title: Sindbis vectors suppress secretion of subviral particles of Japanese encephalitis virus from mammalian cells infected with SIN-JEV recombinants.
abstract: Double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding prM-E or a C-terminally truncated form of E of Japanese encephalitis virus (JEV) were constructed. The products were efficiently expressed in both mammalian and mosquito cell lines infected with the dsSIN recombinants. However, suppression of prM-E secretion from mammalian cells infected with dsSIN-prM-E recombinants was observed. This suppression was more pronounced late in infection (< 5% of total product was secreted during an 8-hr chase) than early in infection (15% secretion during a 6-hr chase). In comparison, a vaccinia virus-prM-E recombinant (vP829) described previously (E. Konishi et al. (1991) Virology 185, 401-410) was shown to secrete 35-50% of total product during a 6- to 8-hr chase both early and late in infection. In contrast, secretion of prM-E from dsSIN-prM-E-infected mosquito (C6/36) cells was found to be efficient (> 50% during an 8-hr chase). The prM-E secreted from both mammalian and mosquito cells was in the form of subviral particles as determined by velocity gradient centrifugation, sensitivity to nonionic detergent, and analysis of processing of N-linked glycans. The truncated E protein expressed by the dsSIN recombinants was secreted efficiently from both mammalian and mosquito cells. Coinfection experiments with the dsSIN-JEV recombinants + wild-type vaccinia virus and vP829 + SIN demonstrated that the reduced level of secretion of subviral particles exhibited by the dsSIN-JEV recombinants was due to an inhibitory effect of the dsSIN vectors. Furthermore, this inhibitory effect was accounted for by the SIN nonstructural proteins since SIN replicons that express prM-E cassette in place of the SIN structural protein open reading frame exhibited a low level of subviral particle secretion. No self-propagating infectious particles were produced in cells transfected with SIN replicons that encode the JEV prM-E cassette. The suppression of subviral particle secretion was apparently correlated with the inhibition of cell protein synthesis which is mediated in SIN-infected vertebrate cells by expression of the SIN nonstructural proteins.
authors.affiliation: Department of Biology, Georgia State University, Atlanta 30303, USA.
authors.forename: K V
authors.initials: KV
authors.lastname: Pugachev
authors.forename: P W
authors.initials: PW
authors.lastname: Mason
authors.forename: T K
authors.initials: TK
authors.lastname: Frey
mesh_terms: D000330:Aedes; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004664:Encephalitis Virus, Japanese / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D012845:Sindbis Virus / Q000235:genetics*; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D003513:Cycloheximide