Identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones

AbstractIn order to determine virulence associated genes in porcine reproductive and respiratory syndrome virus (PRRSV), a series of chimeric viruses were generated where specific genomic regions of a highly virulent PRRSV infectious clone (FL12) were replaced with their counterparts of an attenuated vaccine strain (Prime Pac). Initial genome-wide scanning using a sow reproductive failure model indicated that non-structural (ORF 1a and 1b) and structural (ORF2-7) genomic regions appear to be sites where virulence determinants of PRRSV may reside. These results thus confirm the multigenic character of PRRSV virulence. Additional chimeras containing each individual structural ORFs (2 through 7) of Prime Pac and ORF5 of Neb-1 (parental strain of Prime Pac) within the FL12 backbone were generated and tested individually for further mapping of virulence determinants. Our results allow to conclude that NSP3–8 and ORF5 are the location of major virulence determinants, while other virulence determinants may also be contained in NSP1–3, NSP10–12 and ORF2

The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor CD163

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs, resulting in the formation of multiprotein complex. Our results further show that the GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of the CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and, along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell

METHODS AND COMPOSITIONS FOR VACCNATION OF ANIMALS WITH PRRSV ANTIGENS WITH IMPROVED IMMUNOGENICITY

Pigs challenged with hypoglycosylated variants of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) major surface protein GP5 exhibited increased production of PRRSV-neutralizing antibodies relative to the levels of neutralizing antibodies produced by pigs immunized with wild type (wt) or glycosylated GP5. This invention provides for methods of obtaining improved immune responses in pigs to PRRSV, compositions useful for obtaining the improved immune responses as well as isolated polynucleotides that encode hypoglycosylated variants of PRRSV major surface protein GP5

Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy

A Replicon \u3ci\u3eTrans\u3c/i\u3e-Packaging System Reveals the Requirement of Nonstructural Proteins for the Assembly of Bovine Viral Diarrhea Virus (BVDV) Virion

A selective trans-packaging system was developed to produce and isolate bovine viral diarrhea virus (BVDV) pseudo-particles with complementing reporter replicons and their packaging proteins expressed in trans with recombinant vaccinia virus. The encapsidation of replicon rNS3-5B was dependent not only on the in trans expression of structural proteins C, Erns, E1 and E2, but also the nonstructural proteins, p7 and contiguous precursor NS2-3-4A. Nonstructural p7, NS4B, NS5A or NS5B could be expressed in cis and in trans with precursor NS2-3-4A without significantly affecting virion assembly efficiency. NS2-3-4A was identified as an in trans functional precursor in virion assembly. BVDV genomes with mutant NS5B, which did not undergo active replication, were packaged 5-fold less efficiently than the intact genomes demonstrating the importance of replication in virion packaging. These results suggest that genome replication and assembly are closely associated, consistent with a model in which these two steps are coupled for maximum efficiency

Involvement of a Bovine Viral Diarrhea Virus NS5B Locus in Virion Assembly

A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-to-proline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans-packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly

Establishment of a Subgenomic Replicon for Bovine Viral Diarrhea Virus in Huh-7 Cells and Modulation of Interferon-Regulated Factor 3-Mediated Antiviral Response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2′-β-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system