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

2009 ◽  
Vol 83 (21) ◽  
pp. 11166-11174 ◽  
Author(s):  
Ariel Rodriguez ◽  
Alicia Pérez-González ◽  
M. Jaber Hossain ◽  
Li-Mei Chen ◽  
Thierry Rolling ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Influenza A ◽  
Influenza A Viruses ◽  
Recombinant Viruses ◽  
Ann Arbor ◽  
Rnap Ii ◽  
Specific Degradation

ABSTRACT 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.

scholarly journals Influenza Virus Infection Causes Specific Degradation of the Largest Subunit of Cellular RNA Polymerase II

2007 ◽  
Vol 81 (10) ◽  
pp. 5315-5324 ◽  
Author(s):  
A. Rodriguez ◽  
A. Pérez-González ◽  
A. Nieto
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Proteolytic Activity ◽  
Influenza Virus Infection ◽  
Infected Cells ◽  
Proteasome Pathway ◽  
Rnap Ii ◽  
Specific Degradation

ABSTRACT It has been described that influenza virus polymerase associates with RNA polymerase II (RNAP II). To gain information about the role of this interaction, we explored if changes in RNAP II occur during infection. Here we show that influenza virus causes the specific degradation of the hypophosphorylated form of the largest subunit of RNAP II without affecting the accumulation of its hyperphosphorylated forms. This effect is independent of the viral strain and the origin of the cells used. Analysis of synthesized mRNAs in isolated nuclei of infected cells indicated that transcription decreases concomitantly with RNAP II degradation. Moreover, this degradation correlated with the onset of viral transcription and replication. The ubiquitin-mediated proteasome pathway is not involved in virally induced RNAP II proteolysis. The expression of viral polymerase from its cloned cDNAs was sufficient to cause the degradation. Since the PA polymerase subunit has proteolytic activity, we tested its participation in the process. A recombinant virus that encodes a PA point mutant with decreased proteolytic activity and that has defects in replication delayed the effect, suggesting that PA's contribution to RNAP II degradation occurs during infection.

scholarly journals Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Benjamin E. Nilsson ◽  
Aartjan J. W. te Velthuis ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Influenza A Viruses ◽  
Viral Polymerase ◽  
Virus Rna ◽  
Rna Genome ◽  
Transcription And Replication

ABSTRACT The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome. IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

scholarly journals Influenza Virus RNA-Dependent RNA Polymerase and the Host Transcriptional Apparatus

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Tim Krischuns ◽  
Maria Lukarska ◽  
Nadia Naffakh ◽  
Stephen Cusack
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Annual Review ◽  
Publication Date ◽  
Rna Dependent Rna Polymerase ◽  
Open Questions ◽  
Virus Rna ◽  
Cap Binding Complex ◽  
Rnap Ii

Influenza virus RNA-dependent RNA polymerase (FluPol) transcribes the viral RNA genome in the infected cell nucleus. In the 1970s, researchers showed that viral transcription depends on host RNA polymerase II (RNAP II) activity and subsequently that FluPol snatches capped oligomers from nascent RNAP II transcripts to prime its own transcription. Exactly how this occurs remains elusive. Here, we review recent advances in the mechanistic understanding of FluPol transcription and early events in RNAP II transcription that are relevant to cap-snatching. We describe the known direct interactions between FluPol and the RNAP II C-terminal domain and summarize the transcription-related host factors that have been found to interact with FluPol. We also discuss open questions regarding how FluPol may be targeted to actively transcribing RNAP II and the exact context and timing of cap-snatching, which is presumed to occur after cap completion but before the cap is sequestered by the nuclear cap-binding complex. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Association of the Influenza A Virus RNA-Dependent RNA Polymerase with Cellular RNA Polymerase II

2005 ◽  
Vol 79 (9) ◽  
pp. 5812-5818 ◽  
Author(s):  
Othmar G. Engelhardt ◽  
Matt Smith ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Influenza A ◽  
Large Subunit ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Virus Rna ◽  
Transcription Sites

ABSTRACT Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

scholarly journals Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Jeremy R. Keown ◽  
Zihan Zhu ◽  
Loïc Carrique ◽  
Haitian Fan ◽  
Alexander P. Walker ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Structural Data ◽  
Influenza Viruses ◽  
Viral Rna ◽  
Influenza A Viruses ◽  
Seasonal Epidemics ◽  
1918 Influenza

AbstractInfluenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.

scholarly journals Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 2198-2206 ◽  
Author(s):  
M G Katze ◽  
R M Krug
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Hela Cells ◽  
Influenza Virus Infection ◽  
Beta Actin ◽  
Chicken Embryo Fibroblasts

Influenza virus infection has adverse effects on the metabolism of two representative RNA polymerase II transcripts in chicken embryo fibroblasts, those coding for beta-actin and for avian leukosis virus (ALV) proteins. Proviral ALV DNA was integrated into host cell DNA by prior infection with ALV. Within 1 h after influenza virus infection, the rate of transcription of beta-actin and ALV sequences decreased 40 to 60%, as determined by labeling the cells for 5 min with [3H]uridine and by in vitro, runoff assays with isolated nuclei. The transcripts that continued to be synthesized did not appear in the cytoplasm as mature mRNAs, and the kinetics of labeling of these transcripts strongly suggest that they were degraded in the nucleus. By S1 endonuclease assay, it was confirmed that nuclear ALV transcripts disappeared very early after infection, already decreasing ca. 80% by 1 h postinfection. A plausible explanation for this nuclear degradation is that the viral cap-dependent endonuclease in the nucleus cleaves the 5' ends of new polymerase II transcripts, rendering the resulting decapped RNAs susceptible to hydrolysis by cellular nucleases. In contrast to the nuclear transcripts, cytoplasmic beta-actin and ALV mRNAs, which are synthesized before infection, were more stable and did not decrease in amount until after 3 h postinfection. Similar stability of cytoplasmic host cell mRNAs was observed in infected HeLa cells, in which the levels of actin mRNA and two HeLa cell mRNAs (pHe 7 and pHe 28) remained at undiminished levels for 3 h of infection and decreased only slightly by 4.5 h postinfection. The cytoplasmic actin and pHe 7 mRNAs isolated from infected HeLa cells were shown to be translated in reticulocyte extracts in vitro, indicating that host mRNAs were not inactivated by a virus-induced modification. Despite the continued presence of high levels of functional host cell mRNAs, host cell protein synthesis was effectively shut off by about 3 h postinfection in both chicken embryo fibroblasts and HeLa cells. These results are consistent with the establishment of an influenza virus-specific translational system that selectively translates viral and not host mRNAs.

scholarly journals Avian Influenza A Virus Polymerase Association with Nucleoprotein, but Not Polymerase Assembly, Is Impaired in Human Cells during the Course of Infection

2008 ◽  
Vol 83 (3) ◽  
pp. 1320-1331 ◽  
Author(s):  
Marie-Anne Rameix-Welti ◽  
Andru Tomoiu ◽  
Emmanuel Dos Santos Afonso ◽  
Sylvie van der Werf ◽  
Nadia Naffakh
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Infected Cell ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Influenza A Virus ◽  
Influenza A ◽  
Human Cells ◽  
Cell Extracts ◽  
Core Proteins

ABSTRACT Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle.

scholarly journals Cyclin T1/CDK9 Interacts with Influenza A Virus Polymerase and Facilitates Its Association with Cellular RNA Polymerase II

2010 ◽  
Vol 84 (24) ◽  
pp. 12619-12627 ◽  
Author(s):  
Junjie Zhang ◽  
Gang Li ◽  
Xin Ye
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Influenza A ◽  
Viral Transcription ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Cyclin T1

ABSTRACT Influenza virus RNA-dependent RNA polymerase scavenges the 5′ cap from host pre-mRNA to prime viral transcription initiation. It is also well established that viral RNA-dependent RNA polymerase (vRNP) associates with cellular RNA polymerase II (Pol II), on which viral replication depends. Here we report that cyclin T1/CDK9 can interact with influenza virus polymerase and facilitate its association with cellular Pol II. The immunodepletion of cyclin T1/CDK9 totally abolished the association of vRNP with the C-terminal domain (CTD) Ser-2-phosphorylated form of RNA polymerase II. Further studies showed that overexpression of cyclin T1/CDK9 increased the transcription activity of vRNP, while knockdown of cyclin T1/CDK9 impaired viral replication. Our results suggest that cyclin T1/CDK9 serves as an adapter to mediate the interaction of vRNP and RNA Pol II and promote viral transcription.

Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells

1984 ◽  
Vol 4 (10) ◽  
pp. 2198-2206
Author(s):  
M G Katze ◽  
R M Krug
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Hela Cells ◽  
Influenza Virus Infection ◽  
Beta Actin ◽  
Chicken Embryo Fibroblasts

Influenza virus infection has adverse effects on the metabolism of two representative RNA polymerase II transcripts in chicken embryo fibroblasts, those coding for beta-actin and for avian leukosis virus (ALV) proteins. Proviral ALV DNA was integrated into host cell DNA by prior infection with ALV. Within 1 h after influenza virus infection, the rate of transcription of beta-actin and ALV sequences decreased 40 to 60%, as determined by labeling the cells for 5 min with [3H]uridine and by in vitro, runoff assays with isolated nuclei. The transcripts that continued to be synthesized did not appear in the cytoplasm as mature mRNAs, and the kinetics of labeling of these transcripts strongly suggest that they were degraded in the nucleus. By S1 endonuclease assay, it was confirmed that nuclear ALV transcripts disappeared very early after infection, already decreasing ca. 80% by 1 h postinfection. A plausible explanation for this nuclear degradation is that the viral cap-dependent endonuclease in the nucleus cleaves the 5' ends of new polymerase II transcripts, rendering the resulting decapped RNAs susceptible to hydrolysis by cellular nucleases. In contrast to the nuclear transcripts, cytoplasmic beta-actin and ALV mRNAs, which are synthesized before infection, were more stable and did not decrease in amount until after 3 h postinfection. Similar stability of cytoplasmic host cell mRNAs was observed in infected HeLa cells, in which the levels of actin mRNA and two HeLa cell mRNAs (pHe 7 and pHe 28) remained at undiminished levels for 3 h of infection and decreased only slightly by 4.5 h postinfection. The cytoplasmic actin and pHe 7 mRNAs isolated from infected HeLa cells were shown to be translated in reticulocyte extracts in vitro, indicating that host mRNAs were not inactivated by a virus-induced modification. Despite the continued presence of high levels of functional host cell mRNAs, host cell protein synthesis was effectively shut off by about 3 h postinfection in both chicken embryo fibroblasts and HeLa cells. These results are consistent with the establishment of an influenza virus-specific translational system that selectively translates viral and not host mRNAs.

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