DEDUCED CONSENSUS SEQUENCE OF SINDBIS VIRUS-STRAIN AR339 - MUTATIONS CONTAINED IN LABORATORY STRAINS WHICH AFFECT CELL-CULTURE AND IN-VIVO PHENOTYPES

The consensus sequence of the Sindbis virus AR339 isolate, the prototy pe alphavirus, has been deduced. The results presented here suggest (i ) that a substantial proportion of the sequence divergence evident bet ween the consensus sequence and sequences of laboratory strains of AR3 39 has resulted from selection for efficient growth in cell culture, ( ii) that many of these changes affect the virulence of the virus in an imal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic stu dies of virus pathogenesis and host range, A laboratory strain of Sind bis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was d educed from the complete sequences of pTRSB and HR(sp) (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from parti al sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of four other alphaviru ses closely related to Sindbis virus. The sequence of neither HR(sp) n or TRSB was representative of the consensus Sindbis virus AR339 sequen ce, HR(sp) differed from the consensus sequence by eight coding change s, and TRSB differed by three coding changes, In the 5' untranslated r egion, HR(sp) differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate, At three of the difference loci (one in T RSB and two in HR(sp)), selection of cell-culture-adaptive mutations w as documented with Sindbis virus or other alphaviruses. Selection in c ell culture often results in attenuation of virulence in animals, Cons idering the TRSB and HR(sp) sequences together, one noncoding differen ce from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes ( at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significant ly affected alphavirus virulence in mice, Although the levels of virul ence of isogenic strains Containing either nt 5 A or nt 5 G did not di ffer significantly in neonatal mice, the presence of nt 5 A greatly en hanced the effect of a second attenuating mutation in the E2 gene. The se results suggest that minimal differences in the ''wild-type'' genet ic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes, A cDNA clone of the consensus AR339 sequence, a sequence devoid of occ ult attenuating mutations introduced by cell culture passage, will all ow the molecular genetic examination of cell culture and in vivo pheno types of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation.