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.