Miguel Angel Sanz

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.

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 ∼10% that of wild-type 6K. These findings show the importance of the interfacial 6K sequence for virus budding and modification of membrane permeability.

Genetic analysis of poliovirus protein 3A: characterization of a non-cytopathic mutant virus defective in killing Vero cells.

A mutational and genetic analysis of the poliovirus protein 3A has led to the identification of a single amino acid mutant virus with a restrictive phenotype to form plaques in Vero cells. This mutant (I46T 3A) can be grown and amplified in HeLa cells, where virus replication takes place at wild-type levels. However, Vero cells infected with this virus cannot complete the growth cycle. I46T 3A virus has a defect in the ability to kill Vero cells, as estimated by FACS analysis of propidium iodide uptake by dead cells. Since these defects are observed under conditions where no abnormalities in the rate of synthesis and processing of the mutant polyprotein occur, the inability to induce the cytopathic effect in infected Vero cells denotes the existence of a defect in the activity of 3A, but not the level of expression of the viral genome. As a consequence of this impaired capability to generate the cytopathic effect, I46T 3A mutant viruses cannot be titrated by plaque assay in Vero cells. Only revertant viruses with the wild-type sequence arise and form lysis plaques in Vero cells. Our results suggest a role for the 3A protein (or a precursor thereof) in the virus-induced cytopathic effect. The mutant virus characterized in this work may be a useful tool to understand how poliovirus kills infected cells and carries out the final step of its life-cycle, the release of virus progeny.

Dual Mechanism for the Translation of Subgenomic mRNA from Sindbis Virus in Infected and Uninfected Cells

Infection of BHK cells by Sindbis virus (SV) gives rise to a profound inhibition of cellular protein synthesis, whereas translation of viral subgenomic mRNA that encodes viral structural proteins, continues for hours. To gain further knowledge on the mechanism by which this subgenomic mRNA is translated, the requirements for some initiation factors (eIFs) and for the presence of the initiator AUG were examined both in infected and in uninfected cells. To this end, BHK cells were transfected with different SV replicons or with in vitro made SV subgenomic mRNAs after inactivation of some eIFs. Specifically, eIF4G was cleaved by expression of the poliovirus 2A protease (2Apro) and the alpha subunit of eIF2 was inactivated by phosphorylation induced by arsenite treatment. Moreover, cellular location of these and other translation components was analyzed in BHK infected cells by confocal microscopy. Cleavage of eIF4G by poliovirus 2Apro does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems. SV infection induces phosphorylation of eIF2α, a process that is increased by arsenite treatment. Under these conditions, translation of subgenomic mRNA occurs to almost the same extent as controls in the infected cells but is drastically inhibited in uninfected cells. Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells. Finally, immunolocalization of different eIFs reveals that eIF2 α and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions. These findings support the notion that canonical initiation takes place when the subgenomic mRNA is translated out of the infection context, while initiation can occur without some eIFs and even at non-AUG codons in infected cells.

Viral Translation Is Coupled to Transcription in Sindbis Virus-Infected Cells

ABSTRACT During the late phase of Sindbis virus infection, the viral subgenomic mRNA is translated efficiently in BHK cells, whereas host protein synthesis is inhibited. However, transfection of in vitro-generated Sindbis virus subgenomic mRNA leads to efficient translation in uninfected BHK cells, whereas it is a poor substrate in infected cells. Therefore, the structure of the subgenomic mRNA itself is not sufficient to confer its translatability in infected cells. In this regard, translation of the subgenomic mRNA requires synthesis from the viral transcription machinery. The lack of translation of transfected viral mRNAs in infected cells is not due to their degradation nor is it a consequence of competition between viral transcripts and transfected mRNAs, because a replicon that cannot produce subgenomic mRNA also interferes with exogenous mRNA translation. Interestingly, subgenomic mRNA is translated more efficiently when it is transfected into uninfected cells than when it is transcribed from a transfected replicon. Finally, a similar behavior was observed for other RNA viruses, such as vesicular stomatitis virus and encephalomyocarditis virus. These findings support the notion that translation is coupled to transcription in cells infected with different animal viruses.

Sindbis Virus Variant with a Deletion in the 6K Gene Shows Defects in Glycoprotein Processing and Trafficking: Lack of Complementation by a Wild-Type 6K Gene in trans

ABSTRACT A Sindbis virus (SV) variant with a 6K gene partially deleted has been obtained. This SV Del6K virus is defective in the proteolytic processing of virus glycoprotein precursor, transport of glycoproteins to the plasma membrane, and plaque phenotype. A revertant virus (SV Del6K-revQ21L) containing a point mutation in the deleted 6K gene was isolated and characterized. SV Del6K-revQ21L has corrected the defects of proteolytic processing and transport of virus glycoproteins to the plasma membrane, but it still remains attenuated compared to wild-type (wt) SV, exhibiting defects in virus budding. Neither mutant nor revertant viruses are complemented by the coexpression intrans of a wt SV 6K gene.

Translation without eIF2 Promoted by Poliovirus 2A Protease

Poliovirus RNA utilizes eIF2 for the initiation of translation in cell free systems. Remarkably, we now describe that poliovirus translation takes place at late times of infection when eIF2 is inactivated by phosphorylation. By contrast, translation directed by poliovirus RNA is blocked when eIF2 is inactivated at earlier times. Thus, poliovirus RNA translation exhibits a dual mechanism for the initiation of protein synthesis as regards to the requirement for eIF2. Analysis of individual poliovirus non-structural proteins indicates that the presence of 2Apro alone is sufficient to provide eIF2 independence for IRES-driven translation. This effect is not observed with a 2Apro variant unable to cleave eIF4G. The level of 2Apro synthesized in culture cells is crucial for obtaining eIF2 independence. Expression of the N-or C-terminus fragments of eIF4G did not stimulate IRES-driven translation, nor provide eIF2 independence, consistent with the idea that the presence of 2Apro at high concentrations is necessary. The finding that 2Apro provides eIF2-independent translation opens a new and unsuspected area of research in the field of picornavirus protein synthesis.

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2Apro or Lpro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2Apro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) Lpro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2Apro and Lpro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified Lpro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.

Genetic markers for doubled haploid response in barley

In order to analyse the genetic control of anther culture response in barley, a doubled-haploid (DH) population from the cross between a medium responsive cultivar ‘Dobla’ and the model cultivar ‘Igri’ was produced. A linkage map was constructed with 91 markers. A sub-population of 41 lines was characterised for different components of the anther culture response, and was used for quantitative trait loci (QTL) analysis. The vrs1 locus region on chromosome 2H, which determines inflorescence row type, was coincident with the largest putative QTL for number of embryos (nEMB) and albino plants. A region of chromosome 6H was associated with QTLs for nEMB and green plants. QTLs for number and percentage of green plants were located on the long arm of chromosome 5H. Therefore, new QTLs for main components of barley anther culture response were identified on chromosomes 2H, 5H and 6H, indicating that anther culture response in barley could be controlled by relative few genes of large effect. This work is a useful step towards the identification of new regions on the barley genome that could be associated with fundamental biological process implicated in the anther culture response.

Translation of Viral mRNA without Active eIF2: The Case of Picornaviruses

Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.