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.     

Dual mechanisms of pestiviral superinfection exclusion at entry and RNA replication

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.     

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.     

Brefeldin A inhibits pestivirus release from infected cells, without affecting its assembly and infectivity

The enveloped bovine viral diarrhea virus (BVDV) is a member of the Pestivirus genus within the Flaviviridae family. While considerable information has been gathered on virus entry into the host cell, genome structure and protein function, little is known about pestivirus morphogenesis and release from cells. Here, we analyzed the intracellular localization, N-glycan processing and secretion of BVDV using brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum (ER) and causes disruption of the Golgi complex with subsequent fusion of its cis and medial cisternae with the ER. BFA treatment of infected cells resulted in complete inhibition of BVDV secretion and increased co-localization of the envelope glycoproteins with the cis-Golgi marker GM 130. Processing of the N-linked glycans was affected by BFA, however, virus assembly was not perturbed and intracellular virions were fully infectious, suggesting that trafficking beyond the cis-Golgi is not a prerequisite for pestivirus infectivity.     

The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein

The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx(17)CxCx(20)C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx(22)CxCx(24)C and likely comprises a three-stranded antiparallel beta-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom.     

Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer.

Neutralizing monoclonal antibodies directed against hog cholera virus (HCV) precipitated two HCV-encoded glycoproteins, HCV gp55 and HCV gp33. Immunoassay with bacterial fusion proteins and Western immunoblotting with extracts from infected cells revealed that the antibodies recognized only HCV gp55. Coprecipitation of HCV gp33 was shown to be due to intermolecular disulfide bridges. One of the antibodies also reacted with the major glycoprotein of another pestivirus, bovine viral diarrhea virus (BVDV). The analogous BVDV glycoproteins exhibited a distribution of cysteine residues which was almost identical to that of HCV gp55 and gp33. The two BVDV glycoproteins were also linked by disulfide bridges.     

Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus

Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.     

Identification of potent bovine viral diarrhea virus inhibitors by a structure-based virtual screening approach

Bovine viral diarrhea virus (BVDV) is a pestivirus whose infection in cattle is globally distributed. The use of antivirals could complement vaccination as a tool of control and reduce economic losses. The RNA-dependent RNA polymerase (RdRp) of the virus is essential for its genome replication and constitutes an attractive target for the identification of antivirals. With the aim of obtaining selective BVDV inhibitors, the crystal structure of BVDV RdRp was used to perform a virtual screening. Approximately 15,000 small molecules from commercial and in-house databases were evaluated and several structurally different compounds were tested in vitro for antiviral activity. Interestingly, of twelve evaluated compounds, five were active and displayed EC₅₀ values in the sub and low-micromolar range. Time of drug addition experiment and measured intracellular BVDV RNA showed that compound 7 act during RNA synthesis. Molecular Dynamics and MM/PBSA calculation were done to characterize the interaction of the most active compounds with RdRp, which will allow future ligand optimization. These studies highlight the use of in silico screening to identify a new class of BVDV inhibitors.     

Genetic analysis of the pestivirus nonstructural coding region: defects in the NS5A unit can be complemented in trans

The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5' and 3' untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cis or in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to be cis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.     

Ribonucleoprotein formation by the ORF1 protein of the non-LTR retrotransposon Tx1L in Xenopus oocytes.

The Tx1L elements constitute a family of site-specific non-LTR retrotransposons found in the genome of the frog Xenopus laevis . The elements have two open reading frames (ORFs) with homology to proteins of retroviruses and other retroelements. This study demonstrates an expected activity of one of the element-encoded proteins. The RNA binding properties of ORF1p, the product of the first ORF of Tx1L, were examined after expression from RNA injected into Xenopus oocytes. Using sucrose gradient sedimentation and non-denaturing gel electrophoresis, we show that ORF1p associates with RNA in cytoplasmic ribonucleoprotein (RNP) particles. Discrete RNPs are formed with well-defined mobilities. The ORF1p RNPs are distinct from endogenous RNPs that contain stored oocyte mRNAs and two specific endogenous mRNAs do not become associated with ORF1p. ORF1p appears to be capable of associating with its own mRNA and with other injected RNAs, independent of specific recognition sequences. Although nuclear localization of ORF1p was anticipated, based both on the supposed mechanism of transposition and on the presence of a potential nuclear localization signal, no significant fraction of the protein was found in the oocyte nucleus. Nonetheless, the RNA binding capability of ORF1p is consistent with the proposed model for transposition of non-LTR retrotransposons.     

Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses

The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-gamma.     

Cell-derived sequences in the N-terminal region of the polyprotein of a cytopathogenic pestivirus

Efficient proteolytic release of nonstructural protein 3 (NS3) from the viral polyprotein is considered to be crucial for the cytopathogenicity of pestiviruses. Here we describe a novel cytopathogenic (cp) bovine viral diarrhea virus strain (BVDV CP8) with a complex insertion composed of viral and cell-derived sequences, including two fragments of the cellular J-domain protein Jiv (J-domain protein interacting with viral protein) located in the N-terminal region of the polyprotein. BVDV CP8 expresses a Jiv fusion protein of 513 amino acids in addition to a complete set of viral proteins. This protein has the capacity to induce NS2-3 cleavage in trans. Accordingly, CP8 is a representative of a novel type of cp pestivirus with a cp-specific mutation located outside of the NS2-3 gene.     

Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.     

Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production.

A protocol to test foetal calf serum (FCS) for contamination with bovine viral diarrhoea virus (BVDV) is described. Following this protocol, which combines cell culture methods and detection of pestivirus RNA, seven batches of FCS were tested. Infectious BVDV was detected in four of those batches. One of the remaining batches contained a relatively high number of non-infectious BVDV particles. A sample of this batch was formulated with aluminium hydroxide and aluminium phosphate as adjuvant into an experimental vaccine preparation. This product was injected twice into BVDV seronegative cattle with a 4 week interval. Blood samples taken 4 weeks after the second application were negative for BVDV specific antibodies. Our data stress that detection of BVDV RNA is not sufficient for a complete risk assessment on FCS. Discrimination between infectious and non-infectious BVDV is essential. This can only be achieved by cell culture methods.