Amelia Nieto

Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein, a Translational Activator of Influenza Virus

ABSTRACT 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.

PA Subunit from Influenza Virus Polymerase Complex Interacts with a Cellular Protein with Homology to a Family of Transcriptional Activators

ABSTRACT 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 galluscalled 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.

Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit

The protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A beta-galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.

Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein

ABSTRACT 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.

Snapshots: Chromatin Control of Viral Infection

Like their cellular host counterparts, many invading viral pathogens must contend with, modulate, and utilize the host cells 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.

The Replication Activity of Influenza Virus Polymerase Is Linked to the Capacity of the PA Subunit To Induce Proteolysis

ABSTRACT The PA subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. The amino-terminal third of the protein is responsible for the induction of proteolysis. We mutated five potential casein kinase II phosphorylation sites located in the amino-terminal third of the protein. Mutations affecting position 157 almost completely abrogated proteolysis induction, whereas a mutation at position 162 produced a moderate decrease and mutations at positions 151, 200, and 224 did not affect proteolysis induction. Reconstitution of the influenza virus polymerase in vivo with viral model RNA containing the chloramphenicol acetyltransferase (CAT) gene indicated that the CAT activity obtained correlated with the capacity of each PA mutant to induce proteolysis. RNA protection assays of the products obtained with viral polymerase, reconstituted in vivo with model RNAs, indicated that mutations at position 157 led to a selective loss of the ability to synthesize cRNA from the viral RNA template but not to transcribe viral RNA, while a mutation affecting position 162 showed an intermediate phenotype. Collectively, these data provide a link between PA-mediated induction of proteolysis and the replication activity of the polymerase.

Influenza Virus mRNA Translation Revisited: Is the eIF4E Cap-Binding Factor Required for Viral mRNA Translation?

ABSTRACT Influenza virus mRNAs bear a short capped oligonucleotide sequence at their 5′ ends derived from the host cell pre-mRNAs by a “cap-snatching” mechanism, followed immediately by a common viral sequence. At their 3′ ends, they contain a poly(A) tail. Although cellular and viral mRNAs are structurally similar, influenza virus promotes the selective translation of its mRNAs despite the inhibition of host cell protein synthesis. The viral polymerase performs the cap snatching and binds selectively to the 5′ common viral sequence. As viral mRNAs are recognized by their own cap-binding complex, we tested whether viral mRNA translation occurs without the contribution of the eIF4E protein, the cellular factor required for cap-dependent translation. Here, we show that influenza virus infection proceeds normally in different situations of functional impairment of the eIF4E factor. In addition, influenza virus polymerase binds to translation preinitiation complexes, and furthermore, under conditions of decreased eIF4GI association to cap structures, an increase in eIF4GI binding to these structures was found upon influenza virus infection. This is the first report providing evidence that influenza virus mRNA translation proceeds independently of a fully active translation initiation factor (eIF4E). The data reported are in agreement with a role of viral polymerase as a substitute for the eIF4E factor for viral mRNA translation.

Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant.

The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype. Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.

Nuclear transport of influenza virus polymerase PA protein

The subcellular distribution of influenza polymerase PA subunit has been studied using a SV40-recombinant virus (SVPA76), which allows the expression and accumulation of this protein in COS-1 cells. In contrast to the complete nuclear localization observed for the PA subunit several hours after influenza virus infection, when COS-1 cells were infected with the SVPA76 recombinant, the PA protein accumulated either in the nucleus, in the cytoplasm or was distributed throughout the cell. When cells were infected with the SVPA76 recombinant and superinfected with influenza virus, a clear increase in the proportion of cells showing nuclear localization of the PA protein was observed, suggesting that some trans-factor may be required to allow complete nuclear accumulation of the protein. Double infections using SVPA76 recombinant and either SVPB1 or SVNS recombinant viruses showed a complete correlation between expression of polymerase PB1 subunit or NS1 protein and nuclear localization of polymerase PA subunit. However, no such correlation was observed in the double infections of SVPA76 and SVNP recombinants. These results suggest that polymerase PB1 subunit and the non-structural proteins could be involved in the nuclear targeting or nuclear retention of influenza polymerase PA protein.

Tau factor polymers are similar to paired helical filaments of Alzheimer's disease

Tau factor, upon urea treatment, is able to polymerize in vitro. These polymers are composed of tau factor as shown by immunogold staining. The structure of tau polymers is very similar to that of paired helical filaments (PHFs) of Alzheimers disease in their dimensions as well as in their periodicity. Metal shadowing of both polymers shows a similar twisting. Also, similar peptide maps were found for tau factor and a 33 kDa protein that is the main component of our PHF preparations.