Expression and Antibody Production of Japanese Encephalitis Virus RNA Polymerase (NS5) Protein

Japanese encephalitis virus (JEV), a member of mosquito-borne flaviviruses, is the leading cause of viral encephalitis in a large geographic area of Southeast Asia and Australia. JEV contains a single-stranded positive-sense RNA genome, which encodes its own RNA-dependent RNA polymerase (NS5) that is required for genomic RNA replication. In this study, we have described a pair of mouse antisera specific to the N- or C-terminal region of the NS5. Initially, two hydrophilic regions corresponding to the N-terminus and C-terminus of the NS5 protein were individually amplified by reverse transcription-PCR from the genomic RNA of JEV K87P39 strain. The amplified DNA fragments were cloned into a prokaryotic expression vector, pGEX-4T-1; the resulting constructs were used for the expression of GST fusion proteins, designated GST/NS5N and GST/NS5C, in E. coli BL-21 strain. Following immunization of three BALB/c mice with each of the purified GST/NS5N and GST/NS5C, we obtained two pools of the antisera, specifically recognizing the ~103-kDa NS5 and several smaller NS5-related proteins in BHK-21 and Vero cells infected with JEV K87P39 strain. Overall, we have successfully expressed the N- and C-terminal regions of JEV NS5 fused to the C-terminus of GST and generated the mouse antisera capable of recognizing the NS5 and its related proteins in JEV-infected cells. This would provide a valuable reagent for the study of JEV NS5 in the viral life cycle.

Intracellular Localization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is a small enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. It causes the porcine reproductive and respiratory syndrome in swine. The virus has 7 structural proteins: Of the seven, the N protein is the nucleocapsid that comprises a core of the virus particle. We have expressed the N protein of PRRSV PL97-1/LP1 strain using a heterologous gene expression vector derived from Sindbis virus, called pSinrep5. Immunofluorescence analysis showed that the N proteins were mainly found in the cytoplasm as well as in the nucleus of BHK-21 cells transfected with pSinrep5-N-derived RNA. Moreover, expression of the N protein did not change the incompetence of RNA replication of Mutant/nt14900 that lacks a 3' cis-acting replication element and the efficiency of RNA replication of Mutant/nt14800 that has a low level of RNA replication. Overall, our findings are consistent with previous results and help to understand a role of the N protein in PRRSV biology.

5' and 3' cis-Acting RNA Elements Required for RNA Replication of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5' and 3' cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5'-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3'-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5' cap structure, 3' poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.

Evidence for the Presence of a cis-acting Element in the Coding Region of the Japanese Encephalitis Virus Capsid Protein

Japanese encephalitis virus (JEV) is a member of mosquito-borne flaviviruses. To investigate whether there is a cis-acting genetic element in the coding region of the JEV C protein, which is required for viral replication, we generated four mutants by introducing a various size of deletions in each structural protein-coding region, designated as pJEV/Rep/deltaCC/LUC, pJEV/Rep/deltaC/LUC, pJEV/Rep/deltaprM/LUC, and pJEV/Rep/deltaE/LUC, of these, all replicons except for pJEV/Rep/deltaCC/LUC were competent in replication. Since pJEV/Rep/deltaCC/LUC is the same as pJEV/Rep/ deltaC/LUC except for an additional 5' deletion (nt 132~201) in the coding region of the C protein, this region appeared to be essential for RNA replication. This is consistent with the proposed cyclization sequence motif in the 5' region of the C gene, which has been recently shown to be required for replication in other mosquito-borne flaviviruses such as DV, YFV, KUN, and WNV. Thus, our results suggest that a cis-acting genetic element in the coding region of the JEV C protein may play an important role in RNA replication. This study will facilitate the current understanding of JEV RNA replication.

Expression and Purification of the Capsid Protein of the Japanese Encephalitis Virus and Production of its Polyclonal Antibody

Japanese encephalitis virus (JEV) is a member of Flaviviruses, transmitted by mosquitoes. The core of JEV is composed of the capsid (C) proteins. In order to produce the recombinant viral C protein and the antiserum specifically recognizing the JEV C protein, we have expressed and purified the JEV C protein as a Glutathion-S-Transferase (GST) fusion protein in E. coli. The JEV C protein-coding region was PCR-amplified using the infectious cDNA of a JEV Korean isolate, and the amplicons were cloned into the pGEX4T-1 E. coli expression vector. GST-C fusion proteins were purified using a glutathione sepharose column. Subsequently, the GST-C fusion proteins were used for immunization of rabbits, and the antisera were obtained from those immunized animals. Western blot analysis using the JEV-infected BHK21 cell lysates showed that these antisera specifically reacted with the JEV C proteins. This study will provide a useful reagent for the diagnosis and understanding of the viral morphogenesis in the JEV-infected cells.

Expression of the Structural Proteins of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is one of the most important human pathogens, which causes the permanent neuropsychiatric sequelae and even fatal diseases with high mortality and morbidity, especially among children. In this study, we expressed the structural proteins (C, prM, and E) of JEV using a Sindbis virus-based heterologous gene expression vector, the pSinRep5. We designed two expression vectors (pSinRep5/JEV C-E and pSinRep5/JEV C-NS1), which encode the precise coding sequence of JEV C-E and JEV C-NS1 proteins, respectively. These cloned JEV structural protein genes were designed to express under the Sindbis virus subgenomic promoter. Upon the transfection and expression of the pSinRep5/JEV C-E or pSinRep5/JEV C-NS1 plasmid, the transfected cells expressed approximately 55 kDa JEV E prtiens. As designed, the JEV NS1 proteins were expressed only in the SinRep5/JEV C-NS1 RNAtransfected cells. In addition, we found in the pSinRep5/JEV C-NS1-transfected cells that the viral proteins were predominantly localized around the perinuclear membranes. On the other hand, cytoplasmic staining was mainly observed in the pSinRep5/JEV C-E RNA-transfected cells in the absence of NS1 protein. Thus, our system will provide a useful tool to dissect intracellular membrane localization signals located in the JEV structural proteins without handling the infectious JEV viral particles and to characterize viral morphogenesis of this pathogen.