Biomedical subjects

Luis Carrasco

Publications and source records attributed to Luis Carrasco.

At least 19 recordsLinked to original sources

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

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G.

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.

Animals↗

PubMed2005

Viroporin activity of murine hepatitis virus E protein.

The viroporin activity of the E protein from murine hepatitis virus (MHV), a member of the coronaviruses, was analyzed. Viroporins are a growing family of viral proteins able to enhance membrane permeability, promoting virus budding. Initially, the MHV E gene was inducibly expressed in Escherichia coli cells, leading to the arrest of bacterial growth, cell lysis and permeabilization to different compounds. Thus, exit of labeled nucleotides from E. coli cells to the cytoplasm was apparent upon expression of MHV E. In addition, enhanced entry of the antibiotic hygromycin B occurred at levels comparable to those observed with the viroporin 6K from Sindbis virus. Mammalian cells are also readily permeabilized by the expression of MHV E protein. Finally, brefeldin A powerfully blocks the viroporin activity of the E protein in BHK cells, suggesting that an intact vesicular system is necessary for this coronavirus to permeabilize mammalian cells.

Animals↗

PubMed2005

Regulation of HIV-1 env mRNA translation by Rev protein.

We have examined the effect of Rev on the regulation of the expression of RRE containing mRNAs when they were synthesised in the nucleus or directly in the cytoplasm. In the nuclear expression system, Rev enhanced env mRNA transport by about 1.6-fold, while translation of this mRNA was increased more than a 100-fold. These findings indicate that the target of Rev activity is located mainly at the translational level. Synthesis of Env using a recombinant vaccinia virus system, which synthesised env mRNA directly in the cytoplasm, is also enhanced by Rev. Finally, RRE functioning was examined using a luciferase mRNA bearing this element. Rev stimulated the synthesis of Luciferase both when the luc mRNA was made in the nucleus or in cytoplasm. Our results indicate that the effect of Rev on env mRNA transport is low compared with the enhancement of translation of this mRNA.

Active Transport, Cell Nucleus↗

PubMed2005

Requirement of the vesicular system for membrane permeabilization by Sindbis virus.

The vast majority of animal viruses enhance membrane permeability at two moments of infection. Herein, we describe that the entry of Sindbis virus (SV) in BHK cells promotes the co-entry of the macromolecule alpha-sarcin into the cytoplasm, thereby blocking translation. At a later stage, this protein toxin cannot enter the cell, while low molecular weight compounds, such as hygromycin B, readily pass through the plasma membrane of Sindbis virus-infected BHK cells. To unveil the participation of the different Sindbis virus structural proteins in late permeabilization, transfection experiments with each late gene by separate have been carried out. Our findings indicate that 6K is the main determinant that enhances membrane permeabilization. The co-expression of both viral glycoproteins employing a Sindbis virus variant that lacks the entire 6K gene partly modifies membrane permeability. Brefeldin A, a macrolide antibiotic that interferes with the proper functioning of the vesicular system, hampers the induction of membrane leakiness without significantly affecting viral protein synthesis. On the other hand, the flavone compound Ro-090179 also diminishes the entry of hygromycin B, while bafilomycin A1 or nocodazole have no effect. These data reveal the requirement of the vesicular system for late viral membrane permeabilization.

Animals↗

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↗

PubMed2005

Isolation of Candida famata from a patient with acute zonal occult outer retinopathy.

The etiology of a number of retinopathies, such as acute zonal occult outer retinopathy (AZOOR), remains undetermined. Candida famata was isolated from conjunctival exudates of a patient diagnosed with AZOOR. This yeast was very abundant, particularly in the more affected eye, while no other pathogens or fungal species were in evidence. Immunological tests revealed the presence of antigen-specific T lymphocytes by using C. famata as a challenge. Moreover, enzyme-linked immunosorbent assay analysis showed the presence of specific antibodies against this yeast in the patient's blood. Delayed hypersensitivity by use of a skin test was also positive. Finally, antifungal treatments led to improvements in several clinical symptoms, including funduscopic analysis. However, despite prolonged treatment with fluconazole and itraconazole, C. famata still appeared in the conjunctival exudates. The new antifungal voriconazole may represent a better choice for treatment.

Acute Disease↗

PubMed2004

Membrane-permeabilizing motif in Semliki forest virus E1 glycoprotein.

Cell infection by alphaviruses is accompanied by membrane permeability changes. New predictive approaches, including the computation of interfacial affinity and corresponding hydrophobic moments, suggest a segmented amphipathic-at-interface domain in the stem region of Semliki Forest virus fusion protein E1. Expression of E1 sequences in Escherichia coli cells confirmed that the membrane proximal plus transmembrane (TM) domain unit permeabilizes cells as efficiently as the 6K viroporin. Both our predictive and experimental data support the involvement of the E1 stem-TM region in membrane insertion and permeabilization. We propose to combine Wimley-White hydrophobicity analysis with expression-coupled permeability assays in order to identify viral products implied in breaching cell membrane barriers during infection.

Animals↗

PubMed2004

Individual expression of poliovirus 2Apro and 3Cpro induces activation of caspase-3 and PARP cleavage in HeLa cells.

The expression of individual viral genes enables the study of their effects on cellular functions. Our group previously generated stable HeLa cell lines that efficiently express poliovirus proteases 2A (clone 2A7d) and 3C (clone 3C7) under the control of tetracycline [Virology 266 (2000a) 352; J. Virol. 74 (2000b) 2383]. Upon induction of these proteases, the cells undergo drastic morphological alterations and eventually die. The present paper characterizes, in detail, the cellular and molecular events that lead to cell death in these lines. Several signs of apoptosis were observed in both 2A7d- and 3C7-induced cells, such as nuclear fragmentation, DNA breakdown (as determined by TUNEL), and phosphatidylserine translocation. Protease 2A induces the cleavage of poly-ADP-ribose-polymerase (PARP). This is blocked by the caspase-3 inhibitor DEVD in both 2A7d-On and 3C7-On cells suggesting that this enzyme might account for PARP cleavage in both cell lines. The results indicate that both poliovirus proteases induce apoptosis by mechanisms involving caspase activation, although the kinetics of apoptosis differs.

3C Viral Proteases↗

PubMed2003

Viroporins.

Viroporins are a group of proteins that participate in several viral functions, including the promotion of release of viral particles from cells. These proteins also affect cellular functions, including the cell vesicle system, glycoprotein trafficking and membrane permeability. Viroporins are not essential for the replication of viruses, but their presence enhances virus growth. Comprising some 60-120 amino acids, viroporins have a hydrophobic transmembrane domain that interacts with and expands the lipid bilayer. Some viroporins also contain other motifs, such as basic amino acid residues or a domain rich in aromatic amino acids that confers on the protein the ability to interact with the interfacial lipid bilayer. Viroporin oligomerization gives rise to hydrophilic pores at the membranes of virus-infected cells. As the list of known viroporins steadily grows, recent research efforts focus on deciphering the actions of the viroporins poliovirus 2B, alphavirus 6K, HIV-1 Vpu and influenza virus M2. All these proteins can enhance the passage of ions and small molecules through membranes depending on their concentration gradient. Future work will lengthen the list of viroporins and will provide a deeper understanding of their mechanisms of action.

Amino Acid Sequence↗

PubMed2003

Mechanisms of membrane permeabilization by picornavirus 2B viroporin.

Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane-protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets.

Amino Acid Sequence↗

PubMed2003

Individual expression of sindbis virus glycoproteins. E1 alone promotes cell fusion.

The envelope of alphavirus particles contains two major glycoproteins, E1 and E2, that participate in virus entry and assembly of new virus particles. Interactions between these glycoproteins determine their correct functioning. The expression of each glycoprotein in the absence of the other counterpart was achieved by means of electroporation of modified Sindbis virus (SV) genomes. In addition, in trans coexpression of both glycoproteins was also tested in BHK cells. Synthesis of the E1 glycoprotein alone gave rise to cell fusion after incubation in low-pH medium. In addition, expression of E1 in the absence of the E2 precursor, PE2 (E3+E2), induced the formation of cytoplasmic vacuoles in the transfected cells. The normal phenotype was recovered when PE2 was coexpressed in trans with E1. Moreover, this coexpression modified the processing of the PE2 glycoprotein. PE2 synthesized in the absence of E1 gave rise to a product, E2', whose migration was slower in SDS-polyacrylamide gel than that of genuine E2 from SV-infected cells. This alteration was corrected upon in trans coexpression of E1 and PE2. These results suggest that the two glycoproteins, E1 and PE2, interact after their expression from two separate SV genomes. Notably, BHK cells cotransfected with the two modified genomes produced SV particles. Our findings suggest that SV E1 and E2 synthesized in trans can interact with each other and participate together with capsid protein in the assembly of new virus particles.

Animals↗

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↗

PubMed2003

The large millimeter telescope/el Gran Telescopio Milimétrico: a new instrument for astrobiology.

The Instituto Nacional de Astrofísica, Optica y Electrónica in Mexico and the University of Massachusetts in the U.S.A. are collaborating to build the world's largest radio telescope that operates at short millimeter wavelengths. This facility, known as the Large Millimeter Telescope (LMT) or el Gran Telescopio Milimétrico (GTM), is being sited at an altitude of 4600 m on Volcan Sierra Negra in the Mexican state of Puebla. The telescope will be a fully steerable dish with a diameter of 50 m and a surface consisting of 180 panels that are actively adjusted under computer control to correct for deformations due to gravity and temperature gradients. Instruments will include focal plane arrays to image both continuum and spectral line emission from celestial sources. The LMT/GTM will be an extremely powerful facility for studies encompassing almost all areas of astronomy, including astrobiology. In particular, the high sensitivity, angular resolution, and mapping speed will enable detailed investigations of the organic chemistry of interstellar molecular clouds, protoplanetary disks, and comets.

Chemistry, Organic↗

PubMed2003

The eukaryotic translation initiation factor 4GI is cleaved by different retroviral proteases.

The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.

Animals↗

PubMed2002

Interfacial domains in Sindbis virus 6K protein. Detection and functional characterization.

Alphavirus 6K is a short, constitutive membrane protein involved in virus glycoprotein processing, membrane permeabilization, and the budding of virus particles. The amino-terminal region that immediately precedes the transmembrane anchor contains a conserved sequence motif consisting of two interfacial domains separated by Asn and Gln residues. The presence of this motif confers on the 6K pretransmembrane region the tendency to partition into the membrane interface. To study the functional importance of the interfacial sequences, three different Sindbis virus 6K variants were obtained with the following modifications: 9YLW11xAAA, 18FWV20xAAA, and 9YLW11xAAA/18FWV20xAAA. Reconstituted mutant viruses were infectious and showed no defects in glycoprotein processing, although virus budding was hampered. Single 6K expression in Escherichia coli cells showed interfacial mutants to have a diminished capacity to modify membrane permeability and to have lower toxicity. In particular, the 9YLW11xAAA/18FWV20xAAA variant was expressed at high levels and did not enhance membrane permeability significantly, although it retained its integral membrane protein condition. Parallel analyses of membrane permeabilization in baby hamster kidney cells were carried out using a Sindbis virus replicon that synthesized both capsid protein and 6K. Transfection of the construct with wild-type 6K strongly increased permeability to the antibiotic hygromycin B. Replicons encoding 6K interfacial mutants induced lower membrane permeabilization. Again, the greatest impairment was observed for the 9YLW11xAAA/18FWV20xAAA variant, permeabilization activity of which was approximately 10% that of wild-type 6K. These findings show the importance of the interfacial 6K sequence for virus budding and modification of membrane permeability.

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↗

PubMed2002

Viroporin-mediated membrane permeabilization. Pore formation by nonstructural poliovirus 2B protein.

Enterovirus nonstructural 2B protein is involved in cell membrane permeabilization during late viral infection. Here we analyze the pore forming activity of poliovirus 2B and several of its variants. Solubilization of 2B protein was achieved by generating a fusion protein comprised of poliovirus 2B attached to a maltose-binding protein (MBP) as an N-terminal solubilization partner. MBP-2B was assayed using large unilamellar vesicles as target membranes. This fusion protein was able to assemble into discrete structures that disrupted the permeability barrier of vesicles composed of anionic phospholipids. The transbilayer aqueous connections generated by MBP-2B were stable over time, allowing the passage of solutes of molecular mass under 1,000 Da. Oligomerization was investigated using fluorescence resonance energy transfer. Our data indicate that MBP-2B aggregation occurs at the membrane surface. Moreover, MBP-2B binding to membranes promoted the formation of SDS-resistant tetramers. We conclude that MBP-2B forms oligomers capable of generating a tetrameric aqueous pore in lipid bilayers. These findings are the first evidence of viroporin activity shown by a protein from a naked animal virus.

Animals↗