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 Human Staufen1 Protein Interacts with Influenza Virus Ribonucleoproteins and Is Required for Efficient Virus Multiplication

2010 ◽  
Vol 84 (15) ◽  
pp. 7603-7612 ◽  
Author(s):  
Susana de Lucas ◽  
Joan Peredo ◽  
Rosa María Marión ◽  
Carmen Sánchez ◽  
Juan Ortín
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Influenza A ◽  
Particle Production ◽  
Nonstructural Protein ◽  
A549 Cells ◽  
Influenza Virus Infection ◽  
Ns1 Protein ◽  
Infected Cells

ABSTRACT The influenza A virus genome consists of 8 negative-stranded RNA segments. NS1 is a nonstructural protein that participates in different steps of the virus infectious cycle, including transcription, replication, and morphogenesis, and acts as a virulence factor. Human Staufen1 (hStau1), a protein involved in the transport and regulated translation of cellular mRNAs, was previously identified as a NS1-interacting factor. To investigate the possible role of hStau1 in the influenza virus infection, we characterized the composition of hStau1-containing granules isolated from virus-infected cells. Viral NS1 protein and ribonucleoproteins (RNPs) were identified in these complexes by Western blotting, and viral mRNAs and viral RNAs (vRNAs) were detected by reverse transcription (RT)-PCR. Also, colocalization of hStau1 with NS1, nucleoprotein (NP), and PA in the cytosol of virus-infected cells was shown by immunofluorescence. To analyze the role of hStau1 in the infection, we downregulated its expression by gene silencing. Human HEK293T cells or A549 cells were silenced using either short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting four independent sites in the hStau1 mRNA. The yield of influenza virus was reduced 5 to 10 times in the various hStau1-silenced cells compared to that in control silenced cells. The expression levels of viral proteins and their nucleocytoplasmic localization were not affected upon hStau1 silencing, but virus particle production, as determined by purification of virions from supernatants, was reduced. These results indicate a role for hStau1 in late events of the influenza virus infection, possibly during virus morphogenesis.

scholarly journals Human RNA Polymerase II Elongation in Slow Motion: Role of the TFIIF RAP74 α1 Helix in Nucleoside Triphosphate-Driven Translocation

2005 ◽  
Vol 25 (9) ◽  
pp. 3583-3595 ◽  
Author(s):  
Chunfen Zhang ◽  
Katie L. Zobeck ◽  
Zachary F. Burton
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transient State ◽  
Slow Motion ◽  
Nucleoside Triphosphate ◽  
Deletion Mutants ◽  
Elongation Complex ◽  
Transcription Factor Iif ◽  
Rnap Ii

ABSTRACT The role of the RAP74 α1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1-227), which includes an intact α1 helix, and RAP74(1-158), in which the α1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the α1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in α1. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF 74(1-227) + TFIIS and TFIIF 74(1-158) + TFIIS. TFIIF 74(1-158) is defective because it fails to promote forward translocation. Deletion of the RAP74 α1 helix results in increased occupancy of the backtracking, cleavage, and restart pathways at a stall position, indicating reverse translocation of the elongation complex. During elongation, TFIIF 74(1-158) fails to support detectable nucleoside triphosphate (NTP)-driven translocation from a stall position and is notably defective in supporting bond completion (NTP-driven translocation coupled to pyrophosphate release) during the processive transition between bonds.

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 Human Cytomegalovirus Infection Induces Specific Hyperphosphorylation of the Carboxyl-Terminal Domain of the Large Subunit of RNA Polymerase II That Is Associated with Changes in the Abundance, Activity, and Localization of cdk9 and cdk7

2005 ◽  
Vol 79 (24) ◽  
pp. 15477-15493 ◽  
Author(s):  
Sama Tamrakar ◽  
Anokhi J. Kapasi ◽  
Deborah H. Spector
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Cytomegalovirus ◽  
Cytomegalovirus Infection ◽  
Large Subunit ◽  
Terminal Domain ◽  
Infected Cells ◽  
Human Cytomegalovirus Infection ◽  
Rnap Ii ◽  
Punctate Pattern

ABSTRACT Human cytomegalovirus infection in the presence of the cyclin-dependent kinase (cdk) inhibitor roscovitine leads to changes in differential splicing and the polyadenylation of immediate early IE1/IE2 and UL37 transcripts (V. Sanchez, A. K. McElroy, J. Yen, S. Tamrakar, C. L. Clark, R. A. Schwartz, and D. H. Spector, J. Virol. 78:11219-11232, 2004). To determine if this was associated with specific phosphorylation of the C-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit by cdk7/cyclin H and cdk9/cyclin T1, we examined the expression and localization of these kinases and the various phosphorylated forms of RNAP II. Infection resulted in increased RNAP II CTD phosphorylated on serines 2 and 5 and increased levels of activity of cdk7 and cdk9. At early times, cdk9 localizes with input viral DNA, and aggregates of cdk9 and cdk7 and a subset of Ser2-phosphorylated RNAP II colocalize with IE1/IE2 proteins adjacent to promyelocytic leukemia protein oncogenic domains. Later, cdk9 and Ser2-phosphorylated RNAP II form a nuclear punctate pattern; cdk7 resides in replication centers, and Ser5-phosphorylated RNAP II clusters at the peripheries of replication centers. Roscovitine treatment leads to decreased levels of hyperphosphorylated RNAP II (RNAP IIo) in infected cells and of hypophosphorylated RNAP II in mock-infected and infected cells. The RNAP IIo decrease does not occur if roscovitine is added 8 h postinfection, as was previously observed for processing of IE transcripts. These results suggest that accurate IE gene expression requires specific phosphorylation of the RNAP II CTD early in infection.

scholarly journals Tomato RNA polymerase II interacts with the rod-like conformation of the left terminal domain of the potato spindle tuber viroid positive RNA genome

2012 ◽  
Vol 93 (7) ◽  
pp. 1591-1600 ◽  
Author(s):  
Teodora Bojić ◽  
Yasnee Beeharry ◽  
Da Jiang Zhang ◽  
Martin Pelchat
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Potato Spindle Tuber Viroid ◽  
Site Directed Mutagenesis ◽  
Terminal Domain ◽  
Non Coding Rna ◽  
Rna Genome ◽  
Rnap Ii ◽  
Carboxy Terminal

Potato spindle tuber viroid (PSTVd) is a small, single-stranded, circular, non-coding RNA pathogen. Host DNA-dependent RNA polymerase II (RNAP II) was proposed to be critical for its replication, but no interaction site for RNAP II on the PSTVd RNA genome was identified. Using a co-immunoprecipitation strategy involving a mAb specific for the conserved heptapeptide (i.e. YSPTSPS) located at the carboxy-terminal domain of the largest subunit of RNAP II, we established the interaction of tomato RNAP II with PSTVd RNA and showed that RNAP II associates with the left terminal domain of PSTVd (+) RNA. RNAP II did not interact with any of several PSTVd (−) RNAs tested. Deletion and site-directed mutagenesis of a shortened model PSTVd (+) RNA fragment were used to identify the role of specific nucleotides and structural motifs in this interaction. Our results provide evidence for the interaction of a RNAP II complex from a natural host with the rod-like conformation of the left terminal domain of PSTVd (+) RNA.

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.

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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