Effects of the nsP2-726 Pro mutation on infectivity and pathogenesis of Sindbis virus derived from a full-length infectious cDNA clone

Abstract

The point mutations at residue 726 Pro in the nonstructural gene 2 (nsP2-726P) could make Sindbis virus (SINV) replicons lacking the structural protein-coding region less cytopathic and capable of persisting in some vertebrate cell lines. However, the effects of nsP2-726P mutations on characteristics of SINV in the context of genomic-RNA are poorly understood. To investigate the effects of point mutations at nsP2-726P on the infectivity and the pathogenesis of SINV, based on the infectious clone (pBR-XJ160) of a Sindbis-like XJ-160 virus, we constructed mutants BR-726L, BR-726S, BR-726V and BR-726A containing point mutations Pro-to-Leu, Pro-to-Ser, Pro-to-Val and Pro-to-Ala. The BR-726V virus and BR-726A virus exhibited similar growth characteristics to the wild-type BR-XJ160 in cultured cells, including cytopathic effects (CPE), plaque morphology and growth kinetics. For the Leu substitution, no CPE or plaques were seen after six passages through BHK-21 cells, although expression of XJ-160 virus-specific protein was detectable by indirect immunofluorescence assay (IFA). The Ser substitutions gave an intermediate phenotype. The mutant viruses exhibited different levels of neurovirulence in 3-day-old suckling mice, which did not match their propagation in cultured cells or in the mouse brain. Compared with BR-XJ160, BR-726A with the Ala substitution showed highly increased neurovirulence, while BR-726V with the Val substitution exhibited an attenuated phenotype. In contrast, BR-726S, with reduced growth capacity in cultured cells and mouse brain, showed intermediate neurovirulence. BR-726L virus produced no lethality or morbidity in suckling mice. Thus, the nsP2-726 Pro residue regulates virus–host cell interactions directly and is also important in viral pathogenesis in suckling mice.