Cellular Human CLE/C14orf166 Protein Interacts with Influenza Virus Polymerase and Is Required for Viral Replication

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Transcription and replication of influenza A virus are carried out in the nuclei of infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed of the PB1, PB2, and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated with the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and its partial relocalization to the cytoplasm. Coimmunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with the viral polymerase and RNPs during infection. Downregulation of NXP2/MORC3 by use of two independent short hairpin RNAs (shRNAs) reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of viral RNA (vRNA) and mRNA after NXP2/MORC3 downregulation. Silencing of NXP2/MORC3 in a recombinant minireplicon system in which virus transcription and replication are uncoupled showed reductions in cat mRNA and chloramphenicol acetyltransferase (CAT) protein accumulation but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription. IMPORTANCE Influenza virus infections appear as yearly epidemics Influenza viruses cause an acute respiratory disease that annually affects millions of people worldwide (Global Influenza Surveillance and Response System [GISRS] [http://www.who .int/influenza/gisrs_laboratory/en/]). The genome of influenza A viruses is about 13 kb long and consists of eight single-stranded negative-sense RNA segments. The viral proteome includes 10 viral proteins that have been studied extensively (1) and another 8 proteins, probably accessory proteins, that were identified more recently (reviewed in reference 2). The transcription and replication of influenza viruses occur in the nuclei of infected cells and are mediated by the viral polymerase, a heterotrimer composed of the PB1, PB2, and PA subunits, in the context of viral ribonucleoprotein complexes (RNPs) (3; reviewed in references 4 to 7). The virus recruits host cell factors to help carry out these processes, and in some specific cases, their roles in virus replication have been determined (reviewed in references 4 and 8 to 10). In one such study, we identified the nuclear matrix NXP2 protein as a factor associated with influenza virus polymerase in vivo by proteomic analysis of recombinant purified polymerase complexes (11). Since influenza virus RNA synthesis is connected to the nuclear matrix (12, 13), we decided to further characterize the role of NXP2 in the virus infection cycle.The NXP2 protein (also called MORC3, ZCW...

Section: Resultsmentioning

confidence: 73%

The Cellular Factor NXP2/MORC3 Is a Positive Regulator of Influenza Virus Multiplication

Ver

Marcos-Villar

Landeras-Bueno

et al. 2015

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“…In agreement with the functional coupling between viral and cellular RNA metabolism, many of these factors are involved in the cellular RNA life cycle, such as the RNAP II itself (11), the cyclin T/CDK9 stimulator of transcription elongation (74), the hCLE protein, a positive modulator of the RNAP II (18,20), the splicing factor SFPQ/ PSF (14,17), or the RNA-associated proteins DDX17 and NPM1 (75). All of the above-reported interactions, as well as the vast majority of the polymerase-interacting host factors reported, positively regulate viral replication or viral polymerase activity.…”

Section: Discussionmentioning

confidence: 99%

“…In agreement with this transcriptional association, interaction of the viral polymerase with host cell transcription-related factors has been reported, among which the interaction with the largest subunit of the RNAP II (11) should be emphasized. Other transcription-related factors found to interact with the viral polymerase are Erb-B3 binding protein 1 (Ebp-1) (12), which represses transcription of cell cycle genes regulated by E2F transcription factors (13); DDX5 protein (14), a transcription coactivator that may play a role in transcription initiation (15); SFPQ/PSF factor (14), which stimulates pre-mRNA processing (16) and is essential for influenza virus transcription increasing the efficiency of viral mRNA polyadenylation (17); and hCLE, a positive modulator of the RNAP II (18,19) which is required for influenza virus replication (20).…”

mentioning

confidence: 99%

CHD6, a Cellular Repressor of Influenza Virus Replication, Is Degraded in Human Alveolar Epithelial Cells and Mice Lungs during Infection

Alfonso

Rodríguez

Rodriguez

et al. 2013

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The influenza virus polymerase associates to an important number of transcription-related proteins, including the largest subunit of the RNA polymerase II complex (RNAP II). Despite this association, degradation of the RNAP II takes place in the infected cells once viral transcription is completed. We have previously shown that the chromatin remodeler CHD6 protein interacts with the influenza virus polymerase complex, represses viral replication, and relocalizes to inactive chromatin during influenza virus infection. In this paper, we report that CHD6 acts as a negative modulator of the influenza virus polymerase activity and is also subjected to degradation through a process that includes the following characteristics: (i) the cellular proteasome is not implicated, (ii) the sole expression of the three viral polymerase subunits from its cloned cDNAs is sufficient to induce proteolysis, and (iii) degradation is also observed in vivo in lungs of infected mice and correlates with the increase of viral titers in the lungs. Collectively, the data indicate that CHD6 degradation is a general effect exerted by influenza A viruses and suggest that this viral repressor may play an important inhibitory role since degradation and accumulation into inactive chromatin occur during the infection. The influenza virus contains a segmented genome of eight negative-sense and single-stranded RNA molecules, whose expression takes place in the nucleus of the infected cell. Genomic RNAs (vRNAs) form ribonucleoprotein complexes (vRNPs) that are constituted by the three subunits of the polymerase (PB1, PB2, and PA) and the nucleoprotein (NP), which are responsible for genome expression (1-5). For viral replication, the vRNAs are copied to form full-length positive-stranded RNAs (cRNAs), which serve as templates for vRNA synthesis. During transcription, capped and polyadenylated viral mRNAs are synthesized by the viral polymerase. The mRNA synthesis is primed by shortcapped oligonucleotides of around 10 to 12 nucleotides scavenged from de novo synthesized host cell pre-mRNAs by a viral endonuclease activity (6). This transcription strategy involves a functional coupling between viral and cellular transcription for the capsnatching process, but this functional association is broader and applies to different steps of viral mRNA metabolism. Indeed, two of the viral transcripts are spliced (7,8), but the influenza virus does not possess a viral splicing system, since it is dependent on the host splicing machinery (9), an activity related to the RNA polymerase II (RNAP II) transcription. Furthermore, the influenza virus uses the mRNA export machinery of the infected cell at least for some of its mRNAs, and an active RNA polymerase II is required to facilitate nuclear export of selected viral mRNAs (10). In agreement with this transcriptional association, interaction of the viral polymerase with host cell transcription-related factors has been reported, among which the interaction with the largest subunit of the RNAP II (11) should be emphasi...

“…This transcription strategy involves functional coupling between viral and cellular transcription for the cap-snatching process. The viral polymerase is reported to interact with host cell transcription-related factors (5)(6)(7)(8)(9), among which is the largest subunit of the RNAP II itself (10).…”

mentioning

confidence: 99%

Influenza Virus and Chromatin: Role of the CHD1 Chromatin Remodeler in the Virus Life Cycle

Marcos-Villar

Pazo

Nieto

2016

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Influenza A virus requires ongoing cellular transcription to carry out the cap-snatching process. Chromatin remodelers modify chromatin structure to produce an active or inactive conformation, which enables or prevents the recruitment of transcriptional complexes to specific genes; viral transcription thus depends on chromatin dynamics. Influenza virus polymerase associates with chromatin components of the infected cell, such as RNA polymerase II (RNAP II) or the CHD6 chromatin remodeler. Here we show that another CHD family member, CHD1 protein, also interacts with the influenza virus polymerase complex. CHD1 recognizes the H3K4me3 (histone 3 with a trimethyl group in lysine 4) histone modification, a hallmark of active chromatin. Downregulation of CHD1 causes a reduction in viral polymerase activity, viral RNA transcription, and the production of infectious particles. Despite the dependence of influenza virus on cellular transcription, RNAP II is degraded when viral transcription is complete, and recombinant viruses unable to degrade RNAP II show decreased pathogenicity in the murine model. We describe the CHD1-RNAP II association, as well as the parallel degradation of both proteins during infection with viruses showing full or reduced induction of degradation. The H3K4me3 histone mark also decreased during influenza virus infection, whereas a histone mark of inactive chromatin, H3K27me3, remained unchanged. Our results indicate that CHD1 is a positive regulator of influenza virus multiplication and suggest a role for chromatin remodeling in the control of the influenza virus life cycle. IMPORTANCEAlthough influenza virus is not integrated into the genome of the infected cell, it needs continuous cellular transcription to synthesize viral mRNA. This mechanism implies functional association with host genome expression and thus depends on chromatin dynamics. Influenza virus polymerase associates with transcription-related factors, such as RNA polymerase II, and with chromatin remodelers, such as CHD6. We identified the association of viral polymerase with another chromatin remodeler, the CHD1 protein, which positively modulated viral polymerase activity, viral RNA transcription, and virus multiplication. Once viral transcription is complete, RNAP II is degraded in infected cells, probably as a virus-induced mechanism to reduce the antiviral response. CHD1 associated with RNAP II and paralleled its degradation during infection with viruses that induce full or reduced degradation. These findings suggest that RNAP II degradation and CHD1 degradation cooperate to reduce the antiviral response. Influenza A virus contains eight single-stranded RNA segments of negative polarity (viral RNA [vRNA]) that form viral ribonucleoproteins (vRNP) by association with a trimeric polymerase complex that consists of the PA, PB1, and PB2 subunits and the nucleoprotein (NP). These vRNP are the functional units for RNA transcription and replication, which are restricted to the nucleus of the infected cell (1). For viral RNA r...