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

2016 ◽  
Vol 90 (7) ◽  
pp. 3694-3707 ◽  
Author(s):  
Laura Marcos-Villar ◽  
Alejandra Pazo ◽  
Amelia Nieto
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase Ii ◽  
Polymerase Activity ◽  
Virus Multiplication ◽  
Viral Transcription ◽  
Chromatin Remodeler ◽  
Viral Polymerase ◽  
Chromatin Dynamics ◽  
Rnap Ii ◽  
Cellular Transcription

ABSTRACTInfluenza 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.

scholarly journals Ubiquitination Upregulates Influenza Virus Polymerase Function

2016 ◽  
Vol 90 (23) ◽  
pp. 10906-10914 ◽  
Author(s):  
James Kirui ◽  
Arindam Mondal ◽  
Andrew Mehle
Keyword(s):  
Influenza Virus ◽  
Life Cycle ◽  
Viral Proteins ◽  
Polymerase Activity ◽  
Viral Gene ◽  
Viral Polymerase ◽  
Ubiquitin Proteasome Pathway ◽  
Virus Life Cycle ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

ABSTRACTThe influenza A virus polymerase plays an essential role in the virus life cycle, directing synthesis of viral mRNAs and genomes. It is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher-order ribonucleoprotein (RNP) complexes. The polymerase is regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including posttranslational modifications; however, the mechanisms are not fully understood. Here we demonstrate that ubiquitination plays an important role in stimulating polymerase activity. We show that all protein subunits in the RNP are ubiquitinated, but ubiquitination does not significantly alter protein levels. Instead, ubiquitination and an active proteasome enhance polymerase activity. Expression of ubiquitin upregulates polymerase function in a dose-dependent fashion, causing increased accumulation of viral RNA (vRNA), cRNA, and mRNA and enhanced viral gene expression during infection. Ubiquitin expression directly affects polymerase activity independent of nucleoprotein (NP) or ribonucleoprotein (RNP) assembly. Ubiquitination and the ubiquitin-proteasome pathway play key roles during multiple stages of influenza virus infection, and data presented here now demonstrate that these processes modulate viral polymerase activity independent of protein degradation.IMPORTANCEThe cellular ubiquitin-proteasome pathway impacts steps during the entire influenza virus life cycle. Ubiquitination suppresses replication by targeting viral proteins for degradation and stimulating innate antiviral signaling pathways. Ubiquitination also enhances replication by facilitating viral entry and virion disassembly. We identify here an addition proviral role of the ubiquitin-proteasome system, showing that all of the proteins in the viral replication machinery are subject to ubiquitination and this is crucial for optimal viral polymerase activity. Manipulation of the ubiquitin machinery for therapeutic benefit is therefore likely to disrupt the function of multiple viral proteins at stages throughout the course of infection.

scholarly journals Amino acid sites related to the PB2 subunits of IDV affect polymerase activity

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yutian Wang ◽  
Weiyang Sun ◽  
Zhenfei Wang ◽  
Menglin Zhao ◽  
Xinghai Zhang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Point Mutations ◽  
Polymerase Activity ◽  
Acid Sites ◽  
Feedlot Cattle ◽  
High Morbidity ◽  
Viral Polymerase ◽  
Random Mutation

Abstract Background In 2011, a new influenza virus, named Influenza D Virus (IDV), was isolated from pigs, and then cattle, presenting influenza-like symptoms. IDV is one of the causative agents of Bovine Respiratory Disease (BRD), which causes high morbidity and mortality in feedlot cattle worldwide. To date, the molecular mechanisms of IDV pathogenicity are unknown. Recent IDV outbreaks in cattle, along with serological and genetic evidence of IDV infection in humans, have raised concerns regarding the zoonotic potential of this virus. Influenza virus polymerase is a determining factor of viral pathogenicity to mammals. Methods Here we take a prospective approach to this question by creating a random mutation library about PB2 subunit of the IDV viral polymerase to test which amino acid point mutations will increase viral polymerase activity, leading to increased pathogenicity of the virus. Results Our work shows some exact sites that could affect polymerase activities in influenza D viruses. For example, two single-site mutations, PB2-D533S and PB2-G603Y, can independently increase polymerase activity. The PB2-D533S mutation alone can increase the polymerase activity by 9.92 times, while the PB2-G603Y mutation increments the activity by 8.22 times. Conclusion Taken together, our findings provide important insight into IDV replication fitness mediated by the PB2 protein, increasing our understanding of IDV replication and pathogenicity and facilitating future studies.

scholarly journals Fundamental Contribution and Host Range Determination of ANP32A and ANP32B in Influenza A Virus Polymerase Activity

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Haili Zhang ◽  
Zhenyu Zhang ◽  
Yujie Wang ◽  
Meiyue Wang ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Cellular Protein ◽  
Human Influenza ◽  
Viral Polymerase

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation.IMPORTANCEThe key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

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 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 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 Fundamental contribution and host range determination of ANP32 protein family in influenza A virus polymerase activity

2019 ◽  
Author(s):  
Haili Zhang ◽  
Zhenyu Zhang ◽  
Yujie Wang ◽  
Meiyue Wang ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Viral Polymerase ◽  
Human Influenza Virus ◽  
Virus Rna

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase, and to both influence polymerase activity and interspecies restriction. Here we report that either ANP32A or ANP32B is indispensable for influenza A virus RNA replication. The contribution of ANP32B is equal to that of ANP32A, and together they play a fundamental role in the activity of mammalian influenza A virus polymerase, while neither human ANP32A nor ANP32B support the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving ANP32A alone to support viral replication. Two amino acid mutations at sites 129-130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A&B as key factors for both virus replication and adaption.IMPORTANCEThe key host factors involved in the influenza A viral the polymerase activity and RNA replication remain largely unknown. Here we provide evidence that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza virus RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily non-functional in supporting viral replication because of a 129-130 site mutation. The 129-130 site plays an important role in ANP32A/B and viral polymerase interaction, therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

scholarly journals Low Polymerase Activity Attributed to PA Drives the Acquisition of the PB2 E627K Mutation of H7N9 Avian Influenza Virus in Mammals

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Libin Liang ◽  
Li Jiang ◽  
Junping Li ◽  
Qingqing Zhao ◽  
Jinguang Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Virus Replication ◽  
Driving Force ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Avian Influenza Viruses ◽  
Viral Polymerase ◽  
Adaptive Mutations ◽  
Highly Sensitive

ABSTRACTAvian influenza viruses (AIVs) must acquire mammalian-adaptive mutations before they can efficiently replicate in and transmit among humans. The PB2 E627K mutation is known to play a prominent role in the mammalian adaptation of AIVs. The H7N9 AIVs that emerged in 2013 in China easily acquired the PB2 E627K mutation upon replication in humans. Here, we generate a series of reassortant or mutant H7N9 AIVs and test them in mice. We show that the low polymerase activity attributed to the viral PA protein is the intrinsic driving force behind the emergence of PB2 E627K during H7N9 AIV replication in mice. Four residues in the N-terminal region of PA are critical in mediating the PB2 E627K acquisition. Notably, due to the identity of viral PA protein, the polymerase activity and growth of H7N9 AIV are highly sensitive to changes in expression levels of human ANP32A protein. Furthermore, the impaired viral polymerase activity of H7N9 AIV caused by the depletion of ANP32A led to reduced virus replication inAnp32a−/−mice, abolishing the acquisition of the PB2 E627K mutation and instead driving the virus to acquire the alternative PB2 D701N mutation. Taken together, our findings show that the emergence of the PB2 E627K mutation of H7N9 AIV is driven by the intrinsic low polymerase activity conferred by the viral PA protein, which also involves the engagement of mammalian ANP32A.IMPORTANCEThe emergence of the PB2 E627K substitution is critical in the mammalian adaptation and pathogenesis of AIV. H7N9 AIVs that emerged in 2013 possess a prominent ability in gaining the PB2 E627K mutation in humans. Here, we demonstrate that the acquisition of the H7N9 PB2 E627K mutation is driven by the low polymerase activity conferred by the viral PA protein in human cells, and four PA residues are collectively involved in this process. Notably, the H7N9 PA protein leads to significant dependence of viral polymerase function on human ANP32A protein, andAnp32aknockout abolishes PB2 E627K acquisition in mice. These findings reveal that viral PA and host ANP32A are crucial for the emergence of PB2 E627K during adaptation of H7N9 AIVs to humans.

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.

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