Effect of the attenuating deletion and of sequence alterations evolving invivo on simian immunodeficiency virus C8-Nef function

The simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and sa fety of live attenuated SIV vaccines in a large number of macaques, The att enuating deletion in the SIVmacC8 aef-long terminal repeat region encompass es only 12 bp and is "'repaired" in a subset of infected animals. It is unk nown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies , we? analyzed the relevance of the deletion, and the duplications and poin t mutations selected in infected macaques for Nef function in vitro. The de letion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic dec rease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity a ssays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibi lity complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions . It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmac nef variants carrying the original 12-bp de letion readily outgrew an otherwise isogenic virus containing a 182-bp dele tion in the nef gene. Thus, although C8-Nef is unstable and severely impair ed in in vitro assays, it maintains some residual activity to stimulate vir al replication.