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 ubiquitinmediated 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.The genome of influenza virus consists of eight singlestranded RNA molecules of negative polarity. The viral RNA polymerase is composed of three subunits, PB1, PB2, and PA (16,26,27), which together with the nucleoprotein perform all the activities required for viral RNA expression (15,18,28,33). The PB2 subunit is able to bind cap 1 structures of host cell hnRNAs (8, 57). The PB1 subunit contains both sequence motifs typical of the viral RNA-dependent RNA polymerases (46), which are essential for RNA synthesis (7), and the endonuclease activity responsible for the cleavage of host mRNA precursors (35). The PA subunit is a phosphoprotein with proteolytic activity (25,40,50,51). The phenotype of viral temperature-sensitive and protease mutants suggests that the PA subunit may be involved in the transition from mRNA transcription to replication (29, 37). The transcription process involves a cap-stealing mechanism by which 5Ј-capped oligonucleotides derived from newly synthesized RNA polymerase II (RNAP II) transcripts are used as primers and elongated by the viral polymerase (9, 45). In line with this transcription strategy, parental virion RNPs colocalize with active RNAP II in the infected-cell nucleus (I. Salanueva, personal communication). Due to the requirements for cellular capped mRNAs, virus transcription is inhibited by actinomycin D or ␣-amanitin (38). Viral RNA replication involves the synthesis of cap-independent, full-length positive-stranded RNAs complementary to the genomic viral RNAs (vRNAs), which serve as templates for amplification of the vRNAs and are not sensitive to actinomycin D or ␣-amanitin (53).Many viruses induce alterations in host cell gene expression. Among these, changes in the transcriptional machinery of the infected cells are broadly documented. RNAP ...

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SummaryThe influenza virus establishes close functional and structural connections with the nucleus of the infected cell. Thus, viral ribonucleoproteins (RNPs) are closely bound to chromatin components and the main constituent of viral RNPs, the nucleoprotein (NP) protein, interacts with histone tails. Using a yeast two-hybrid screening, we previously found that the PA influenza virus polymerase subunit interacts with the CHD6 protein, a member of the CHD family of chromatin remodelers. Here we show that CHD6 also interacts with the viral polymerase complex and colocalizes with viral RNPs in the infected cells. To study the relationships between RNPs, chromatin and CHD6, we have analysed whether NP and CHD6 binds to peptides representing trimethylated lysines of histone 3 tails that mark transcriptionally active or inactive chromatin. Upon infection, NP binds to marks of repressed chromatin and, interestingly an important recruitment of CHD6 to these heterochromatin marks occurs in this situation. Silencing experiments indicate that CHD6 acts as a negative modulator of influenza virus replication. Hence, the CHD6 association with inactive chromatin could be part of a process where the influenza virus triggers modifications of chromatin-associated proteins that could contribute to the pathogenic events used by the virus to induce host cell shut-off.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including RNA polymerase II. We previously described the interaction of influenza virus polymerase subunit PA with human CLE/C14orf166 protein (hCLE), a positive modulator of this cellular RNA polymerase. Here, we show that hCLE also interacts with the influenza virus polymerase complex and colocalizes with viral ribonucleoproteins. Silencing of hCLE causes reduction of viral polymerase activity, viral RNA transcription and replication, virus titer, and viral particle production. Altogether, these findings indicate that the cellular transcription factor hCLE is an important protein for influenza virus replication.Influenza A virus contains eight single-stranded segments of negative-polarity RNA and encodes 11 proteins (9). Four of them are responsible for genome expression, the three polymerase subunits (PA, PB1, and PB2) and the nucleoprotein (NP). These proteins associate with each viral RNA segment to constitute the viral ribonucleoproteins (vRNPs) (9,22). A functional coupling between viral and cellular transcription exists, due to the unusual viral initiation mechanism that uses as primers short-capped oligonucleotides scavenged from newly synthesized RNA polymerase II (RNAP II) transcripts. Within the viral polymerase, subunit PB1 contains the catalytic polymerase activity (4), cap recognition is achieved by subunit PB2 (5, 13, 27), and subunit PA is required to cleave the capped oligonucleotides (8,29). In accordance with the viral transcription mechanism, a number of cellular transcriptionrelated factors have been reported to associate with the viral polymerase complex and/or the polymerase subunits. Among these, the interaction with RNAP II itself should be emphasized (11). Other transcription-related factors found to interact with the viral polymerase are Ebp-1 (Erb-B3 binding protein 1) (14), which represses the transcription of cell cycle genes regulated by E2F transcription factors (21), DDX5 (16), a transcription coactivator that may play a role in cellular transcription initiation (3), and SFPQ/PSF factor (16), which stimulates cellular pre-mRNA processing (25). However, the individual biological mechanisms behind these host-cell interactions remain elusive in most cases.hCLE interacts with the influenza virus polymerase complex. Using a yeast two-hybrid screening assay, we previously reported the interaction of human CLE (hCLE) and the chromatin remodeler factor CHD6 with the influenza virus polymerase subunit PA (15). Further characterization indicated that hCLE associates with and is a positive modulator of RNAP II (20). Hence, we wanted to determine whether hCLE interacted with the entire viral polymerase complex. HEK293T cells were infected with the influenza virus A/WSN/33 (WSN) strain at a multiplicity of infection (MOI) of 3 PFU/cell, and at 6 h postinfection (h.p.i.), coimmunoprecipitation analyses were carried out. The infected cells were collected and lysed in a buffer composed of 150 mM...

Pandemic 2009 H1N1 (pH1N1) influenza viruses caused mild symptoms in most infected patients. However, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. Here we tested whether influenza strains displaying differential virulence could be present among circulating pH1N1 viruses. The biological properties and the genotype of viruses isolated from a patient showing mild disease (M) or from a fatal case (F), both without known co-morbid conditions were compared in vitro and in vivo. The F virus presented faster growth kinetics and stronger induction of cytokines than M virus in human alveolar lung epithelial cells. In the murine model in vivo, the F virus showed a stronger morbidity and mortality than M virus. Remarkably, a higher proportion of mice presenting infectious virus in the hearts, was found in F virus-infected animals. Altogether, the data indicate that strains of pH1N1 virus with enhanced pathogenicity circulated during the 2009 pandemic. In addition, examination of chemokine receptor 5 (CCR5) genotype, recently reported as involved in severe influenza virus disease, revealed that the F virus-infected patient was homozygous for the deleted form of CCR5 receptor (CCR5Δ32).

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...

The viral sense and antisense RNAs were misidentified in Fig. 4. The sense RNA was indicated as vRNA, and the antisense RNA was indicated as mRNA. The corrected Fig. 4 is shown below. The figure legend is correct as originally published.