Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy.

A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.

Synthesis of a eukaryotic virus protein in a prokaryotic viral-cell system: production of the adenovirus type 2 fiber shaft fragment by a tightly regulated T7POL-M13 expression system.

The use of recombinant technology for the production of proteins of interest in biotechnology and medicine has grown immensely during the last decade. A major problem often encountered is the degradation of the recombinant product by host cell proteases. We developed a novel system based on the cloning and expression of an inducible phage T7 RNA polymerase into the main intergenic region of the phage M13-KO7. After infection of permissive bacterial strains with the engineered phage, the polymerase gene is transcribed, subsequently translated and gene fragments cloned under T7 promoter sequences are then transcribed. For the evaluation of this system, the gene encoding the shaft fragment of the adenovirus type 2 fiber was cloned into a pET 3a-based expression vector. Expression was demonstrated in a BL21(DE3) strain (containing one copy of the T7 RNA polymerase gene) and also in several F pili-containing bacterial strains only after infection with the proper bacteriophage. Several important parameters for heterologous gene expression in Escherichia coli were investigated. Different bacterial strains were evaluated for the production of the recombinant protein, following: the expression levels, the growth rates and the stability of the plasmid vector at different time intervals after induction. It was observed that the expression levels as well as division rates and plasmid stability differed between the different bacterial strains. The best expression levels were obtained when using the E. coli Top IOF' strain. Degradation was only observed in BL21(DE3) cells after 6 h of induction, whereas none of the F'-containing cells were shown to degrade the recombinant protein during the time of expression. This system, based on the T7 pol-M13 bacteriophage, was shown to be very tightly regulated for most of the bacterial strains evaluated with no expression before induction of the T7 RNA polymerase.

Epidemiological study of prevalence of genogroup II human calicivirus (Mexico virus) infections in Japan and Southeast Asia as determined by enzyme-linked immunosorbent assays.

Mexico virus (MXV) is a genogroup II human calicivirus (HuCV). We conducted an epidemiological study to determine the prevalence of MXV infection in infants and adults in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs) developed by using baculovirus-expressed recombinant MXV (rMXV) capsids. Of 155 stool specimens obtained from children younger than 10 years old with acute clinical gastroenteritis (diarrhea and vomiting) associated with small, round-structured viruses in Japan from 1987 to 1989, only 2 were positive for MXV antigen. In 42 outbreaks of acute gastroenteritis in Japan from 1986 to 1994, 1 in an infant home and 1 among adults were positive for MXV antigen. The pattern of acquisition of antibody to rMXV was different from that of acquisition of antibody to group A rotavirus, the prototype HuCV Sapporo virus, and Norwalk virus. The prevalence of antibody to rMXV remained low for the first 3 years of life, showed a steep rise during nursery school age, reaching a prevalence of 50%, and another steep rise during adolescence, reaching 80%; and steadily increased thereafter. A high prevalence of antibody (82 to 88%) was observed in adult populations in Japan and Southeast Asia, suggesting that MXV infection is common in these areas. The discrepancy between the high prevalence of antibody to MXV and a low rate of detection of MXV antigen may be explained by a high specificity of the antigen ELISA for the prototype and closely related MXV strains while serological responses can detect responses to a broader group of viruses.

Deletion mapping of functional domains in the rotavirus capsid protein VP6.

VP6, the major capsid protein of rotavirus, oligomerizes into trimers that constitute the intermediate shell of the virions. In order to map functional domains in this protein, we introduced seven internal in-frame deletions within the coding region of gene 6 of human rotavirus strain Wa. Regions of homology among the VP6 proteins of group A and group C rotaviruses were targeted for deletion mutagenesis. The mutant VP6 proteins were expressed in mammalian cells using the recombinant vaccinia virus system and were examined for their ability to oligomerize into trimers as well as to assemble into double-layered virus-like particles upon coexpression with the rotavirus core protein VP2. Deletions that abolished trimerization defined a domain (residues 246 to 314) that maps within a larger region previously found to be critical for oligomerization (amino acids 105 to 328). When the capacity of each mutant to assemble into double-layered virus-like particles was analysed, three different assembly phenotypes were observed. Phenotype I was represented by two deletion mutants lacking residues 246 to 250 and 308 to 314 that produced particles with efficiencies similar to that of wild-type VP6. Phenotype II, characterized by a moderate decrease in the efficiency of particle assembly with respect to that of wild-type VP6, included two mutants with deletions at the C terminus of the protein. Phenotype III was exhibited by three mutants whose abilities to assemble into double-layered virus-like particles were drastically impaired. Two of these mutants define a previously unidentified assembly domain (amino acids 122 to 147) at the N terminus of rotavirus VP6.

Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo.

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.

Identification of domains in the simian immunodeficiency virus matrix protein essential for assembly and envelope glycoprotein incorporation.

The matrix domain (MA) of the simian immunodeficiency virus (SIV) is encoded by the amino-terminal region of the Gag polyprotein precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. To define domains of the SIV MA protein that are involved in virus morphogenesis, deletion and substitution mutations were introduced in this protein in the context of a gag-protease construct and expressed in the vaccinia virus vector system. The MA mutants were characterized with respect to synthesis and processing of the Gag precursor, assembly and release of virus-like particles, and incorporation of the envelope (Env) glycoprotein into particles. We have identified two regions of the SIV MA which are critical for particle formation. Both domains are located in a central hydrophobic alpha-helix of the SIV MA, according to data on the structure of this protein. In addition, we have characterized a domain whose mutation impairs the incorporation of SIV Env glycoproteins with long transmembrane cytoplasmic tails into particles. Interestingly, these mutant particles retained the ability to associate with SIV Env proteins with short cytoplasmic tails.

Strong sequence conservation of African swine fever virus p72 protein provides the molecular basis for its antigenic stability.

The major capsid protein p72 of African swine fever virus (ASFV) has long been considered an important immunodominant antigen for serologic diagnosis. Here we describe the cloning and sequence analysis of two p72-coding genes from ASFV strains Uganda (UGA) and Dominican Republic-2 (DR2). Sequence comparison of these genes, together with those from two other ASFV strains (BA71V and E70), demonstrated that the p72 proteins are highly conserved (97.8% to 100% amino acid sequence identity) in strains isolated from different parts of the world. These results support previous observations indicating that p72 is antigenically stable, and provide a useful molecular basis for further development of ASFV serologic tests using this important antigenic molecule.

Assembly of double-layered virus-like particles in mammalian cells by coexpression of human rotavirus VP2 and VP6.

Development in mammalian cells of a recombinant expression system that mimics the rotavirus capsid assembly process would be advantageous for studying the structural requirements for particle formation. To this end, we investigated the ability of a recombinant vaccinia virus system to produce double-layered virus-like particles. The genes coding for VP2 and VP6 proteins of the human rotavirus strain Wa were cloned and used to generate recombinant vaccinia viruses. Metabolic labelling of CV-1 cells infected with these recombinant viruses followed by immunoprecipitation with a polyclonal antiserum directed to Wa virus showed that VP2 and VP6 were efficiently expressed. The recombinant proteins were similar in size and immunoreactivity to authentic rotavirus proteins. Biochemical and electron microscopy analyses demonstrated that simultaneous expression of VP2 and VP6 in mammalian cells resulted in the formation of intracellular spherical particles resembling double-layered rotavirus particles.

Polyamines modulate streptomycin-induced mistranslation in Escherichia coli.

The effects of intracellular levels of polyamines on both the in vivo inhibition of protein synthesis and the decrease of translation accuracy induced by streptomycin have been studied in polyamine-auxotrophic strains of Escherichia coli infected with the MS2 bacteriophage. The amount of viral coat protein formed was strongly reduced upon addition of increasing concentrations of streptomycin to polyamine-supplemented bacteria. In contrast, the antibiotic almost did not inhibit coat protein synthesis in polyamine-starved cells. The increase of mistranslation frequency elicited by streptomycin was only observed in bacteria grown with putrescine. In these cells several coat protein-satellites were detected after two-dimensional gel electrophoresis. These proteins, more basic than the normal MS2 coat protein, contain multiple substitutions of lysine for asparagine.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells.

The cloned genes for the nucleocapsid proteins N of Junín and LCM (lymphocytic choriomeningitis) arenaviruses were inserted into the SV40-derived expression vector designated pKG4. When BHK-21 (baby hamster kidney fibroblasts) and CV-1 (African green monkey kidney fibroblasts) cell lines were transfected using these constructions, the transient expression yielded a polypeptide that could not be distinguished either by size nor by immunoreactivity from the N protein synthesized during the viral infection. The immunofluorescence analysis showed a pattern of intracellular localization similar to that observed in virus infected cells, i.e. varying from a diffuse cytoplasmic staining to granules, either distributed throughout the cytoplasm or concentrated in the perinuclear region. The association of the N protein with basophilic granules is similar to that observed in the cytopathic effect caused by arenaviruses, and could be related to the physicochemical properties of this polypeptide containing numerous basic amino acid sequences, that would allow for the interaction with cellular RNAs.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells

Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells

Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares (AU)

Involvement of structural and nonstructural polypeptides on rotavirus RNA synthesis.

Rotavirus are segmented double stranded RNA viruses with a double protein capsid around a central core. The virus replicates in the cell cytoplasm. After infection, eleven mRNAs are transcribed from the viral genome. To characterize further the infection cycle, viral polypeptide synthesis and RNA replication were studied using labelled precursors. The involvement of nonstructural polypeptides NS34 and NS35 was determined by the kinetics of the appearance of viral polypeptides in infected cells. Experiments in which cycloheximide was used showed that the synthesis of both polypeptides was required to begin RNA replication. The isolation of subviral particles at 8 hours postinfection indicates that there is a particle containing the nonstructural polypeptides and the structural polypeptides VP1, VP2, and VP6 that seem to be able to transcribe the viral genome to produce other RNA species. The results suggest that there is a core-like particle similar to one obtained in vitro which upon the addition of VP6 is able to transcribe the virus genome. This seems to indicate that core-like particles may alter their specificity for plus or minus RNA synthesis depending upon the polypeptides that interact with it. The interaction between VP6 and the viral core was analyzed by means of antibodies raised against the viral core and VP6. The results suggest that VP6 contains a specific binding site to the core complex and this interaction allows the synthesis of mRNA.