Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes.
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Noncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), beta-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3'-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications. (+info)
Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus.
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We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans. (+info)
Degradation of Japanese encephalitis virus by neutrophils.
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The ability of neutrophils to degrade the phagocytosed Japanese encephalitis (JE) virion, via triggering of the respiratory burst and generation of toxic radicals has been investigated. JEV or JEV-induced macrophage derived factor (MDF) induces increase in intracellular oxidative signals with generation of superoxide anion (O2-), via activation of cytosolic NADPH and subsequent formation of hydrogen peroxide, with maximum activity on day 7 post infection. The response was sensitive to anti-MDF antibody treatment. Further, the study revealed rapid degradation of phagocytosed JE viral protein and nucleic acid. The viral protein degradation was partially dependent on the generation of toxic oxygen species as it could be abrogated by pretreatment of the cells with staurosporine. (+info)
Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing.
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Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been implicated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg-Arg) in the P1 through P4 positions of this cleavage site. Multiple alanine substitutions were made for these residues in order to investigate the substrate specificity and biological significance of this cleavage. Mutants were analyzed by several methods: (i) a cell-free trans processing assay for direct analysis of NS2B-NS3-mediated cleavage; (ii) a trans processing assay in BHK-21 cells, using a C-prM polyprotein, for analysis of prM production; (iii) an infectivity assay of full-length transcripts to determine plaque-forming ability; and (iv) analysis of proteins expressed from full-length transcripts to assess processing in the context of the complete genome. Mutants that exhibited severe defects in processing in vitro and in vivo were incapable of forming plaques. Mutants that contained two adjacent basic residues within the P1 through P4 region were processed more efficiently in vitro and in vivo, and transcripts bearing these mutations were fully infectious. Furthermore, two naturally occurring plaque-forming revertants were analyzed and shown to have restored protein processing phenotypes in vivo. Finally, the efficient production of prM was shown to be dependent on the proteolytic activity of NS3. These data support a model of two coordinated cleavages, one that generates the carboxy terminus of C and another that generates the amino terminus of prM. A block in the viral protease-mediated cleavage inhibits the production of prM by the signal peptidase, inhibits particle release, and eliminates plaque formation. (+info)
Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo.
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The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule. (+info)
Phylogeny of the genus flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector.
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Attempts to define the evolutionary relationships and origins of viruses in the genus Flavivirus are hampered by the lack of genetic information particularly amongst the non-vectored flaviviruses. Using a novel protocol for sequence determination, the first complete coding sequence of St Louis encephalitis virus and those of two representative non-vectored flaviviruses, Rio Bravo (isolated from bat) and Apoi (isolated from rodent), are reported. The encoded polyproteins of Rio Bravo and Apoi virus are the smallest described to date within the genus FLAVIVIRUS: The highest similarities with other flaviviruses were found in the NS3 and NS5 genes. The proteolytic cleavage sites for the viral serine protease were highly conserved among the flaviviruses completely sequenced to date. Comparative genetic amino acid alignments revealed that p-distance cut-off values of 0.330-0.470 distinguished the arthropod-borne viruses according to their recognized serogroups and Rio Bravo and Apoi virus were assigned to two distinct non-vectored virus groups. Within these serogroups, cladogenesis based on the complete ORF sequence was similar to trees based on envelope and NS5 sequences. In contrast, branching patterns at the deeper nodes of the tree were different from those reported in the previous study of NS5 sequences. The significance of these observations is discussed. (+info)
cis- and trans-acting elements in flavivirus RNA replication.
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Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functional relationships of these components in RNA replication, we used trans-complementation analysis of full-length infectious RNAs of Kunjin (KUN) virus with a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KUN RNAs with large carboxy-terminal deletions including the entire RNA polymerase region in the NS5 gene, representing 34 to 75% of the NS5 coding content, could be complemented after transfection into repBHK cells. In this study we have demonstrated that KUN RNAs with deletions of 84 to 97% of the NS1 gene, or of 13 to 63% of the NS3 gene including the entire helicase region, were also complemented in repBHK cells with variable efficiencies. In contrast, KUN RNAs with deletions in any of the other four nonstructural genes NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstrated successful trans complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural coding content. Based on these and our previous complementation results, we have generated a map of cis- and trans-acting elements in RNA replication for the nonstructural coding region of the flavivirus genome. These results are discussed in the context of our model on formation and composition of the flavivirus replication complex, and we suggest molecular mechanisms by which functions of some defective components of the replication complex can be complemented by their wild-type counterparts expressed from another (helper) RNA molecule. (+info)
A novel model for the study of the therapy of flavivirus infections using the Modoc virus.
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The murine Flavivirus Modoc replicates well in Vero cells and appears to be as equally sensitive as both yellow fever and dengue fever virus to a selection of antiviral agents. Infection of SCID mice, by either the intracerebral, intraperitoneal, or intranasal route, results in 100% mortality. Immunocompetent mice and hamsters proved to be susceptible to the virus only when inoculated via the intranasal or intracerebral route. Animals ultimately die of (histologically proven) encephalitis with features similar to Flavivirus encephalitis in man. Viral RNA was detected in the brain, spleen, and salivary glands of infected SCID mice and the brain, lung, kidney, and salivary glands of infected hamsters. In SCID mice, the interferon inducer poly IC protected against Modoc virus-induced morbidity and mortality and this protection was associated with a reduction in infectious virus content and viral RNA load. Infected hamsters shed the virus in the urine. This allows daily monitoring of (inhibition of) viral replication, by means of a noninvasive method and in the same animal. The Modoc virus model appears attractive for the study of chemoprophylactic or chemotherapeutic strategies against Flavivirus infections. (+info)