It has previously been shown that influenza virus NS1 protein enhances the translation of viral but not cellular mRNAs. This enhancement occurs by increasing the rate of translation initiation and requires the 59UTR sequence, common to all viral mRNAs. In agreement with these findings, we show here that viral mRNAs, but not cellular mRNAs, are associated with NS1 during virus infection. We have previously reported that NS1 interacts with the translation initiation factor eIF4GI, next to its poly(A)-binding protein 1 (PABP1)-interacting domain and that NS1 and eIF4GI are associated in influenza virus-infected cells. Here we show that NS1, although capable of binding poly(A), does not compete with PABP1 for association with eIF4GI and, furthermore, that NS1 and PABP1 interact both in vivo and in vitro in an RNA-independent manner. The interaction maps between residues 365 and 535 in PABP1 and between residues 1 and 81 in NS1. These mapping studies, together with those previously reported for NS1-eIF4GI and PABP1-eIF4GI interactions, imply that the binding of all three proteins would be compatible. Collectively, these and previously published data suggest that NS1 interactions with eIF4GI and PABP1, as well as with viral mRNAs, could promote the specific recruitment of 43S complexes to the viral mRNAs. INTRODUCTIONInfluenza virus infection efficiently shuts off the expression of the host cell genes (Skehel, 1972), while maintaining an efficient translation of viral proteins. During influenza virus infection, the virus evades the inhibition of protein synthesis through the inhibition of the double-stranded RNAactivated kinase (Lee et al., 1992;Lu et al., 1995;Polyak et al., 1996). Cellular protein synthesis shutoff may be the result of several alterations induced by the virus during infection. These include: (i) cap-snatching of cellular premRNAs (Krug et al., 1979), which probably contributes towards decreasing the synthesis of cellular mRNAs; (ii) inhibition of cleavage and polyadenylation of cellular premRNAs (Chen & Krug, 1999;Nemeroff et al., 1998); (iii) nuclear retention of poly(A)-containing cellular mRNAs (Fortes et al., 1994); (iv) degradation of cytoplasmic cellular mRNAs (Beloso et al., 1992;Inglis, 1982; Zürcher et al., 2000); and (v) preferential utilization of the translation machinery by the viral-specific mRNAs (Katze et al., 1986).Influenza virus mRNAs have a capped 59 end followed by a 10-12 nt long untranslated region of cellular, heterogeneous sequences generated by cap-snatching, which precede a viral-encoded, highly conserved sequence that is common to all influenza virus genes. The 39 end of the viral mRNAs is polyadenylated by a reiterative copy of a U 5-7 track present near the 59 end of the viral RNA (Luo et al., 1991;Poon et al., 1998Poon et al., , 1999Robertson et al., 1981). Although viral mRNAs are formally equivalent to cellular ones, influenza virus infection specifically enhances viral mRNA translation, with the conserved sequences contained within the 59-untranslated region (59UTR...

Like their cellular host counterparts, many invading viral pathogens must contend with, modulate, and utilize the host cell’s chromatin machinery to promote efficient lytic infection or control persistent-latent states. While not intended to be comprehensive, this review represents a compilation of conceptual snapshots of the dynamic interplay of viruses with the chromatin environment. Contributions focus on chromatin dynamics during infection, viral circumvention of cellular chromatin repression, chromatin organization of large DNA viruses, tethering and persistence, viral interactions with cellular chromatin modulation machinery, and control of viral latency-reactivation cycles.

Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5 untranslated region (5 UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

The PA subunit of the influenza virus polymerase complex is a phosphoprotein that induces proteolytic degradation of coexpressed proteins. Point mutants with reduced proteolysis induction reconstitute viral ribonucleoproteins defective in replication but not in transcriptional activity. To look for cellular factors that could associate with PA protein, we have carried out a yeast two-hybrid screen. Using a human kidney cDNA library, we identified two different interacting clones. One of them was identified as the human homologue of a previously described cDNA clone from Gallus gallus called CLE. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiquitously in all human organs tested. The interaction of PA and hCLE was also observed with purified proteins in vitro by using pull-down and pep-spot experiments. Mapping of the interaction showed that hCLE interacts with PA subunit at two regions (positions 493 to 512 and 557 to 574) in the PA protein sequence. Immunofluorescence studies showed that the hCLE protein localizes in both the nucleus and the cytosol, although with a predominantly cytosolic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggesting that PA-hCLE interaction is functionally relevant. Searches in the databases showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with transactivators. Similar homologies were found with the other members of the Cdc68 homologue family of transcriptional activators, including the human FACT protein.The genome of influenza A virus consists of a set of eight single-stranded RNA segments of negative polarity. These RNAs form ribonucleoproteins (RNPs) with four viral proteins: the nucleoprotein (NP) and the three subunits of the polymerase (PB1, PB2, and PA). These elements are required for both transcription and replication of the viral genome (10,16,18,29).The roles of the polymerase subunits have been partly outlined. The PB1 subunit contains sequence motifs typical of the viral RNA-dependent RNA polymerases (43), which have been shown to be essential for RNA synthesis (3), suggesting that this subunit is the polymerase itself. PB2 protein binds to CAP1 structures (4, 51) and is involved in the endonucleolytic cleavage of cellular mRNAs to generate the precursors used as primers for the viral transcription (6,22). PA is a phosphoprotein in vivo and is a substrate of casein kinase II in vitro (47). This subunit induces a proteolytic process when expressed individually, affecting both coexpressed proteins and PA protein itself (46). The amino-terminal third of the molecule is sufficient to activate this proteolysis (48). Recently, we have reconstituted RNPs in vivo from cloned genes using PA point mutants deficient in proteolytic activity. These mutant RNPs are as active as the wild type in their transcription activity but have a lower capacity to support replication of model vRNA ...

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.

Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.The genome of influenza A virus consists of eight singlestranded RNA molecules of negative polarity associated with nucleoprotein (NP) molecules and the polymerase in the form of ribonucleoprotein (RNP) complexes (for reviews, see references 40, 43, and 66). The first step in viral gene expression is primary transcription from the incoming viral RNPs (28). The expression of virus proteins, at least NP, leads to the shift from transcription to the synthesis of complete positive-polarity RNAs (cRNAs) (29, 73), which serve as templates for the synthesis of virion RNAs (vRNAs). Transcription and replication of vRNA take place in the nucleus of the infected cell (30,34) and require at least the activity of the three subunits of the polymerase (PB1, PB2, and PA) and the NP (9,31,38,55,64). The syntheses of the various vRNAs are not simultaneous during the infection cycle. Thus, NS1 or NP vRNAs are replicated earlier than M1 or hemagglutinin (HA) vRNAs (72). Since transcription is coupled to replication at the beginning of vRNA synthesis, the NS1 protein and NP are expressed earlier than the M1 protein and HA (72). However, later in the process of vRNA synthesis, transcription is discontinued and viral protein synthesis rests on previously synthesized mRNAs (72). In the course of the infection, viral gene expression takes over the cell machinery, leading to the shutoff phenomenon. Several alterations induced by the virus in the infected cell may be connected to shutoff: nuclear retention and degradation of polymerase II transcripts in the nucleus (35), inhibition of cellular pre-mRNA cleavage and polyadenylation (56, 74), cytoplasmic degradation of preexisting cellular mRNAs (3, 32), and preferential utilization of the translation machinery by the virus-specific mRNAs (36).Influenza A virus encodes a nonstructural protein (NS1) that is translated from the unspliced transcript of segment 8 (33, 44). NS1 is a nuclear protein, both in the infected cell (5, 41) and when expressed from cDNA (23,45,6...

A collection of C-terminal deletion mutants of the influenza A virus NS1 gene has been used to define the regions of the NS1 protein involved in its functionality. Immunofluorescence analyses showed that the NS1 protein sequences downstream from position 81 are not required for nuclear transport. The capacity of these mutants to bind RNA was studied by in vitro binding tests using a model vRNA probe. These experiments showed that the N-terminal 81 amino acids of NS1 protein are sufficient for RNA binding activity. The collection of mutants also served to map the NS1 sequences required for nuclear retention of mRNA and for stimulation of viral mRNA translation, using the NP gene as reporter. The results obtained indicated that the N-terminal 113 amino acids of NS1 protein are sufficient for nuclear retention of mRNA and stimulation of viral mRNA translation. The possibility that this region of the protein may be sufficient for virus viability is discussed in relation to the sequences of NS1 genes of field isolates and to the phenotype of known viral mutants affected in the NS1 gene.

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 ...