Biomedical subjects

Iván Ventoso

Publications and source records attributed to Iván Ventoso.

5 recordsLinked to original sources

PubMed2006

Antiviral effect of the mammalian translation initiation factor 2alpha kinase GCN2 against RNA viruses.

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

Alphavirus Infections↗

PubMed2006

Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR.

The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2alpha in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2alpha. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2alpha. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2alpha. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2alpha phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. Our findings show the existence of an alternative way to locate the ribosomes on the initiation codon of mRNA that is exploited by a family of viruses to counteract the antiviral effect of PKR.

Alphavirus Infections↗

PubMed2005

Involvement of HIV-1 protease in virus-induced cell killing.

Acute infection of human CD4+ cells with cytopathic strains of HIV-1 causes rapid cell death. The role played by HIV-1 protease (PR) in virus-induced cell killing was investigated by subjecting C8166 cells to a single round of infection. The presence of HIV-1 PR inhibitor saquinavir from 24h post-infection prevented virus-induced cell lysis. This inhibitor caused only a small reduction in the number of infected cells and in the expression of HIV-1-specific proteins. Moreover, treatments that block HIV-1 reinfection, such as AZT or the anti-CD4 antibody leu3.a, exerted little effect on virus-induced cell death. Thus, the specific inhibition of HIV-1 protease reduced the extent of both necrosis and apoptosis in C8166 cells such that most cells survived HIV-1 infection. Continued treatment of the infected cells with saquinavir led to the progressive suppression of HIV-1 expression; no viral proteins being detected 10 days after primary infection. Notably, reactivation of HIV-1 protease in these cells by removing the saquinavir triggered virus replication and cell lysis. These findings may contribute towards a better understanding of HIV-1 pathogenesis, and emphasise the potential of the virus protease as a key therapeutic target in AIDS treatment.

Apoptosis↗

PubMed2003

Cleavage of eIF4G by HIV-1 protease: effects on translation.

We have recently reported that HIV-1 protease (PR) cleaves the initiation factor of translation eIF4GI [Ventoso et al., Proc. Natl. Acad. Sci. USA 98 (2001) 12966-12971]. Here, we analyze the proteolytic activity of HIV-1 PR on eIF4GI and eIF4GII and its implications for the translation of mRNAs. HIV-1 PR efficiently cleaves eIF4GI, but not eIF4GII, in cell-free systems as well as in transfected mammalian cells. This specific proteolytic activity of the retroviral protease on eIF4GI was more selective than that observed with poliovirus 2A(pro). Despite the presence of an intact endogenous eIF4GII, cleavage of eIF4GI by HIV-1 PR was sufficient to impair drastically the translation of capped and uncapped mRNAs. In contrast, poliovirus IRES-driven translation was unaffected or even enhanced by HIV-1 PR after cleavage of eIF4GI. Further support for these in vitro results has been provided by the expression of HIV-1 PR in COS cells from a Gag-PR precursor. Our present findings suggest that eIF4GI intactness is necessary to maintain cap-dependent translation, not only in cell-free systems but also in mammalian cells.

Amino Acid Sequence↗

PubMed2002

Cell killing by HIV-1 protease.

The human immunodeficiency virus protease (HIV-1 PR) was expressed both in the yeast Saccharomyces cerevisiae and in mammalian cells. Inducible expression of HIV-1 PR arrested yeast growth, which was followed by cell lysis. The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium. Given that neither poliovirus 2A protease nor 2BC protein, both being highly toxic for S. cerevisiae, were able to produce similar effects, it seems that this lytic phenotype is specific of HIV-1 PR. Drastic alterations in membrane permeability preceded the lysis in yeast expressing HIV-1 PR. Cell killing and lysis provoked by HIV-1 PR were also observed in mammalian cells. Thus, COS7 cells expressing the protease showed increased plasma membrane permeability and underwent lysis by necrosis with no signs of apoptosis. Strikingly, the morphological alterations induced by HIV-1 PR in yeast and mammalian cells were similar in many aspects. To our knowledge, this is the first report of a viral protein with such an activity. These findings contribute to the present knowledge on HIV-1-induced cytopathogenesis.

Animals↗