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...