hCLE/CGI-99, a Human Protein that Interacts with the Influenza Virus Polymerase, Is a mRNA Transcription Modulator

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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...

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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“…In this study, we showed that DR1 directly interacts with all three subunits of the viral RdRp complex and enhances the viral RdRp activity; thus, DR1 directly plays a positive role in IAV RNA replication. So far, IAV RNA replication has been shown to be coordinated by several host factors; for instance, (i) hCLE and cyclin T1/CDK9 interact with Pol II and the viral RdRp, thus enhancing Pol II activity to promote viral transcription (31,32); and (ii) the MCM complex and Tat-SF1 associate with the viral RdRp and NP, respectively, to promote viral replication (33,34). Intriguingly, unlike cyclin T1/CDK9 and Tat-SF1, which are positive cellular transcription elongation factors, DR1 is thought to be a cellular transcription repressor by precluding the assembly of the PIC to inhibit Pol II initiation (15).…”

Section: Discussionmentioning

confidence: 99%

A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

Hsu

Jeng

et al. 2015

Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-␤) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication. IMPORTANCE Investigations of virus-host interactions involved in influenza A virus (IAV)replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection.

“…To analyze RNA synthesis, cultures of HEK293T cells were mock infected or infected with the VIC or PR8 strains, and their nuclei were isolated and frozen as described previously (54). These nuclei were used to detect in vitro RNA synthesis by incorporation of [ 32 P]␣-GTP (250 Ci/ml) during a 30-min pulse, in the presence or absence of ␣-amanitin (5 g/ml), as described previously (58).…”

Section: Methodsmentioning

confidence: 99%

Attenuated Strains of Influenza A Viruses Do Not Induce Degradation of RNA Polymerase II

Rodríguez

Pérez-González

Hossain

et al. 2009

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We have previously shown that infection with laboratory-passaged strains of influenza virus causes both specific degradation of the largest subunit of the RNA polymerase II complex (RNAP II) and inhibition of host cell transcription. When infection with natural human and avian isolates belonging to different antigenic subtypes was examined, we observed that all of these viruses efficiently induce the proteolytic process. To evaluate whether this process is a general feature of nonattenuated viruses, we studied the behavior of the influenza virus strains A/PR8/8/34 (PR8) and the cold-adapted A/Ann Arbor/6/60 (AA), which are currently used as the donor strains for vaccine seeds due to their attenuated phenotype. We have observed that upon infection with these strains, degradation of the RNAP II does not occur. Moreover, by runoff experiments we observe that PR8 has a reduced ability to inhibit cellular mRNA transcription. In addition, a hypervirulent PR8 (hvPR8) variant that multiplies much faster than standard PR8 (lvPR8) in infected cells and is more virulent in mice than the parental PR8 virus, efficiently induces RNAP II degradation. Studies with reassortant viruses containing defined genome segments of both hvPR8 and lvPR8 indicate that PA and PB2 subunits individually contribute to the ability of influenza virus to degrade the RNAP II. In addition, recently it has been reported that the inclusion of PA or PB2 from hvPR8 in lvPR8 recombinant viruses, highly increases their pathogenicity. Together, the data indicate that the capacity of the influenza virus to degrade RNAP II and inhibit the host cell transcription machinery is a feature of influenza A viruses that might contribute to their virulence.The genome of the influenza A viruses consists of eight single-stranded RNA segments of negative polarity, encoding a total of 11 proteins. Upon entry into susceptible cells, infecting ribonucleoprotein complexes (RNPs) are transported to the nucleus, where transcription and replication take place. Replication of viral RNAs (vRNAs) involves the synthesis of positivestrand replicative intermediates (cRNAs) that are exact copies of the virion RNAs (for a review, see reference 15). A functional link between viral and cellular transcription has been proposed since influenza virus mRNA transcription is initiated using short capped RNA oligonucleotides as primers that are obtained by endonucleolytic cleavage of de novo-synthesized cellular premRNAs (6, 56). This cap-snatching process is performed by the viral polymerase, a heterotrimeric complex comprised of the PB1, PB2, and PA subunits (15,30,40).The carboxy-terminal domain (CTD) of the largest subunit of the RNA polymerase II (RNAP II) complex plays an essential role in cellular transcription. This domain is differentially phosphorylated during the transcription cycle, dynamically permitting or impeding its association with a large number of factors (27). Two major forms of RNAP II can be found in cells when the CTD of its largest subunit is hyperphosphorylated or hyp...