Recombinant viral RdRps can initiate RNA synthesis from circular templates

The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage phi6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the phi6 RdRp could only use circular RNAs made from the 3'-terminal sequence of the phi6 genome.     

Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed.     

Temperature-sensitive mutants of Japanese encephalitis virus.

Ten stable temperature-sensitive mutants of Japanese encephalitis virus were isolated after mutagenesis by growth of cloned wild-type virus in the presence of the nucleic acid precursor analogs 5-fluorouracil and 5-azacytidine. Mutants were selected which grew at least 100-fold better at 33 degrees C than at 41 degrees C. The 5-fluorouracil was found to be more effective at inducing temperature-sensitive mutations than was 5-azacytidine. Analysis of the virus-specific RNA and proteins synthesized by each mutant at the nonpermissive temperature was used to determine biochemical phenotypes. The mutants were analyzed for abilities to complement in mixed infections. Although inefficient and sometimes nonreciprocal, complementation occurred at higher levels than previously reported for flavivirus mutants. Interference between mutants in some mixed infections was also observed. Seven complementation groups were defined. Three groups contained mutants incapable of synthesizing virus-specific RNA at the nonpermissive temperature, whereas the remaining complementation groups displayed an RNA+ phenotype. Levels of protein synthesis comparable to that of wild type were observed at the nonpermissive temperature in three groups. Two other groups were represented by mutants which synthesized only low levels of virus-specific proteins at the higher temperature. Mutants in the remaining two groups did not produce detectable levels of proteins under nonpermissive conditions.     

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

Background  

Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.     

Japanese encephalitis virus: from genome to infectome

Japanese encephalitis virus (JEV) is an arbovirus belonging to the family Flaviviridae. It is maintained in a zoonotic cycle involving pigs, ardeid birds and Culex species of mosquitoes. Humans are accidental/dead end hosts of JEV infection because they cannot sustain high viral titers. Factors affecting the clinical manifestations and pathogenesis of JEV infection are not well understood. Though, vaccines are currently available against JEV, it has to be further improved. Here we review the literature on the JEV life cycle, pathogenesis and host immune responses to JEV infection.     

Agglutination of Japanese encephalitis virus with concanavalin A.

Results of experiments have indicated that reduction in biological activities at high concentrations of Japanese encephalitis virus is caused by aggregates of the virus by concanavalin A. The possibility exists that the concanavalin A binding site is different from hemagglutination and antireceptor sites of Japanese encephalitis virus.     

Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies

In order to evaluate the possibility of developing an oral vaccine against Japanese encephalitis virus (JEV), mice were fed with recombinant JEV envelope (E) protein synthesized in Escherichia coli. The protein was administered orally to mice with or without an immunostimulatory cytosine-phosphate-guanosine (CpG) motif containing synthetic oligodeoxynucleotide (ODN) as an adjuvant. The immunized mice made high-titered anti-E and anti-JEV antibodies. Mice immunized with JEV E protein along with the ODN adjuvant produced higher antibody titers and these were predominantly IgG2a type. These antibodies, however, failed to neutralize JEV activity in vitro, and the immunization did not protect the mice against lethal JEV challenge. Splenocytes from the immunized mice secreted large amounts of interferon (IFN)-gamma and showed proliferation in the presence of JEV E protein. Our results indicate that JEV E protein delivered orally to mice together with ODN generated both humoral and cellular immune responses to JEV, and these were of the Th1 type.     

Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication.

The antiviral effects of nitric oxide (NO) on Japanese encephalitis virus (JEV), a member of the family Flaviviridae, were investigated in this study. In vitro, inhibition of replication of JEV in gamma interferon-activated RAW 264.7 murine macrophages was correlated to cellular NO production. When cocultured with infected murine neuroblastoma N18 cells, gamma interferon-activated RAW 264.7 cells also efficiently hindered JEV replication in contiguous bystanders, and this anti-JEV effect could be reversed by an NO synthase (NOS) inhibitor, N-monomethyl-L-arginine acetate. In vivo, the mortality rate increased as the NOS activity of JEV-infected mice was inhibited by its competitive inhibitor, N-nitro-L-arginine methyl ester. Moreover, when an organic donor, S-nitro-N-acetylpenicillamine (SNAP), was used, the NO-mediated antiviral effect was also observed in primarily JEV-infected N18, human neuronal NT-2, and BHK-21 cells, as well as in persistently JEV-infected C2-2 cells. These data reaffirm that NO has an effective and broad-spectrum antimicrobial activity against diversified intracellular pathogens. Interestingly, the antiviral effect of NO was not enhanced by treatment of N18 cells with SNAP prior to JEV infection, a measure which has been shown to greatly increase the antiviral effect of NO in infection by vesicular stomatitis virus. From biochemical analysis of the impact of NO on JEV replication in cell culture, NO was found to profoundly inhibit viral RNA synthesis, viral protein accumulation, and virus release from infected cells. The results herein thus suggest that NO may play a crucial role in the innate immunity of the host to restrict the initial stage of JEV infection in the central nervous system.     

Sequence of 3000 nucleotides at the 5' end of Japanese encephalitis virus RNA

The 5' region of the Japanese encephalitis virus (JEV) RNA was cloned and 3000 nucleotides (nt) were determined by sequencing DNA complementary to viral RNA, and genomic RNA, using oligodeoxynucleotide primers and the dideoxy chain-termination reaction. Comparison of the nt sequence and the reduced amino-acid sequence of JEV with those of other flaviviruses showed significant homologies, which allowed locations to be assigned for three structural proteins.     

Autoimmunity-related demyelination in infection by Japanese encephalitis virus

Japanese encephalitis (JE) virus is the most common cause of epidemic viral encephalitis in the world. The virus mainly infects neuronal cells and causes an inflammatory response after invasion of the parenchyma of the brain. The death of neurons is frequently observed, in which demyelinated axons are commonly seen. The mechanism that accounts for the occurrence of demyelination is ambiguous thus far. With a mouse model, the present study showed that myelin-specific antibodies appeared in sera, particularly in those mice with evident symptoms. Meanwhile, specific T cells proliferating in response to stimulation by myelin basic protein (MBP) was also shown in these mice. Taken together, our results suggest that autoimmunity may play an important role in the destruction of components, e.g., MBP, of axon-surrounding myelin, resulting in demyelination in the mouse brain after infection with the JE virus.