MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF SIMIAN IMMUNODEFICIENCY VIRUS MACAQUE STRAIN 32H PROVIRAL CLONES CONTAINING NEF SIZE VARIANTS

The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both lo ng terminal repeats, it is 10277 base pairs in length and contains ope n reading frames for all known SIV genes (gag, poi, vif, vpx, vpr, tat , rev, env and nef). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in seque nce only within their last 1.2 kb. Two of the molecular clones, SIVmac 32H(pJ5) and SIVmac32H (pC8), differ in the nef coding region by an in -frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3' LTR were not associat ed with known functionally critical motifs. The third clone, SIVmac32H (pB1), contains the last 1.2 kb of the SIVmac251 clone pBK28. The biol ogical properties of virus produced after electroporation of these clo nes into C8166 cells has been assessed by infection of rhesus and cyno molgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymph ocytes with C8166 cells. The viruses obtained from these clones have i dentical growth kinetics in vitro but differ in their ability to persi st in macaques. Macaques infected with pJ5 derived virus remain viraem ic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nef gene evolved over time in pJ5 virus-infected macaques, whereas late in infection in pC8 virus -infected macaques the nef gene remained invariant in sequence. These results support the observation that a nef deletion mutant of SIVmac23 9 lost its pathogenic potential and resulted in low-level viraemia whe n rhesus macaques were infected. Virus challenge pools for vaccine stu dies have been prepared for pJ5 using both human and monkey cell subst rates and these stocks have been titrated both in vitro and in vivo. V irus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaque s. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDS-like symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequ ence features are important for the pathogenesis of AIDS.