(Taken from Abstract) Infection of macaques with simian immunodeficiency virus (SIV) provides incisive opportunities to analyze the in vivo function of viral genes for induction of simian AIDS (SAIDS). Such studies in juvenile and adult macaques revealed that viral accessory genes, including nef, are important for pathogenesis. Accordingly, SIV clones with deletions in nef, as well as replication-competent SIV vectors expressing cytokines in place of nef, were tested as live-attenuated viral vaccines in juveniles and adults. We have found that juvenile, as well as newborn macaques, can develop SAIDS after inoculation with an SIV clone with a large deletion in nef, or with SIV-interleukin-2 vectors. Viruses recovered from several of these animals with disease produced either (i) a novel truncated form of Nef protein, designated tNef, or (ii) no detectable form of Nef protein (Sawai et al. 1999-Appendix) These findings indicate strong selection pressure to restore pathogenic potential in live attenuated primate lentivirus vaccines containing large deletions in viral accessory genes. Accordingly, the hypothesis for this project is that SIV pathogenesis can be mediated by truncated forms of Nef and/or changes in other viral genes that compensate for the loss of Nef function(s). This project proposes a genetic study of SIV Nef, both in vitro (tissue culture systems) and in vivo (experimental infection of macaques).
Specific Aim 1 characterizes Nef mutants in two newly identified domains required for interaction with the Nef-associated kinase in vitro.
Specific Aim 2 determines whether the Nef domains described in Aim 1 are important for pathogenesis by analysis of SIV nef mutants in vivo in macaques.
Specific Aim 3 explores viral phenotypes in pathogenic viruses recovered from macaques infected with SIV clones containing large deletions in the nef genes.
Specific Aim 4 searches for viral gene(s), which compensate for loss of Nef function, by testing novel recombinant viruses for pathogenic potential in macaques. The proposed in vitro and in vivo studies will define the regions of multifunctional SIV Nef that are critical for pathogenesis. Furthermore, it may be possible to identify viral gene changes that compensate for loss of one or more functions ascribed to this viral protein. Accordingly, the proposed studies will provide critical insight into molecular changes in the viral genome that are necessary for pathogenic conversion of live-attenuated viral vaccines.
