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

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The worst known H1N1 influenza pandemic in history resulted in more than 20 million deaths in 1918 and 1919. Although the underlying mechanism causing the extreme virulence of the 1918 influenza virus is still obscure, our previous functional genomics analyses revealed a correlation between the lethality of the reconstructed 1918 influenza virus (r1918) in mice and a unique gene expression pattern associated with severe immune responses in the lungs. Lately, microRNAs have emerged as a class of crucial regulators for gene expression. To determine whether differential expression of cellular microRNAs plays a role in the host response to r1918 infection, we compared the lung cellular "microRNAome" of mice infected by r1918 virus with that of mice infected by a nonlethal seasonal influenza virus, A/Texas/36/91. We found that a group of microRNAs, including miR-200a and miR-223, were differentially expressed in response to influenza virus infection and that r1918 and A/Texas/36/91 infection induced distinct microRNA expression profiles. Moreover, we observed significant enrichment in the number of predicted cellular target mRNAs whose expression was inversely correlated with the expression of these microRNAs. Intriguingly, gene ontology analysis revealed that many of these mRNAs play roles in immune response and cell death pathways, which are known to be associated with the extreme virulence of r1918. This is the first demonstration that cellular gene expression patterns in influenza virus-infected mice may be attributed in part to microRNA regulation and that such regulation may be a contributing factor to the extreme virulence of the r1918.

Section: Discussionmentioning

confidence: 99%

MicroRNA Expression and Virulence in Pandemic Influenza Virus-Infected Mice

Yu¹,

Chan²,

et al. 2010

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160

“…3B). Thus, at 4 and 6 h p.i., coinciding with the nuclear redistribution of RNA Pol II, there was a reduction in the hyperphosporylated form of RNA Pol II (recognized by the H5 antibody) and a proportional increase in the hypophosphorylated band of RNA Pol II (a smaller band detected with the 8WG16 antibody) (32). The dramatic reduction of all forms of RNA Pol II was evident from 8 h p.i., as shown with the N-20 RNA Pol II polyclonal antibody (Fig.…”

Section: Resultsmentioning

confidence: 90%

Disruption of Nuclear Organization during the Initial Phase of African Swine Fever Virus Infection

Ballester

Rodríguez-Cariño

Pérez

et al. 2011

African swine fever virus (ASFV), the causative agent of one of the most devastating swine diseases, has been considered exclusively cytoplasmic, even though some authors have shown evidence of an early stage of nuclear replication. In the present study, an increment of lamin A/C phosphorylation was observed in ASFV-infected cells as early as 4 h postinfection, followed by the disassembling of the lamina network close to the sites where the viral genome starts its replication. At later time points, this and other nuclear envelope markers were found in the cytoplasm of the infected cells. The effect of the infection on the cell nucleus was much more severe than previously expected, since a redistribution of other nuclear proteins, such as RNA polymerase II, the splicing speckle SC-35 marker, and the B-23 nucleolar marker, was observed from 4 h postinfection. All this evidence, together with the redistribution, dephosphorylation, and subsequent degradation of RNA polymerase II after ASFV infection, suggests the existence of sophisticated mechanisms to regulate the nuclear machinery during viral infection.Viruses are obligate intracellular parasites that have evolved many diverse strategies to remodel the infected cell, thus providing an ideal environment for their replication and optimal virus production.The nucleus of the infected cell plays an essential role during most viral infections. While some viruses, such as retroviruses, replicate entirely within the nucleus (42), some others are considered nucleocytoplasmic viruses due to the fact that they have an early stage of nuclear replication (20). New evidence demonstrates the relevance of the nucleus and/or its components, even for viruses traditionally considered cytoplasmic (19,29,41). This is the case for one of the most complex viruses found in the animal kingdom, African swine fever virus (ASFV). Despite ASFV being the sole member of the family Asfarviridae (9), it has been phylogenetically incorporated within the nucleocytoplasmic large DNA virus clade, together with iridoviruses, phycodnaviruses, mimiviruses, and poxviruses, forming an individual lineage with poxviruses (20, 21). ASFV and poxviruses share several characteristics that have caused them to be considered purely cytoplasmic viruses, including their capacity to encode a wide range of enzymes that could allow self-replication and transcription, theoretically without needing the cell machinery. In spite of the abovementioned data, there is some evidence indicating an early stage of nuclear replication during ASFV infection (13,30,35,40).In situ hybridization and autoradiography experiments revealed ASF viral DNA in the nuclei of infected cells (macrophages and Vero cells) at early times of viral DNA synthesis (13), confirming largely ignored pioneer studies done 20 years ago showing, for the first time, ASFV DNA within the nuclei of infected macrophages (40) or the incapability of ASFV to replicate in enucleated cells (30). Today, we know that small DNA fragments are synthesized intranuclearly i...

“…At different times postinfection, cells were collected in phosphate-buffered saline with protease and phosphatase inhibitors (500 M sodium orthovanadate, 500 M ␤-glycerophosphate, and 500 M sodium molybdate), and the cell pellet was resuspended in Laemmli sample buffer. Western blotting was carried out as described previously (58). The following primary antibodies were used: for RNAP II, monoclonal antibodies 8WG16 (1:500), H14 (1:500), and H5 (1:500) from BabCo; for PA, monoclonal antibody 14 (1:250) (2); for PB1, a rabbit polyclonal antibody (1:1,000) (20); for nucleoprotein (NP), a rat polyclonal antibody generated using as antigen a His-NP protein expressed and purified from bacteria (1:2,000); and for ␤-tubulin, a mouse monoclonal antibody (1: 15,000) from Sigma.…”

Section: Methodsmentioning

confidence: 99%

“…For example, cytomegalovirus (1, 67), Bunyamwera virus (70), Epstein-Barr virus (3), human immunodeficiency virus type 1 (12,34,74), or adenovirus (51) induce changes in the phosphorylation state of the CTD of the RNAP II. Other viruses, such as herpes simplex virus type 1 (13) or influenza virus (58), provoke the degradation of the largest subunit of the RNAP II complex. Both the dephosphorylation of the CTD and the degradation of the RNAP II could be considered as a potential mechanism in viral pathogenesis.…”

mentioning

confidence: 99%

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

Rodríguez

Pérez-González

Hossain

et al. 2009

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...