Use of the vaccinia virus/t7 expression system for studying HCV protein processing.

HCV and related viruses are now classified as a separate genus in the family Flaviviridae (1), which includes two other genera, Flavivirus (2), and Pestivirus (3). The positive-strand HCV genome RNA is approx 9.4 kb in length and contains a highly conserved 5' noncoding region followed by a long open reading frame encoding a polyprotein of 3010-3033 amino acids (4,5) Recently, it was determined that the 3' end contains another highly conserved noncoding region approx 100 bp in length (6-8). Because a cell-culture system supporting efficient HCV replication is lacking, efforts to define potential HCV-encoded polypeptides have utilized expression of HCV cDNA in cell-free translation systems and in cell cultures. The HCV polyprotein appears to be cleaved at multiple sites to produce at least 10 structural and nonstructural (NS) proteins (9). The order and nomenclature of these cleavage products for the HCV-H strain are NH(2)-C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH, where C, El, and E2 are putative structural proteins and the remaining NS proteins are believed to be replicase components (9-12). Host signal peptidase in the endoplasmatic reticulum lumen appears to catalyze cleavages in the structural-NS2 region (C/El, E1/E2, E2/p7, and p7/NS2 sites) (9,13), whereas an HCV-encoded serine proteinase located in the N-termmal one-third of the NS3 protein is responsible for four cleavages in the NS region (3/4A, 4A/4B, 4B/5A, and 5A/5B sites) (11,14-16).

Noncytopathic Sindbis virus RNA vectors for heterologous gene expression.

Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.

Molecular virology of hepatitis C virus: an update with respect to potential antiviral targets.

Hepatitis C virus (HCV), a positive-strand enveloped RNA virus, is a major cause of chronic liver disease worldwide. Cis-acting RNA elements and virus-encoded polypeptides required for HCV replication represent attractive targets for the development of antiviral therapies. Internal ribosome entry site-directed translation of HCV genome RNA produces a long polyprotein which is co- and post-translationally processed to yield at least 10 viral proteins. A host signal peptidase is responsible for maturation of the structural proteins located in the N-terminal one-third of the polyprotein. Thus far, four enzymatic activities encoded by the non-structural (NS) proteins have been reported. The NS2-3 region encodes an autoproteinase responsible for cleavage at the 2/3 site. The N-terminal one-third of NS3 functions as the catalytic subunit of a serine proteinase which cleaves at the 3/4A, 4A/4B, 4B/5A and 5A/5B sites, and NS4A is an essential cofactor for some of these cleavages. NS3 also encodes an RNA-stimulated NTPase/RNA helicase at its C terminus, and NS5B has been shown to possess an RNA-dependent RNA polymerase activity. To date, no functions have been reported for NS4B or NS5A in RNA replication, however, NS5A has been implicated in modulating the sensitivity of HCV to interferon. Sequence and structural conservation within the 3' terminal 98 bases of genomic RNA suggest a functional importance in the virus life-cycle and hence another target for antiviral intervention. Recently, HCV infection was shown to be initiated in chimpanzees following intrahepatic inoculation of RNA transcribed from cloned HCV cDNA. The ability to generate large quantities of infectious HCV RNA may facilitate the development of reliable cell culture replication systems useful for the evaluation of antiviral drugs.

Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.

More than 1% of the world's population is chronically infected with hepatitis C virus (HCV). HCV infection can result in acute hepatitis, chronic hepatitis, and cirrhosis, which is strongly associated with development of hepatocellular carcinoma. Genetic studies of HCV replication have been hampered by lack of a bona fide infectious molecular clone. Full-length functional clones of HCV complementary DNA were constructed. RNA transcripts from the clones were found to be infectious and to cause disease in chimpanzees after direct intrahepatic inoculation. This work defines the structure of a functional HCV genome RNA and proves that HCV alone is sufficient to cause disease.

[Preparation and study of recombinant strains of Venezuelan equine encephalomyelitis virus expressing the PreS2-S gene of the hepatitis B virus]. / Poluchenie i issledovanie rekombintnykh shtammo-v virusa venesuél'skogo éntsefalomielita loshadei, ékspressiruiushchikh gen preS2-S virusa gepatita B.

Venezuelan equine encephalomyelitis (VEE) virus-based vectors for expression of heterologous genes have been constructed using full-length cDNA copy of the interstrain recombinant virus (Trinidad Donkey and 230 strain). Gene cassettes carrying the subgenomic mRNA promoter of VEE virus and the preS2-S gene of hepatitis B virus (HBV) were inserted before or after the genes of structural viral proteins. Live virus stocks were obtained by transfection of chick embryo fibroblasts with in vitro transcribed full-length RNA. Insertions of gene cassettes before the structural region resulted in expression of HBsAg (VEHB-25 and VEHB-361 viruses), whereas insertions in the 3' region did not. Recombinant virus VEHB-25 expressed HBsAg during 5 passages in Vero cells. VEHB-25 stimulated immune response to HBsAg and was less virulent than the parental virus.

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.

Localization of four antigenic sites involved in Venezuelan equine encephalomyelitis virus protection.

Stable neutralization and protection escape variants of a virulent strain (Trinidad Donkey) of the VEE virus were selected by monoclonal antibodies (MAbs). Determination of nucleotide sequences of nine variants revealed a clustering of single mutations in four regions of the E1 and E2 glycoproteins. Involvement of amino acid residues 206 (site E1-1), 57 and 59 (site E2-2), 180, 182, 213, 214 and 216 (site E2-6) and 232 (site E2-3) in protective epitopes was demonstrated.

Analysis of the hemagglutination activity domains of the Venezuelan equine encephalomyelitis and eastern equine encephalomyelitis viruses.

The hemagglutination (HA) domains of the Venezuelan equine encephalomyelitis (VEE) and the eastern equine encephalomyelitis (EEE) viruses providing the interaction of virions and red blood cells were studied with the use of a panel of 17 hemagglutination inhibition (HI) monoclonal antibodies (MAbs). A highly conserved domain (C domain) forming alphavirus-group-reactive MAbs was identified in the E2 protein of the VEE and EEE viruses. These MAbs inhibited HA of the western equine encephalomyelitis, Semliki Forest, Sindbis, Getah, Aura, Chikungunya and Pixuna viruses. The involvement of amino acid residues 59 and 232 in the formation of the C region was demonstrated by sequencing the gene encoding the E2 protein of three escape variants of the VEE virus.

[The immunogenic properties of a recombinant vaccinia virus with an incorporated DNA copy of the 26S RNA of the Venezuelan equine encephalomyelitis virus]. / Immunogennye svoistva rekombinantnogo virusa ospovaktsiny so vstroennoi DNK-kopiei 26S RNK virusa venesuél'skogo éntsefalomielita loshadei.

A recombinant strain of vaccinia virus (VR26) containing a DNA-copy of the subgenomic 26S RNA of Venezuelan equine encephalomyelitis virus (VEE) inserted into the coding region of thymidine kinase (TK) gene was produced. This subgenomic RNA contained the genes for all structural proteins of the VEE virus, the strain Trinidad donkey (TRD). VR26 effectively expressed VEE virus glycoproteins on the membranes of the infected cells. Blood sera of VR26-immunized animals were found to contain VEE virus-specific antibodies. VR26-immunized mice and rabbits showed a high level of resistance to subcutaneous inoculation with the pathogenic TRD strain of VEE virus. VR26 also provided a high level of protection in animals against aerogenic infection. The absence of virus-neutralizing antibodies in most VR26-immunized animals resistant to inoculation with high doses of VEE suggests the dominant role of the cell component in the immune response. The immune response induced by the recombinant VR26 strain was stable as demonstrated by the resistance of the animals to a challenge with VEE virus 7 months after immunization. The experimental results suggest that this recombinant strain may be considered as a candidate for vaccine preparation.

[The peptide mapping of the E2-2 and E2-6 sites of the E2 glycoprotein in the Venezuelan equine encephalomyelitis virus]. / Kartirovanie s pomoshch'iu peptidov saitov E2-2 i E2-6 glikoproteina E2 virusa venesuél'skogo éntsefalomielita loshadei.

Nine peptides were synthesized for detailed mapping of VEE virus E2-2 and E2-6 sites responsible for the formation of protective antibodies that neutralize the virus and block hemagglutination. The sequence of the peptides over-lapped the regions of amino acid residues 30-75 and 202-250 of VEE virus E2 protein in which antigenic mutations caused by monoclonal antibodies to E2-2 and E2-6 sites had been mapped. None of the synthesized peptides reacted in the enzyme immunoassay with a panel of 17 Mabs to VEE virus E2 protein. However, eight peptides reacted with polyclonal antiviral serum and two of them elicited antiviral antibody production. The E2-2 site might be associated with amino acid residues 30-45, and the region of E2 residues 57-62 in which antigenic mutations are observed is not a linear type antigenic determinant, but participates in the formation of antigenic determinants of the conformational type. The mapping of residues 202-250 demonstrated that all the peptides in this region were well recognized by polyclonal antiviral serum. The residues 235-240 were shown to form a linear epitope which provided a crossover between VEE and EEE viruses and was not recognized by 19 types of monoclonal antibodies cross-reacting with VEE and EEE viruses.

[The antigenic structure of the eastern equine encephalomyelitis virus studied by using monoclonal antibodies]. / Issledovanie antigennoi struktury virusa vostochnogo éntsefalomielita loshadei s pomoshch'iu monoklonal'nykh antitel.

Thirty-three monoclonal antibodies (Mabs) interacting with the structural proteins of Eastern equine encephalomyelitis (EEE) virus were prepared. The mutual arrangement of the antigenic sites on the E1 and E2 glycoproteins was studied by competitive radioimmunoassay. At least four nonoverlapping sites were found on the E1. The E2 glycoprotein contained at least seven partially overlapping antigenic sites. Mabs to the sites E2-2 and E2-3 neutralized viral infectivity and blocked hemagglutination. Mabs to the site E2-1 blocked hemagglutination. Mabs to sites E2-2, 3, and 7 protected mice against lethal infection although the protective Mabs to sites E2-2b and E2-7 did not neutralize the virus. The antibodies to the other three sites of E2 and to all sites of E1 did not have any biological activity. The experimental results indicate the dominant role of E2 in antiviral immunity, over 98% of the observed protective effect being associated with the E2-2 site.

[A monoclonal antibody study of the receptor area of the Venezuelan encephalitis virus]. / Izuchenie retseptornoi oblasti virusa venesuél'skogo éntsefalita loshadei pri pomoshchi monoklonal'nykh antitel.

The receptor region for virus-cell interaction in Venezuelan equine encephalomyelitis (VEE) and Eastern equine encephalomyelitis (EEE) viruses was studied using a panel of 17 monoclonal antibodies (MCA). They were able to block agglutination of goose erythrocytes. The dominant role of glycoprotein E2 in the formation of viral receptor for EEE and VEE viruses was demonstrated. Competitive radioimmunoassay identified three antigenic sites in this region. These sites were also responsible for virus neutralization. MCAs to these sites protected outbred mice against lethal infection. The presence of a highly conservative region in VEE (site E2-3) and EEE (site E2a) which produced cross-reacting antibodies blocking hemagglutination of Western equine encephalomyelitis, Semliki Forest, Sindbis, Getah, Aura, Chikungunya, and Pixuna viruses was established. A hypothesis is suggested concerning the existence of similar regions for the entire alphavirus genus, and the role of this region in virus-cell interaction.

[Study of the antigenic structure of the E1 glycoprotein of the Venezuelan equine encephalomyelitis virus using monoclonal antibodies]. / Izuchenie antigennoi struktury glikoproteina E1 virusa venesuél'skogo éntsefalomielia loshadei s pomoshch'iu monoklonal'nykh antitel.

The collection of eight rat and mouse hybridomas secreting the high affinity monoclonal antibodies to glycoprotein E1 of the Venezuelan equine encephalomyelitis has been obtained. The antigenic structure of E1 protein has been studied with the use of these antibodies for the strains Trinidad, TC-83 and 230 of the virus. Antigenic map of glycoprotein E1 based on competition radioimmunoanalysis is proposed. Five sites are mapped including eight epitopes binding monoclonal antibodies. Antibodies to sites E1-1, E1-3 and E1-5 are crossreactive in interaction with the virus of Venezuelan equine encephalomyelitis, while antibodies to site E1-5 interact also with the virus of tick-borne encephalitis. Antibodies to site E1-1 possess the protective effect and lack the neutralizing effect in tissue cultures. Antibodies to all sites of E1 protein are devoid of ability to neutralize the Venezuelan equine encephalitis virus.

[The antigenic structure of glycoprotein E2 in the Venezuelan equine encephalomyelitis virus studied using rat monoclonal antibodies]. / Izuchenie antigennoi struktury glikoproteida E2 virusa venesuél'skogo éntsefalomielita loshadei s pomoshch'iu krysinykh monoklonal'nykh antitel.

A collection of 21 rat hybridomas secreting high-affinity monoclonal antibodies to Venezuelan equine encephalomyelitis (VEE) virus was generated. Using a panel of 15 monoclonal antibodies to glycoprotein E2, the antigenic structure of this protein of VEE strains TC-83 and 230 was studied. A competitive radioimmunoassay suggested a new map of the antigenic structure of glycoprotein E2 in which 5 sites including 11 epitopes of monoclonal antibody binding were distinguished. Antibody to E2-2 site neutralized virus infectivity and blocked hemagglutination test and antibody to E2-3 site could only block hemagglutination. Antibodies to other E2 protein sites lacked any biological activity.

Investigation of antigenic structure of attenuated and virulent Venezuelan equine encephalomyelitis virus by means of monoclonal antibodies.

A comparative study of the antigenic structure of virulent strains and attenuated vaccine strains of Venezuelan equine encephalomyelitis virus (VEEV) by means of monoclonal antibodies has made it possible to investigate the antigenic structure of the envelope glycoproteins E1 and E2, and to specify their role in the development of antiviral immunity. On the E1 glycoprotein there are five nonoverlapping antigenic sites consisting of eight epitopes that are recognized by monoclonal antibodies; six sites consisting of twenty epitopes were found on the E2 glycoprotein. The monoclonal antibodies against four sites protect the animals from lethal infection with the virulent strain, Trinidad donkey. Out of the thirteen epitopes identified as being responsible for antiviral immunity, three are changes in the TC-83 strain, and six belong to two sites in the strain 230. The results obtained indicate the necessity for further improvement of the available vaccine preparations against this dangerous infectious disease.