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 ...

hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.

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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...

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.

hCLE/C14orf166/RTRAF, DDX1, and HSPC117 are components of cytoplasmic mRNA-transporting granules kinesin-associated in dendrites. They have also been found in cytoplasmic ribosome-containing RNA granules that transport specific mRNAs halted for translation until specific neuronal signals renders them accessible to the translation machinery. hCLE associates to DDX1, HSPC117, and FAM98B in HEK293T cells and all four proteins bind to cap analog-containing resins. Competition and elution experiments indicate that binding of hCLE complex to cap resins is independent of eIF4E; the cap-binding factor needed for translation. Purified hCLE free of its associated proteins binds cap with low affinity suggesting that its interacting proteins modulate its cap association. hCLE silencing reduces hCLE accumulation and that of its interacting proteins and decreases mRNA translation. hCLE-associated RNAs have been isolated and sequenced; RNAs involved in mRNA translation are specifically associated. The data suggest that RNA granules may co-transport RNAs encoding proteins involved in specific functions together with RNAs that encode proteins needed for the translation of these specific RNAs and indicate an important role for hCLE modulating mRNA translation.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.