Enrique Tabarés

Characterization of regions in the GP5 protein of porcine reproductive and respiratory syndrome virus required to induce apoptotic cell death

Expression of the GP5 protein of porcine reproductive and respiratory syndrome virus in mammalian cells using a recombinant vaccinia virus has been shown to induce strong cytotoxicity due to apoptotic death. We have now developed a transient expression system that allows the observation and quantitation of the cell death due to GP5 synthesis, taking advantage of the reduction that this protein induces in the expression of two different co-transfected reporter genes. In this way, we are able to study the regions in GP5 implicated in apoptosis induction. The first 119 aminoacids constitute a region capable of fully inducing apoptosis, aminoacids 90-119 having a fundamental role. On the contrary, the C-terminal region is unable by itself of cell death induction and, moreover, is dispensable for this phenotype. We have also observed that induction of apoptosis is independent of cleavage of the N-terminal putative signal sequence in GP5 or N-glycosylation of this protein.

High susceptibility of a human oligodendroglial cell line to herpes simplex type 1 infection

More than 20 infectious agents, ranging from retroviruses to mycobacteria, have been associated with multiple sclerosis onset or relapses in which oligodendrocytes, the myelin-forming cells of the central nervous system, are the initial target of the pathogenic status. In this work, the nature of the susceptibility of the human precursor oligodendroglial KG-1C cell line to herpes simplex virus type 1 (HSV-1) was investigated. Infection of KG-1C cells was characterized by a high level of virus production and a notable progression of the cytopathic effect. After infection, there was a significant shut-off of host mRNA translation, which was correlated with evident synthesis of viral proteins. An examination by electron microscopy of the infected cells revealed the presence of large clusters of mitochondria located in the proximity of intracellular HSV-1 particle groups. In addition, transmission electron microscopy and nuclear fluorescence analysis showed neither signs of chromatin condensation nor of apoptotic bodies. Furthermore, procaspase-3 remained uncleaved, suggesting that apoptosis does not take place, at least in this system. Finally, expression and localization of MAL2, a subpopulation of detergent-insoluble lipid raft protein, was studied. Detection of MAL2 significantly increased after infection and it was colocalized with HSV-1 proteins. From these findings the authors conclude that human oligodendrocyte-like cells are highly susceptible to HSV-1 infection. The implications of this for central nervous system viral infection are discussed.

DIVA diagnostic of Aujeszky's disease using an insect-derived virus glycoprotein E

Commercial vaccines against Aujeszkys disease are mainly formulated using deleted versions of attenuated or inactivated Pseudorabies virus (PRV) particles lacking of the structural glycoprotein E (gE). Complementary diagnostic assays used to differentiate infected from vaccinated animals (DIVAs), are based on the detection of serum antibodies against gE. A recombinant version of the PRV gE protein was expressed in a baculovirus vector system in Trichoplusia ni insect larvae in order to obtain this diagnostic reagent for large scale diagnosis at reduced costs. A recombinant gE gene (gEr), lacking of signal peptide and transmembrane domains, was cloned into a modified baculovirus vector to allow glycosylation of the protein and its subsequent exportation to the extracellular space. Analysis by SDS-PAGE, Western-blotting and glycoprotein staining revealed that a glycosylated protein of the expected electrophoretic mobility was obtained in infected larvae. Time course experiments revealed that maximum expression levels were reached 72h post-infection using 10(4)pfu of the recombinant baculovirus (BACgEr) per inoculated larva. An indirect PRV gE-ELISA was developed using gEr as a coating antigen. A comparison between larvae-derived PRV gE-ELISA and two commercially available PRV diagnostic kits showed good correlation between assays and better sensitivity when testing certain sera pig samples using the gEr ELISA. More than 30,000 ELISA determinations could be performed from crude extracts obtained from a single larva infected with the recombinant baculovirus, indicating the feasibility of this strategy for inexpensive production of glycosylated antigens for PRV diagnosis.

Glycoprotein G from pseudorabies virus binds to chemokines with high affinity and inhibits their function

Pseudorabies virus (PRV), also known as suid herpesvirus, is the aetiological agent of Aujeszkys disease in swine. In other animals, except higher-order primates, PRV infection is often fatal. The mechanisms of PRV pathogenesis and immune modulation are largely unknown. PRV codes for 11 glycoproteins. Among them, glycoprotein G (gG) is the most abundant PRV protein found in the supernatant of PRV-infected cell cultures. PRV-gG has low amino acid sequence similarity with gG from other animal alphaherpesviruses and its function is unknown. gG from other animal alphaherpesviruses, with the exception of at least equine herpesvirus 4, binds to chemokines. We show here that PRV-gG binds to the human chemokine CL1 and several CC and CXC human chemokines with high affinity. Chemokine-binding activity can be detected in the supernatants of PRV-infected cell cultures, and insertional inactivation of the gene encoding gG from the PRV genome results in loss of chemokine-binding activity. Binding of PRV-gG to chemokines inhibits chemokine-mediated cell migration, suggesting a role for PRV-gG in immune evasion.

Loss of pseudorabies virus thymidine kinase activity due to a single base mutation and amino acid substitution

The nucleotide sequences of the coding region of the thymidine kinase (TK) gene of pseudorabies virus strain NIA3 and a TK- mutant (ATK5) were determined. The coding region of the TK gene consists of 320 codons capable of producing a polypeptide with an Mr of 34979. The mutant expressed an inactive TK polypeptide when translated in vitro; a unique base substitution was detected in the mutant TK modifying amino acid position 13, at which aspartic acid replaces glycine. This modification affects the nucleotide-binding site, thus explaining the expression of an inactive TK polypeptide.

Role of the small GTPase Rab27a during Herpes simplex virus infection of oligodendrocytic cells

BackgroundThe morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection.ResultsHerein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells.ConclusionsAltogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.

Evidence of Muller's ratchet in herpes simplex virus type 1

Population bottlenecks can have major effects in the evolution of RNA viruses, but their possible influence in the evolution of DNA viruses is largely unknown. Genetic and biological variation of herpes simplex virus type 1 (HSV-1) has been studied by subjecting 23 biological clones of the virus to 10 plaque-to-plaque transfers. In contrast to large population passages, plaque transfers led to a decrease in replicative capacity of HSV-1. Two out of a total of 23 clones did not survive to the last transfer in 143 TK(-) cells. DNA from three genomic regions (DNA polymerase, glycoprotein gD and thymidine kinase) from the initial and passaged clones was sequenced. Nucleotide substitutions were detected in the TK and gD genes, but not in the DNA polymerase gene. Assuming a uniform distribution of mutations along the genome, the average rate of fixation of mutations was about five mutations per viral genome and plaque transfer. This value is comparable to the range of values calculated for RNA viruses. Four plaque-transferred populations lost neurovirulence for mice, as compared with the corresponding initial clones. LD(50) values obtained with the populations subjected to serial bottlenecks were 4- to 67-fold higher than for their parental clones. These results equate HSV-1 with RNA viruses regarding fitness decrease as a result of plaque-to-plaque transfers, and show that population bottlenecks can modify the pathogenic potential of HSV-1. Implications for the evolution of complex DNA viruses are discussed.

Conversion of US3-encoded protein kinase gene from pseudorabies virus in a diploid gene located within inverted repeats by genetic recombination between the viral genome isomers

The pseudorabies virus (PRV) genome consists of two components, long (U(L)) and short (U(S)) regions. The U(S) region is the only one capable of inverting itself relative to the U(L) region during productive infection, generating two equimolecular isomeric forms of viral DNA. Here we describe a recombinant virus (gIp2) generated by genetic recombination between pseudorabies viral isomers. This recombination event was observed in the parental virus gIS8, which was obtained by insertion of the alpha4-TK herpes simplex virus type 1 (HSV1) gene. The growth of gIS8 virus in the presence of 5-bromodeoxyuridine (BrdU) yielded gIp2. This was generated by nonhomologous recombination either between the two viral genomic isomers of gIS8, P and I(U/S), or between the same P isomer using nonhomologous and homologous recombination, with loss of the HSV1 sequences and duplication of the PRV US3-encoded protein kinase gene. Virus gIp2 is negative for TK, gI, gE, 11K and 28K and shows an in vitro replication capacity in neuronal cells approximately 22 times lower than that of parental virus gIS8, and similar to that of the Bartha vaccine virus strain in monkey kidney and human neuronal cells.

Development of new expression vector based on Pseudorabies virus amplicons: application to human insulin expression

Pseudorabies virus (PrV), a herpesvirus from the Alphaherpesvirinae subfamily, is suitable for amplicon vector replication and packaging into virions, with helper virus for trans replication and cleavage-packaging functions. PrV amplicon vectors were developed in a bacterial plasmid construction using PrV ori(s) and pac signals as the required cis elements. Human insulin cDNA was then cloned in the amplicon vector for human proinsulin expression. In the same construction, green fluorescent protein was used as a marker. PrV amplicons may have several advantages over herpes simplex virus type 1 (HSV1) amplicons in human gene therapy because it can infect human cells in vitro and in vivo, it is not pathogenic for primates and there is no pre-existing immunity and risk of recombination with latent PrV as occurs with HSV1.

The Effect of Cellular Differentiation on HSV-1 Infection of Oligodendrocytic Cells

Herpes simplex type 1 (HSV-1) is a neurotropic virus that infects many types of cells. Previous studies have demonstrated that oligodendrocytic cells are highly susceptible to HSV-1 infection. Here we analysed HSV-1 infection of a human oligodendrocytic cell line, HOG, and oligodendrocyte precursor cells (OPCs) cultured under growth or differentiation conditions. In addition to cell susceptibility, the role of the major cell receptors for viral entry was assessed. Our results revealed that OPCs and HOG cells cultured under differentiation conditions became more susceptible to HSV-1. On the other hand, viral infection induced morphological changes corresponding to differentiated cells, suggesting that HSV-1 might be inducing cell differentiation. We also observed colocalization of HVEM and nectin-1 with viral particles, suggesting that these two major HSV-1 receptors are functional in HOG cells. Finally, electron microscopy assays indicated that HSV-1 may be also entering OLs by macropinocytosis depending on their differentiation stage. In addition, vesicles containing intracellular enveloped virions observed in differentiated cells point to an endocytic mechanism of virus entry. All these data are indicative of diverse entry pathways dependent on the maturation stage of OLs.