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.Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae within the order Nidovirales which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus. The viral genome is a linear, positive-stranded RNA molecule of approximately 15.0 kb in length and possesses a cap structure at the 5Ј end and a poly(A) tail at the 3Ј end. Eight open reading frames (ORFs) are in the viral genome (9, 34). The first two open reading frames (ORF1a and ORF1ab) encode viral nonstructural (NS) polyproteins that are involved in polyprotein processing and genome transcription and replication (47). The viral structural proteins, encoded in ORFs 2 to 7, are expressed from six subgenomic capped and polyadenylated mRNAs that are synthesized as a 3Ј-coterminal nested set of mRNAs with a common leader sequence at the 5Ј end.The major viral envelope protein is glycoprotein 5 (GP5), which is encoded in ORF5 of the viral genome (29,35,36). GP5 is a glycosylated transmembrane protein of approximately 25 kDa (10,16,35). It has a putative N-terminal signal peptide and possesses three potential N-linked glycosylation sites which are located in a small ectodomain comprising the first 40 residues of the mature protein (28,35). In EAV and LDV, the major envelope glycoprotein forms a disulfide-l...

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.

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with Hepatitis E virus (HEV) is established as an etiological agent of the epidemic and sporadic forms of waterborne hepatitis (6, 25). The first well-characterized HEV epidemic was reported in Delhi, India, in 1955 (35). Several other epidemics have since been described in developing countries on nearly every continent (19,21). HEV has a positive-strand polyadenylated RNA genome ϳ7.2 kb in length (27) containing three open reading frames (ORFs). Nonstructural ORF1 (5Ј end) codes for a polyprotein of 1,693 amino acids (pORF1) and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicase, and an RNAdependent RNA polymerase (RdRp). The second ORF (3Ј end) codes for the major viral capsid protein of 660 amino acid (pORF2), while the third and smallest ORF (ORF3) codes for a 123-amino-acid-long polypeptide (pORF3) whose function is unknown (32). Apart from its coding region, the viral genome has 27-and 68-nucleotide (nt)-long noncoding regions at its 5Ј and 3Ј ends, respectively (32). The genome sequences of HEV have been reported from different geographical isolates and show a high degree of homology at both the nucleotide and amino acid levels (3,4,12,32,33). Expression of structural proteins pORF2 and pORF3 in prokaryotic and eukaryotic systems has been reported by different investigators (7,11,14,23,29). Earlier, we have expressed and characterized pORF2 and pORF3 in animal cells. pORF2 is an 88-kDa glycoprotein which is expressed intracellularly, as well as on the cell surface (14, 37). The ORF3 protein (pORF3) is a 13.5-kDa phosphoprotein which is phosphorylated by the cellular mitogen-activated protein kinase and associates with the cytoskeleton (36). We have also recently expressed the ORF1 polyprotein in both prokaryotic and eukaryotic systems (2).In the absence of a reliable in vitro culture system...

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BackgroundHepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations.Methodology/Principal FindingsWe examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture.Conclusions/SignificanceCollectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.