Identification of amino acid residues involved in the interaction between peste-des-petits-ruminants virus haemagglutinin protein and cellular receptors.

Peste-des-petits-ruminants virus (PPRV) haemagglutinin (H) protein mediates binding to cellular receptors and then initiates virus entry. To identify the key residues of PPRV H (Hv) protein of the Nigeria 75/1 strain involved in binding to receptors, interaction of the Hv and mutated Hv (mHv) proteins with receptors (SLAM and Nectin 4) and their mutants (mSLAM1, mSLAM2, mSLAM3 and mNectin 4) was investigated using surface plasmon resonance imaging (SPRi) and coimmunoprecipitation (co-IP) assays. The results showed that the Hv protein failed to interact with mSLAM3, but interacted at a strong or medium intensity with SLAM, mSLAM2, Nectin 4 and mNectin 4, and at a low level with mSLAM1. The mHv protein was unable to interact with SLAM and its mutants, but bound to Nectin 4 and mNectin 4 with medium and weak intensity, respectively. Further analysis showed that the Hv protein could precipitate mSLAM1, mSLAM2 and mNectin 4, but not mSLAM3. The mHv protein failed to coprecipitate with SLAM and its mutants. The binding activities of mNectin 4 and Nectin 4 to mHv were less than 30.36 and 51.94 % of the wild-type levels, respectively. Based on the results obtained, amino acids at positions R389, L464, I498, R503, R533, Y541, Y543, F552 and Y553 of H protein and I61, H62, L64, K76, K78, E123, H130, I210, A211, S226 and R227 in SLAM were identified to be essential for the specificity of H-SLAM interaction, while the critical residues of H-Nectin 4 interaction require further study. These findings would improve our understanding of the invasive mechanisms of PPRV.

Peste des petits ruminants virus hemagglutinin (H) induces lysosomal degradation of host cyclophilin A to facilitate viral replication.

Peste des petits ruminants virus (PPRV) is a highly contagious disease that affects sheep and goats. To better understand PPRV replication and virulence, cyclophilin A (CypA), a multifunctional goat host protein, was selected for further studies. CypA has been reported to inhibit or facilitate viral replication. However, the precise roles of CypA during PPRV infection remain unclear. Our data show for the first time that CypA suppressed PPRV replication by its PPIase activity, and PPRV infection decreased CypA protein levels. Detailed analysis revealed that PPRV H protein was responsible for the reduction of CypA, which was dependent on the lysosome pathway. No interaction was identified between H and CypA. Furthermore, the 35-58 region of H was essential for the reduction of CypA. In conclusion, our findings identify the antiviral role of CypA against PPRV and provide key insights into how PPRV H protein antagonizes host antiviral response.

Baculovirus expression and purification of peste-des-petits-ruminants virus nucleocapsid protein and its application in diagnostic assay.

Peste-des-petits-ruminants (PPR) is a contagious and highly devastating disease of small ruminants. For control of endemic PPR, adequate supply of affordable and reliable diagnostics is critical for effective surveillance, along with the use of highly efficacious live vaccines that are currently available. The nucleocapsid (N) protein of PPR virus (PPRV) is an important candidate antigen for developing specific diagnostic, as it is a major viral protein being highly immunogenic and conserved among the structural proteins. In the present study, we expressed the N protein of PPRV (Sungri/96 strain), in baculovirus expression system and purified using affinity column chromatography. The recombinant protein reacted well with PPRV anti-N monoclonal antibodies and PPRV-specific polyclonal antiserum, suggesting that the expressed protein was authentic and in native form. The recombinant protein was evaluated as antigen in the diagnostic ELISA as reference positive control in place of whole virus antigen. The utility of recombinant PPRV N protein circumvents the need to use live PPRV antigen in the routinely used diagnostics targeting 'N' protein of PPRV, thus allowing large-scale field application of the test.

The 135 Gene of Goatpox Virus Encodes an Inhibitor of NF-κB and Apoptosis and May Serve as an Improved Insertion Site To Generate Vectored Live Vaccine.

Goatpox virus (GTPV) is an important member of the Capripoxvirus genus of the Poxviridae Capripoxviruses have large and complex DNA genomes encoding many unknown proteins that may contribute to virulence. We identified that the 135 open reading frame of GTPV is an early gene that encodes an ∼18-kDa protein that is nonessential for viral replication in cells. This protein functioned as an inhibitor of NF-κB activation and apoptosis and is similar to the N1L protein of vaccinia virus. In the natural host, sheep, deletion of the 135 gene from the GTPV live vaccine strain AV41 resulted in less attenuation than that induced by deletion of the tk gene, a well-defined nonessential gene in the poxvirus genome. Using the 135 gene as the insertion site, a recombinant AV41 strain expressing hemagglutinin of peste des petits ruminants virus (PPRV) was generated and elicited stronger neutralization antibody responses than those obtained using the traditional tk gene as the insertion site. These results suggest that the 135 gene of GTPV encodes an immunomodulatory protein to suppress host innate immunity and may serve as an optimized insertion site to generate capripoxvirus-vectored live dual vaccines.IMPORTANCE Capripoxviruses are etiological agents of important diseases in sheep, goats, and cattle. There are rare reports about viral protein function related to capripoxviruses. In the present study, we found that the 135 protein of GTPV plays an important role in inhibition of innate immunity and apoptosis in host cells. Use of the 135 gene as the insertion site to generate a vectored vaccine resulted in stronger adaptive immune responses than those obtained using the tk locus as the insertion site. As capripoxviruses are promising virus-vectored vaccines against many important diseases in small ruminants and cattle, the 135 gene may serve as an improved insertion site to generate recombinant capripoxvirus-vectored live dual vaccines.

Prokaryotic expression of f protein from PPRV and characterization of its polyclonal antibody.

The goal of this study was to evaluate the specificity of a polyclonal antibody against the F protein from Peste des petits ruminants virus (PPRV). A pET30a/F prokaryotic expression vector was successfully constructed and its recombinant protein was expressed. The result of Western blot analysis showed that the fusion protein pET30a/F possessed good immunoreactivity and the purified recombinant protein was then used as the antigen to raise anti-pET30a/F polyclonal antibody in rabbits. The polyclonal antibody titer against the recombinant F protein was confirmed by indirect ELISA, and the protein's specificity against pET30/F polyclonal antibody was confirmed by both Western blot and indirect immunofluorescence assay in transfected cells. In short, we obtained the high-level expression of recombinant F protein as well as high titers of rabbit polyclonal antibody specificity against F protein in pCAGGS/F transfected cells. This special polyclonal antibody offers a valuable and useful tool for further study of the pathogenesis of PPRV early infection and the structural and functional characterization of PPRV F protein.

Complete genome sequence of a Peste des petits ruminants virus recovered from wild bharal in Tibet, China.

For the first time, here we announce the complete genome sequence of a field isolate of Peste des petits ruminants virus (PPRV) derived from macerated rectal tissue of a free living bharal (Pseudois nayaur) that displayed clinical disease consistent with severe infection with PPRV. Further, we compare the full genome of this isolate, termed PPRV Tibet/Bharal/2008, with previously available PPRV genomes, including those of virus isolates from domestic small ruminants local to the area where the reported isolate was collected. The current sequence is phylogenetically classified as a lineage IV virus, sharing high levels of sequence identity with previously described Tibetan PPRV isolates. Indeed, across the entire genome, only 26 nucleotide differences (0.16% nucleotide variation) and, consequently, 9 amino acid changes were present compared to sequences of locally derived viruses.

[Sequence analysis of the phosphoprotein gene of peste des petits ruminants virus of Chinese origin].

The nucleotide sequences of P gene from a field strain of peste des petits ruminants virus (PPRV) ("China/Tib/Gej/07-30") was firstly determined. The P gene is 1,655 nucleotides long with two overlapping open reading frames (ORFs). The first ORF is 1530 nucleotides long and would produce P protein of 509 amino acid residues. The second ORF is 534 nucleotides long and would produce C protein of 177 amino acid residues. The first ORF produces a second mRNA transcript of 897 nucleotides long with an extra G nucleotide at position 751. Translation from this mRNA would produce V protein of 298 amino acid residues. The nucleotide and deduced amino acid sequence were compared with the homologous region of other PPRV isolates. At the amino acid level, the "China/Tib/Gej/07-30" shares homology of 86.10%-97.3%, 84.3%-94.9%, and 82.9%-96.3% for P, C, and V proteins respectively. Several sequence motifs in the P genes were identified on the basis of conservation in the PPRVs and the morbilliviruses.

[Sequence analysis of the matrix protein and fusion protein genes of a field peste des petits ruminants virus strain from Tibet, China].

The nucleotide sequences of M and F genes from a field strain of peste des petits ruminants virus (PPRV) ("China/Tib/Gej/07-30") was firstly determined. The M gene was 1 483 nucleotides in length with a single open reading frame (ORF), encoding a protein of 335 amino acids. The F gene was 2411 nucleotides in length, encoding a protein of 546 amino acids. The resulting nucleotide sequence and the deduced amino acid sequences were compared with the homologous regions of other PPRV isolates. The nucleotide sequences of M and F genes of the "China/Tib/Gej/07-30" was 92.4%-97.7% and 85.5%-96.1% identical to other PPRV isolates, respectively, while a homology of 97.0%-98.2% and 94.3%-98.2% could be observed at the amino acids level respectively. Several sequence motifs in the M and F genes had been identified on the basis of conservation in the PPRVs and the morbilliviruses. The 3' untranslated region of M gene was 443 nucleotides in length with 82.4%-93.5% identical to other PPRV isolates. The 5' untranslated region of F gene was 634 nucleotides in length with 76.2%-91.7% identical to other PPRV isolates.

Identification of functional domains of phosphoproteins of two morbilliviruses using chimeric proteins.

The paramyxovirus P protein is an essential component of the transcriptase and replicase complex along with L protein. In this article, we have examined the functional roles of different domains of P proteins of two closely related morbilliviruses, Rinderpest virus (RPV) and Peste des petits ruminants virus (PPRV). The PPRV P protein physically interacts with RPV L as well as RPV N protein when expressed in transfected cells, as shown by co-immunoprecipitation. The heterologous L-P complex is biologically active when tested in a RPV minigenome replication/transcription system, only when used with PPRV N protein but not with RPV N protein. Employing chimeric PPRV/RPV cDNAs having different coding regions of P protein in the minigenome replication/transcription system, we identified a region between 290 and 346 aa in RPV P protein necessary for transcription of the minigenome.

Mapping the Peste des Petits Ruminants virus nucleoprotein: identification of two domains involved in protein self-association.

For Mononegavirales, the template for transcription and replication is not the naked RNA but the nucleoprotein (N) encapsidated genomic and anti-genomic RNA. Because of this central role in the replication of these viruses, N has been the subject of numerous structural and functional mapping studies. Here, we report on the cloning of the Peste des Petits Ruminants virus (PPRV) N gene into the baculovirus vector and the expression of the protein in insect cells. By electron microscopy observation, we have shown that this recombinant PPRV N forms nucleocapsid-like particles in insect cells in the absence of other PPRV proteins, as reported for other paramyxoviruses. As it is known that the formation of these particles is first linked to the self-assembly of N, we have made several deletions in the PPRV N gene and expressed these mutants in insect cells. Analysis of these proteins by immunoprecipitation and electron microscopy observation enabled us to map the N-N interaction domains into two regions of PPRV N: aa 1-120 and 146-241. The fragment aa 121-145, which is not conserved within the morbillivirus group, is also required for the formation/stability of the nucleocapsid helical structure.

[Sequence analysis of the nucleocapsid gene and genome promoter region of peste des petits ruminants virus of Chinese origin].

The N gene and genome promoter nucleotide sequence of a Chinese Peste des petits rumiants virus (PPRV) ("China/Tib/Gej/07-30") was firstly determined. The length of N gene was 1689 nucleotides with a single open reading frame (ORF). The nucleotide and deduced amino acid sequence was compared with the homologous region of other PPRV isolates. The nucleotide sequence of the "China/Tib/Gej/07-30" was 91.7%-97.6% identical to other PPRV isolates, while a homology of 94.9%-98.5% could be observed at the amino acids level. The N gene encoded a protein of 525 amino acids. Several sequence motifs were identified on the basis of conservation in the PPRVs and the morbilliviruses. The genome length of promoter region was 107 nucleotides with 91.8%-98.2% identity to other PPRV isolates. Phylogenetic analysis showed that the "China/Tib/Gej/07-30" belonged to the Asian lineage.

The fusion core complex of the peste des petits ruminants virus is a six-helix bundle assembly.

We describe the properties of the two heptad repeats (HR1 and HR2) of the Peste des petits ruminants virus (PPRV) fusion protein (F) to obtain insights into the mechanism by which these repeats influence PPRV-mediated cell fusion. Both HR1 and HR2 inhibit PPRV-mediated syncytia formation in Vero cells in vitro. Of these, HR2 was found to be more effective than HR1. We studied the mechanism of fusion inhibition by these two repeats by using various biophysical and biochemical methods either separately or together. CD spectral analysis of these repeats revealed that the alpha-helical content of HR1 and HR2 when used together is higher than that of their simulated spectrum in the mixture, suggesting the formation of a highly structured complex by these repeats. Protease protection assays confirmed that such a complex is highly stable. Electrospray mass spectrometry of protease-digested products of the HR1-HR2 complex showed protection of fragments corresponding to both HR1 and HR2 sequences involved in complex formation. By employing size-exclusion chromatography and chemical cross-linking experiments, we show that three units each of HR1 and HR2 form a complex in which HR1 is a trimer and HR2 is a monomer. Homology-based three-dimensional modeling of this complex showed that HR1 and HR2 together form a six-helix and trimeric coiled-coil bundle. In this model, the HR1 trimer forms the core whereas HR2, while interacting with HR1 in an antiparallel orientation, forms a two-stranded coiled-coil structure and lies at the periphery of the structure. These results are discussed in the context of a common fusion mechanism among paramyxoviruses.

Protection of rabbits against lapinized rinderpest virus with purified envelope glycoproteins of peste-des-petits-ruminants and rinderpest viruses.

Haemagglutinin (HA) and fusion (F) proteins of peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) were purified by immunoaffinity chromatography. The purified proteins were characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Rabbit hyperimmune sera were raised against the purified HA and F proteins and assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination-inhibition (HAI) and virus neutralization (VN) tests. The immunized animals were challenged with a virulent lapinized (rabbit-adapted) strain of RPV. Both HA and F proteins of PPRV protected rabbits against a lethal challenge with lapinized RPV. As expected, RPV HA and F proteins also conferred a similar protection against the homologous challenge. The postchallenge antibody responses were of a true anamnestic type.