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.