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Juan Ortín

Publications and source records attributed to Juan Ortín.

10 recordsLinked to original sources
PubMed2006

Structure and function of RNA replication.

Contrary to their host cells, many viruses contain RNA as genetic material and hence encode an RNA-dependent RNA polymerase to replicate their genomes. This review discusses the present status of our knowledge on the structure of these enzymes and the mechanisms of RNA replication. The simplest viruses encode only the catalytic subunit of the replication complex, but other viruses also contribute a variable number of ancillary factors. These and other factors provided by the host cell play roles in the specificity and affinity of template recognition and the assembly of the replication complex. Usually, these host factors are involved in protein synthesis or RNA modification in the host cell, but they play roles in remodeling RNA-RNA, RNA-protein, and protein-protein interactions during virus RNA replication. Furthermore, viruses take advantage of and modify previous cell structural elements, frequently membrane vesicles, for the formation of RNA replication complexes.

Genome, Viral↗
PubMed2005

Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins.

It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.

Animals↗
PubMed2005

Genetic analysis of influenza virus NS1 gene: a temperature-sensitive mutant shows defective formation of virus particles.

To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 mutants was generated by PCR-mediated mutagenesis. A collection of mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus mutant library by a novel RNP competition virus rescue procedure. Several temperature-sensitive (ts) mutant viruses were obtained by screening of the mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.

Amino Acid Sequence↗
PubMed2004

The composition of Staufen-containing RNA granules from human cells indicates their role in the regulated transport and translation of messenger RNAs.

hStaufen is the human homolog of dmStaufen, a double-stranded (ds)RNA-binding protein involved in early development of the fly. hStaufen-containing complexes were purified by affinity chromatography from human cells transfected with a TAP-tagged hStaufen gene. These complexes showed a size >10 MDa. Untagged complexes with similar size were identified from differentiated human neuroblasts. The identity of proteins present in purified hStaufen complexes was determined by mass spectrometry and the presence of these proteins and other functionally related ones was verified by western blot. Ribosomes and proteins involved in the control of protein synthesis (PABP1 and FMRP) were present in purified hStaufen complexes, as well as elements of the cytoskeleton (tubulins, tau, actin and internexin), cytoskeleton control proteins (IQGAP1, cdc42 and rac1) and motor proteins (dynein, kinesin and myosin). In addition, proteins normally found in the nucleus, like nucleolin and RNA helicase A, were also found associated with cytosolic hStaufen complexes. The co-localization of these components with hStaufen granules in the dendrites of differentiated neuroblasts, determined by confocal immunofluorescence, validated their association in living cells. These results support the notion that the hStaufen-containing granules are structures essential in the localization and regulated translation of human mRNAs in vivo.

ATP-Binding Cassette Transporters↗
PubMed2004

Defective RNA replication and late gene expression in temperature-sensitive influenza viruses expressing deleted forms of the NS1 protein.

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.

Active Transport, Cell Nucleus↗
PubMed2003

3D structure of the influenza virus polymerase complex: localization of subunit domains.

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP-mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP.

Image Processing, Computer-Assisted↗
PubMed2003

PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes.

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 5'UTR 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.

Animals↗
PubMed2003

Threonine 157 of influenza virus PA polymerase subunit modulates RNA replication in infectious viruses.

Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo. In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.

Animals↗
PubMed2003

Mutations in the N-terminal region of influenza virus PB2 protein affect virus RNA replication but not transcription.

PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

Amino Acid Sequence↗