronavirus (SCoV) belonging to betacoronavirus family, is reported to induce host mRNA degradation by binding to 40S ribosome [27], [28]. Nsp1 also prevents Sendai virus-induced endogenous IFN-β mRNA accumulation. Later Kamitani et al. found that Nsp1 bound to host 40S ribosomal subunit and inactivated the translational activity of the 40S subunits. Meanwhile, Nsp1-40S ribosome complex induces the modification of the 5′ region of capped mRNA template and renders the template RNA translationally incompetent [1].

RNA endoribonuclease designated as PA-X, which is encoded in the genome of Influenza A virus (IAV), stimulates the decay of cellular mRNA [29]. Host RNA degradation was also reported in IAV-infected cells, though the sensitivity of host mRNAs to IAV-induced host shutoff varies. For example, host proteins that facilitate viral propagation (such as the proteins that maintain oxidative phosphorylation) are less attacked [4], [30]. This suggests that like IAV, virus infection in fact effectively and strictly control the total pool of mRNAs in virus-infected cells, allowing selection of host mRNAs that are important for virus replication to persist and be translated [4], [31].

As illustrated in Fig. 1B, poxviruses use virally encoded decapping proteins to remove the 5′-cap of the mRNA resulting in destabilization of the mRNA. A typical poxvirus, vaccinia virus (VACV) encodes two decapping proteins, D9 and D10, and these proteins likely maintain viral stage-specific protein synthesis as well as host cellular mRNA turnover [32], [33]. Other viruses including bunyaviruses and orthomyxoviruses use cap-snatching mechanisms to remove the 5′-cap and use the removed cap to protect viral RNAs [8].

Intriguingly, prokaryotic viruses such as bacteriophages also trigger rapid host mRNA degradation. As shown in Fig. 1C, bacteriophage T4 Srd is demonstrated to stimulate the activity of E. coli essential endoribonuclease E via binding to its N-terminal in order to induce host mRNA degradation immediately after infection [34]. Though there is no homolog of Srd found yet in bacteriophage T2 and T7, host bulk mRNA degradation was also observed in T2- and T7- infected cells [7]. Little is known in the case of other bacteria such as B. subtilis yet, but host shutoff was observed in AR9 phage infected B. subtilis cells [35]. Further research is expected on their mRNA elimination after phage infection.

Conclusion and discussion

Influence of functional deficiency of viral factors that raises host mRNA turnover on viral replication has been studied for years. Functional loss of viral factors mentioned above usually is not fatal for viral growth, but several pieces of evidence have been also obtained that global host mRNA turnover should be required for rapid transition of gene expression from host to viruses and efficient viral replication [16], [34], [36], [37]. For instance, vhs-deficient mutant of HSV-1 results in low virulence and Vhs plays a role in evasion from non-specific host defense mechanisms during primary infection [16], [36]. In addition, Matsuura's group showed that RNA replication of ScoV was low when nsp1 was mutated in an in vitro RNA replica system [37]. Furthermore, from our own previous work, bacteriophage T4 Srd could stimulate the activity of host E. coli essential endonuclease E inducing bulk mRNA degradation and a deletion of srd causes inefficient propagation [34]. Therefore, it is likely to be a common mechanism in both prokaryote and eukaryote that infection with viral organisms activates the host mRNA degradation machinery for the shift of gene expression from host to virus and thus facilitates efficient viral propagation. How this contributes to offense immune response in mammalian cells is also an unanswered question so far.

Perspective

In this concise review, we mainly focus on virus-induced degradation of host RNAs during host shutoff, which helps to maximize viral gene expression and thus their replication.

Mammalian viruses as well as bacteriophages evolved unique mechanisms to propagate efficiently during infection. Host cells respond in order to create a hostile environment for viral replication, leading to the shutoff of mRNA translation (protein synthesis) and the assembly of RNA granules [38].

Recently, the HIV-1 unspliced mRNA has been shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6 methyladenosine RNA binding protein 2 (YTHDF2), which is involved in mRNA decay. This indicates that HIV-1 interacts with host mRNA decay components to accomplish viral replication successfully [39]. This finding implicates a novel mechanism involved in infection-induced host RNA degradation. In addition, viruses also employ numerous strategies to regulate the gene expression of both host and themselves despite of host shutoff. Hepatitis B Virus (HBV) mRNA interacting with host microRNA-122 (miR122) was reported to have an indispensable role for viral replication [40], [41]. The interaction between viral mRNA and host microRNA is also known to be able to facilitate Hepatitis C Virus (HCV) replication [42].

Under development of imaging technologies, converting virus-host interaction signals to imaging visualization will help decipher the underlying cellular mechanisms. Single-molecule fluorescence imaging has been reported to monitor the entry, transport and virus-host cell interaction for some viruses like polio virus (PV) and Adeno-associated virus (AAV) [43]. Moreover, live cell imaging technology has also been reported for mRNA-protein interaction, which makes it possible to study cellular and viral gene expression directly. Yin et al. has constructed red color mCherry-based trimolecular fluorescence complementation (TriFC) systems, visualized influenza A viral mRNA-protein interaction in living cells and helped understand more about IAV mRNA nuclear export mechanism [44]. They show that adapter proteins Aly and UAP56 are able to interact with three kinds of viral mRNAs while splicing factor 9G8 only interacts with one mRNA [44]. Therefore, it is expectable that application of advanced live cell imaging technology will facilitate in-depth research on virus-induced host mRNA degradation.

In summary, comprehensive understanding of virus-induced host shutoff and its connection in the whole process of viral replication as well as its interaction with host response through multiple technologies (e.g. molecular cell biological techniques and advanced imaging methods) is crucial to struggle with viruses. This could facilitate decoding virus-raised diseases and may provide new insights for both novel research tool and therapeutic strategy design for disease.