Mechanism of action of a pestivirus antiviral compound.

We report here the discovery of a small molecule inhibitor of pestivirus replication. The compound, designated VP32947, inhibits the replication of bovine viral diarrhea virus (BVDV) in cell culture at a 50% inhibitory concentration of approximately 20 nM. VP32947 inhibits both cytopathic and noncytopathic pestiviruses, including isolates of BVDV-1, BVDV-2, border disease virus, and classical swine fever virus. However, the compound shows no activity against viruses from unrelated virus groups. Time of drug addition studies indicated that VP32947 acts after virus adsorption and penetration and before virus assembly and release. Analysis of viral macromolecular synthesis showed VP32947 had no effect on viral protein synthesis or polyprotein processing but did inhibit viral RNA synthesis. To identify the molecular target of VP32947, we isolated drug-resistant (DR) variants of BVDV-1 in cell culture. Sequence analysis of the complete genomic RNA of two DR variants revealed a single common amino acid change located within the coding region of the NS5B protein, the viral RNA-dependent RNA polymerase. When this single amino acid change was introduced into an infectious clone of drug-sensitive wild-type (WT) BVDV-1, replication of the resulting virus was resistant to VP32947. The RNA-dependent RNA polymerase activity of the NS5B proteins derived from WT and DR viruses expressed and purified from recombinant baculovirus-infected insect cells confirmed the drug sensitivity of the WT enzyme and the drug resistance of the DR enzyme. This work formally validates NS5B as a target for antiviral drug discovery and development. The utility of VP32947 and similar compounds for the control of pestivirus diseases, and for hepatitis C virus drug discovery efforts, is discussed.

Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity.

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10(5) PFU/microg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts.

Bovine viral diarrhea virus (BVDV) is the most insidious and devastating viral pathogen of cattle in the United States. Disease control approaches must be based on detailed knowledge of virus biology. To develop reverse-genetic systems to study the molecular biology of the virus, we first constructed a plasmid containing the entire genome of BVDV cloned as cDNA. Subsequently, we showed that infectious BVDV was produced by cells transfected with uncapped RNA transcribed in vitro from the cDNA clone. This result defined functional 5' and 3' termini in viral genomic RNA and established the biological importance of the proposed internal ribosome entry site element in the 5' untranslated region of the viral genome. BVDV rescued from the infectious cDNA clone has an in vitro phenotype similar to that of the wild-type parent, the National Animal Disease Laboratory strain of BVDV. A deletion of a single codon in the full-length genomic BVDV cDNA clone, encoding glutamic acid at position 1600, gave rise to sequence-tagged virus easily identified by restriction fragment length polymorphism analysis of reverse transcription-PCR amplicons. Suitability of the molecular clone of BVDV for genomic manipulations was shown by substitution of the major envelope glycoprotein E2/gp53 with that of the Singer strain, giving rise to a chimeric virus. The predicted change in antigenic structure of the chimeric virus could be readily identified with strain-specific monoclonal antibodies by neutralization and immunofluorescence assays. Immediate applications of this system include development of safe and effective live vaccine strains possessing predetermined defined attenuating mutations.

Pestivirus NS3 (p80) protein possesses RNA helicase activity.

The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed.

Pestivirus translation initiation occurs by internal ribosome entry.

The role of the 385 nucleotide 5' noncoding region (NCR) in the translation of the pestivirus genome was investigated. In vitro translation of an RNA transcript containing the 5' NCR of the bovine viral diarrhea virus (BVDV) genome followed by the coding sequence of the first gene product (p20) of the BVDV large open reading frame resulted in the synthesis of a 20-kDa polypeptide. Results from hybrid-arrest translation studies identified a region involving a predicted RNA stem-loop structure spanning nucleotides 154-261 within the 5' NCR that was important for p20 synthesis. An additional inhibitory oligonucleotide was complementary to the sequence at the base of this stem-loop and encompassed the initiating AUG at nucleotide 386. Antisense oligonucleotides both upstream and downstream of those that were inhibitory had no effect on p20 translation. RNA from a dicistronic expression vector in which the BVDV 5' NCR was inserted between two reporter genes, CAT and LUC, showed strong expression of the second (LUC) cistron upon in vitro translation. This expression was dramatically reduced in an analogous construct in which nucleotides 173-236 of the 5' NCR were deleted. Similar results were obtained when RNA from these same vectors was evaluated for expression after transfection into BHK cells. These results suggest that the BVDV 5' NCR contains an internal ribosome entry site for translation initiation. This translational mechanism is similar to that shown for hepatitis C virus, further demonstrating the close relationship between viruses of these two genera within the family Flaviviridae.

Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.

Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses.

Generation of neutralizing anti-B19 parvovirus human monoclonal antibodies from patients infected with human immunodeficiency virus.

The prevalence of IgG antibodies to human B19 parvovirus (anti-B19) is elevated in individuals infected with human immunodeficiency virus (HIV), especially during the later stages of HIV infection. In subjects with high titers of IgG anti-B19, 86% (19 of 22) had circulating B cells producing anti-B19. Immortalization of these cells with Epstein-Barr virus and generation of heterohybridomas by fusion with a mouse X human heteromyeloma resulted in the production of two cell lines producing IgG1 kappa monoclonal antibodies (MAbs). Both of these MAbs were specific for conformational epitopes on the VP2 capsid protein of B19 parvovirus and both were capable of neutralizing 50% of the viral infectivity in a human erythroid colony-forming unit assay at < or = 1 micrograms of MAb/mL. These human MAbs are potentially useful in the treatment of acute B19 parvovirus infection.

Candidate recombinant vaccine for human B19 parvovirus.

Recombinant baculoviruses were used to produce human B19 parvovirus empty capsids composed of only VP2 and VP2 capsids containing 4%, 25%, 35%, or 41% VP1 protein. Immunogenicity of the purified capsids, formulated with or without adjuvant, was evaluated in mice, guinea pigs, and rabbits. Sera were analyzed for total anti-B19 parvovirus antibodies, antibodies specific to the region unique to the VP1 capsid protein, and virus neutralizing antibodies. A relationship was observed between the development of antibodies specific to sequences unique to the VP1 protein and virus neutralization. The polypeptide composition of the empty capsid immunogens appeared to be important for elicitation of potent virus neutralizing activity. VP2 capsid immunogens devoid of VP1 protein, or consisting of only 4% VP1, the composition of naturally occurring virions, were generally poor at eliciting high levels of virus neutralizing activity. Capsids consisting of > or = 25% VP1 protein efficiently and consistently provoked vigorous B19 virus neutralizing responses. Recombinant empty capsids enriched for the VP1 protein should serve as the basis for a human B19 parvovirus vaccine.

RNA-stimulated NTPase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus.

The genomic RNA of pestiviruses contains a single large open frame coding for virion structural proteins and viral nonstructural polypeptides. Based on the presence of specific amino acid sequence motifs, pestivirus nonstructural protein p80 was predicted to be both a serine-type proteinase and a nucleoside triphosphatase (NTPase)/RNA helicase. We previously demonstrated p80 possesses the former activity (Wisherchen and Collett, Virology 184, 341-350, 1991). Here, we provide experimental evidence that this protein is also an RNA-stimulated NTPase. Employing immunoaffinity chromatography, we partially purified a p80 protein analog (p87) from recombinant baculovirus-infected insect cells. We show this preparation contained a specific NTPase activity. This activity was not found in material similarly purified from lysates of baculovirus-infected insect cells not expressing the p87 protein. That the NTPase activity was associated with the p87 polypeptide was demonstrated in two ways. First, the NTPase activity was shown to be completely inhibited by monoclonal antibodies specific to the p80 polypeptide, but was unaffected by monoclonal antibodies to unrelated antigens. Second, radiolabeled ATP could be specially cross-linked to the p87 polypeptide. NTP hydrolysis by the p87 protein was stimulated by the presence of particular single-strand RNA molecules. Initial enzymologic characterization of the pestivirus p80 NTPase is presented, and the presumptive role of this activity in pestivirus replication is discussed.

RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria.

The nonstructural protein NS3 of the prototypic flavivirus, yellow fever virus, was investigated for possession of an NTPase activity. The entire NS3 protein coding sequence and an amino-terminal truncated version thereof were engineered into Escherichia coli expression plasmids. Bacteria harboring these plasmids produced the expected polypeptides, which upon cell disruption were found in an insoluble aggregated material considerably enriched for the NS3-related polypeptides. Solubilization and renaturation of these materials, followed by examination of their ability to hydrolyze ATP, revealed an ATPase activity present in both the full-length and amino-terminal truncated NS3 preparations but not in a similarly prepared fraction from E. coli cells engineered to express an unrelated polypeptide. The amino-terminal truncated NS3 polypeptide was further enriched to greater than 95% purity by ion-exchange and affinity chromatography. Throughout the purification scheme, the ATPase activity cochromatographed with the recombinant NS3 polypeptide. The enzymatic activity of the purified material was shown to be a general NTPase and was dramatically stimulated by the presence of particular single-stranded polyribonucleotides. These results are discussed in view of similar activities identified for proteins of other positive-strand RNA viruses.

Rearrangement of viral sequences in cytopathogenic pestiviruses.

Two cytopathogenic isolates of bovine viral diarrhea virus (cpBVDV) have been analyzed. For both viruses two regions of their genomic RNAs were found to be duplicated and rearranged. The viral genomes contain a small duplicated element (SD) derived from the genomic 5' end far downstream of its original context. This sequence is followed by a larger duplication which encompasses the region coding for the protein p80(LD), a molecular marker for cpBVDV. The SD element codes for the viral protease p20. In the case of the viruses analyzed here the aminoterminus of p80 is generated by autoproteolytic removal of the preceding SD-encoded protease. For one of the cpBVDV isolates a specific fusion protein (p28) could be identified which is composed of p20 and part of p10, another viral protein. Molecular characterization of the respective noncytopathogenic counterpart revealed that duplication and rearrangement of sequences as well as the expression of p28 and p80 are specific for the cytopathogenic virus.

Dipeptide backbone conformation and antibody recognition of a viral octapeptide epitope.

The peptide YKGTMDSG (Tyr-Lys-Gly-Thr-Met-Asp-Ser-Gly) represents an important antigenic determinant from the glycoprotein G2 of the pathogenic Rift Valley fever virus. By preparing a series of single-residue substitution peptides, the importance to antigenicity of individual residues within this octapeptide has been determined. Here, we investigated a simple and rapid computational analysis to test for correlations between the observed antigenicity of the substitution analogue peptides and the calculated conformational preferences in local regions of the peptides. Conformational energy analyses were carried out on all dipeptide combinations represented in the wild-type octapeptide and in the single-residue substitution analogue peptides. Conformational similarities and differences between wild-type and substitution dipeptide pairs were determined. The results of these computational analyses were then compared with the data on the relative antigenicity of the wild-type octapeptide and the substitution analogues. This comparison revealed a positive correlation. Substitution peptides showing changes in antigenicity possessed significant changes in the calculated backbone conformation relative to wild type in the dipeptides encompassing the residue substitution. Substitution peptides showing no change in antigenicity similarly showed no significant changes in dipeptide conformation. The potential utility of dipeptide conformational energy analyses and this preliminary structure-activity correlation are discussed.

Molecular genetics of pestiviruses.

Recent developments in understanding the molecular genetics of pestiviruses are reviewed. The genomic RNA of several pestiviruses has been molecularly cloned and sequenced. The genetic organization of the viral protein coding region has been refined, and the complete complement of virus-encoded proteins has been elucidated. Viral gene expression involves both co-translational and post-translational precursor polyprotein processing. Biogenesis of the virion structural proteins requires the proteolytic activity of the first protein of the open reading frame (p20), as well as host cell enzymes. A second virus-encoded proteinase (p80) is required for processing viral nonstructural proteins. Molecular and biochemical data indicate pestiviruses share many features with both the flaviviruses and human hepatitis C viruses, while at the same time, also reveal their distinct nature.

Baculovirus expression of pestivirus non-structural proteins.

Bovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3'-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.

Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface.

Capsids of the B19 parvovirus are composed of major (VP2; 58 kD) and minor (VP1; 83 kD) structural proteins. These proteins are identical except for a unique 226 amino acid region at the amino terminus of VP1. Previous immunization studies with recombinant empty capsids have demonstrated that the presence of VP1 was required to elicit virus-neutralizing antibody activity. However, to date, neutralizing epitopes have been identified only on VP2. Crystallographic studies of a related parvovirus (canine parvovirus) suggested the unique amino-terminal portion of VP1 assumed an internal position within the viral capsid. To determine the position of VP1 in both empty capsids and virions, we expressed a fusion protein containing the unique region of VP1. Antisera raised to this protein recognized recombinant empty capsids containing VP1 and VP2, but not those containing VP2 alone, in an enzyme-linked immunosorbent assay. The antisera immunoprecipitated both recombinant empty capsids and human plasma-derived virions, and agglutinated the latter as shown by immune electron microscopy. The sera contained potent neutralizing activity for virus infectivity in vitro. These data indicate that a portion of the amino terminus of VP1 is located on the virion surface, and that this region contains intrinsic neutralizing determinants. The external location of the VP1-specific tail may provide a site for engineered heterologous epitope presentation in novel recombinant vaccines.

Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing.

Bovine viral diarrhea virus (BVDV), the prototypic pestivirus, possesses a positive-strand RNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. We have endeavored to elucidate the mechanisms involved in protein biogenesis by this pestivirus. Here, we present our studies on gene expression from the viral nonstructural protein coding region encompassing the carboxy-terminal 60% of the ORF. Previous sequence and modeling analyses predicted the amino-terminal region of the BVDV nonstructural protein p80 to be a trypsin-like serine proteinase. Using a mammalian cell transient expression system, we show that this region indeed possessed a proteolytic activity and, further, required the serine residue previously predicted to be the putative serine proteinase catalytic site. We found the p80-region proteinase activity was required for proteolytic processing of all viral nonstructural proteins. Cleavage by this activity at the amino and carboxy termini of the p80 protein itself likely occurred intramolecularly (in cis), since we were unable to demonstrate activity in trans at these sites. Cleavages at the three processing sites downstream of the carboxy terminus of p80 were shown to occur in trans. However, p80 proteinase activity alone was not sufficient for cleavage of the last of these sites. Another viral gene product, or specific condition, is implicated as a necessary cofactor for p80 proteinase activity at this site. Pestivirus polyprotein processing can now be compared to similar events by viruses of other groups. Finally, the potential role of p80 proteinase activity in the phenotype of cytopathic biotypes of BVDV is discussed.

Efficiency of various cloned DNA probes for detection of bovine viral diarrhea viruses.

We have evaluated 24 cytopathic (CP) and 37 noncytopathic (NCP) strains of bovine viral diarrhea virus (BVDV) with a dot blot assay using four different genome segments of the NADL strain as hybridization probes (p80, p54, gp53, and gp62). The p80 and p54 probes hybridized to 23/24 (96%) and 22/24 (92%), respectively, of CP strains examined. In contrast, these same two probes only detected 16/37 (43%) and 5/37 (13%), respectively, of the NCP strains examined. The gp53 probe detected 18/24 (75%) and the gp62 probe detected 19/24 (79%) of the CP strains. In contrast, these latter two probes only detected 9/37 (24%) and 7/37 (20%), respectively, of NCP strains. This low detection rate of NCP strains suggests a need for developing a probe based on NCP sequences for identification of NCP strains.

Pestivirus gene expression: the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity.

The positive-strand RNA genome of pestiviruses contains a single large open reading frame (ORF) extending its entire length and is capable of encoding 450 kDa of protein. Studies have been undertaken with the purpose of elucidating the specific mechanisms involved in the biogenesis of the complete complement of pestivirus proteins. Here, we report on gene expression at the 5' end of the genome of the prototype pestivirus, bovine viral diarrhea virus (BVDV). We demonstrate, using both a cell-free transcription-translation system and a mammalian-cell transient-expression system, that the first protein product of the large ORF of BVDV, the p20 protein, possesses a specific proteolytic activity. The p20 proteinase activity acts to release the p20 protein from the nascent polyprotein. The p20 proteinase activity is not, however, required for downstream glycoprotein processing, indicating translocation of the pestivirus glycoprotein precursor is affected by an internal signal sequence.