RNA viruses promote viral replication via disruption of mRNA export pathways
In addition to exploiting host mRNA nuclear export pathways, viruses also block export of host mRNAs. The expression of mRNAs encoding cellular defense proteins is critical for the host to mount a proper immune response to invading pathogens. Therefore, mRNA export pathways are an enticing target for viruses to block host expression of antiviral genes. Similar to most viral infections, influenza A virus (IAV) replication in vertebrate cells is recognized by the innate immune system. Upon recognition, the innate immune system triggers signal transduction pathways that lead to production of type I IFNs, which are antiviral cytokines that induce the expression of mRNAs encoding antiviral factors , including nucleoporins [16, 45]. IAV has evolved several mechanisms to inhibit this response, mainly through non-structural protein 1 (NS1), a multifunctional protein with activities in both the nucleus and cytoplasm [91, 92].
In the nucleus, NS1 inhibits mRNA processing and export . The steps of mRNA processing and export are closely linked, as some proteins that interact with mRNAs remain bound throughout both processes, whereas others are exchanged for factors specific for each step . NS1 inhibits pre-mRNA splicing by binding to the U6 snRNA component of the spliceosome and/or through interactions with the putative splicing protein NS1-BP [94, 95]. NS1 further disrupts host mRNA processing by binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) and the poly(A)-binding protein II (PABII), which are involved in binding the polyadenylation signal and in the elongation of the poly(A) chain of mRNAs, respectively [96, 97]. The interaction of NS1 with these proteins inhibits 3′-end processing of host mRNAs and contributes to inhibition of host gene expression. However, production of viral transcripts is unaffected by NS1-mediated disruption of mRNA processing because poly(A) tail synthesis on viral mRNAs is carried out by the viral polymerase complex [98, 99]. Additionally, NS1 may facilitate splicing of the viral M1 mRNA segment by interacting with the host mRNA-binding proteins, NS1-BP (NS1-binding protein) and hnRNP K . This permits efficient processing of viral mRNA transcripts before nuclear export occurs. In addition to disrupting host mRNA processing, IAV further disrupts expression of host antiviral genes via NS1 interactions with the host mRNA export machinery. NS1 interacts with the mRNA export factors NXF1-NXT1, which form a complex with Rae1 and E1B-AP5, preventing nuclear export of poly(A) RNA . Altogether, these studies show that NS1 of IAV employs several mechanisms to inhibit the connected and highly regulated processes of mRNA processing and export. Interestingly, mRNAs encoding antiviral factors are retained in the nucleus owing to NS1-mediated inhibition of mRNA nuclear export , indicating that disruption of these pathways likely represents a viral strategy to promote viral replication and avoid the host immune response.
Certain RNA viruses that replicate in the cytoplasm also inhibit host mRNA nuclear export. Vesiculoviruses, such as vesicular stomatitis virus (VSV), are negative-stranded RNA viruses that prevent proper mRNA export through the action of the VSV matrix (M) protein [45, 69, 102-106], resulting in inhibition of host gene expression. This effect decreases competition of VSV mRNAs with host mRNAs for use of the translational machinery. Similar to IAV infection, blockage of mRNA export by VSV also prevents expression of mRNAs that encode antiviral factors . The M protein contains NLSs that allow its import into the nucleus, where it exerts its inhibitory function on mRNA export [104, 107]. Once inside the nucleus, M protein interacts with the mRNA export factor Rae1, which is in complex with Nup98 [69, 105]. This prevents export of bulk poly(A) mRNAs during VSV infection. It has been reported that the interaction of M protein with Rae1 and Nup98 inhibits host transcription . However, high levels of polyadenylated RNA are retained inside the nucleus in the presence of M protein indicating complete mRNA synthesis, as shown by various methods including nucleocytoplasmic fractionation followed by microarray analysis and real-time reverse transcriptase polymerase chain reaction , oligo-dT in situ hybridization [45, 69, 105, 106] and mRNA nuclear export assays in Xenopus oocytes [103-106]. These results indicate that M protein utilizes post-transcriptional mechanisms to inhibit gene expression. Transcriptional studies are necessary to investigate whether M protein regulates expression of certain subsets, rather than the general population, of mRNAs. This is possible, as Nup98 has been shown to regulate transcription of subsets of genes [109, 110].
The inhibition of bulk poly(A) mRNA export by M protein may occur via several mechanisms. The VSV M–Rae1–Nup98 complex may inhibit the export of a subset of mRNAs that include major regulators of gene expression, thereby indirectly triggering a shutoff of host gene expression. Another possibility is that Rae1–Nup98 may facilitate export of bulk NXF1-mediated mRNA export. Therefore, M protein inhibition of Rae1–Nup98 would lead to retention of the majority of mRNAs in the nucleus. Interestingly, M protein-mediated block of mRNA export can be antagonized by IFN, which upregulates the expression of Nup98, Nup96 and Rae1 [16, 45]. Genome-wide studies that identify mRNAs that are directly targeted by the Rae1–Nup98 complex at early stages of infection as well as additional biochemical studies on the interaction of M protein with the mRNA export machinery will further reveal the mechanism of action of VSV M protein.
Interestingly, both Rae1 and Nup98 function during mitosis to regulate spindle assembly [111, 112]. VSV M protein interaction with this complex inhibited mitotic progression and triggered substantial cell death . This has several implications for VSV, which is an oncolytic virus that is currently being developed as a cancer therapeutic [114-116]. As tumor cells have an increased mitotic index, VSV-mediated mitotic cell death likely contributes to its oncolytic activity.
As discussed above, picornaviruses are RNA viruses that replicate within the host cell cytoplasm and regulate nucleocytoplasmic trafficking. Many picornaviruses, including the important human pathogens PV and HRV, inhibit nucleocytoplasmic trafficking of host proteins and mRNAs to promote viral protein synthesis and disrupt host expression of antiviral factors. As discussed above, PV and HRV infection results in the mislocalization or degradation of several important nuclear export factors such as Nup62, Nup98 and Nup153 [14-16, 19, 20]. Cleavage and subsequent degradation or mislocalization of these proteins is mediated by the viral 2Apro, which leads to changes in NPC architecture that affects both host protein and mRNA transport [14-17, 19, 20]. Overall, RNA viruses employ a multitude of strategies to inhibit nucleocytoplasmic trafficking, which suppresses the host innate immune response and enhances viral replication.
Disruption of host gene expression by DNA viruses facilitates export of viral RNA
Not only RNA viruses disrupt nucleocytoplasmic trafficking of mRNA. Many DNA viruses selectively inhibit host mRNA export, while ensuring that viral mRNAs are efficiently exported after transcription. Adenoviruses (AdVs) are double-stranded DNA viruses that infect many vertebrate species, including humans. Two adenoviral protein products, E1B-55K and Ad E4 open reading frame 6 (E4orf6), have been shown to mediate the degradation of cellular proteins that may have a deleterious effect on viral propagation . The interaction of E1B-55K and E4orf6 with host proteins results in the formation of a complex with E3 ubiquitin ligase activity that may contribute to inhibition of host mRNA export and promotion of late viral mRNA export from the nucleus [118, 119]. In one model, it was proposed that the E1B-55K and E4orf6 ubiquitin ligase activity promotes the degradation of an as of yet unidentified cellular protein involved in host mRNA export . Another possibility is that E1B-55K disrupts NXF1-mediated host mRNA export by binding to E1B-AP5, a member of the RNP family that interacts with NXF1 . While it is clear that AdV is able to regulate cellular mRNA export to favor export of late AdV mRNAs, more studies are needed to establish how AdV infection promotes nuclear accumulation of host mRNAs.
As discussed above, herpesviruses are experts at hijacking host cell functions to ensure viral replication. These viruses replicate within the nucleus and therefore must export viral transcripts to the cytoplasm for protein synthesis. One of the proteins encoded by the α-herpesvirus HSV-1 is ICP27, which disrupts host mRNA processing [120, 121] while allowing the export of intronless viral transcripts [122-124]. ICP27 binds directly to the RNA export factor ALY/REF and NXF1, which recruits viral mRNAs to export receptors for preferential transport into the cytoplasm [122-124]. Interestingly, other related herpesviruses do not inhibit host mRNA processing during infection, but do encode an ICP27-like protein that favors viral mRNA export. These viruses include human cytomegalovirus (hCMV), Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein–Barr virus (EBV) and varicella-zoster virus (VZV) [125-129].