Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants

Abstract

The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif, vpx, ~pr, tat, rev, env and neJ). 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 sequence only within their last 1.2 kb. Two of the molecular clones, SIVmac32H(pJS) and SIVmae32H (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 associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1.2 kb of the SIVmae251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with p J5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nefgene evolved over time in p J5 virusinfected macaques, whereas late in infection in pC8 virus-infected macaques the nefgene remained invariant in sequence. These results support the observation that a nefdeletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for p J5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDSlike symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.

CD8 + Lymphocytes Do Not Mediate Protection against Acute Superinfection 20 Days after Vaccination with a Live Attenuated Simian Immunodeficiency Virus

“…Macaques were housed and maintained in accordance with United Kingdom Home Office guidelines for the care and maintenance of nonhuman primates. The attenuated SIVmac32H (SIVmacC8) virus clone differs from the wild-type SIVmac32H (SIVmacJ5) clone by a 12-bp deletion and two nonsynonymous nucleotide changes, resulting in conservative amino acid changes in the nef open reading frame (36). For all live attenuated SIV inoculations, macaques were intravenously administered 5,000 50% tissue culture infective doses of the 9/90 pool of SIVmacC8 on day 0 (11,36).…”

“…The attenuated SIVmac32H (SIVmacC8) virus clone differs from the wild-type SIVmac32H (SIVmacJ5) clone by a 12-bp deletion and two nonsynonymous nucleotide changes, resulting in conservative amino acid changes in the nef open reading frame (36). For all live attenuated SIV inoculations, macaques were intravenously administered 5,000 50% tissue culture infective doses of the 9/90 pool of SIVmacC8 on day 0 (11,36). For all wild-type SIV challenges, macaques were intravenously administered 20 50% median infective doses of the 3/92 stock of SIVmacJ5 on day 20 (12,36).…”

“…For all live attenuated SIV inoculations, macaques were intravenously administered 5,000 50% tissue culture infective doses of the 9/90 pool of SIVmacC8 on day 0 (11,36). For all wild-type SIV challenges, macaques were intravenously administered 20 50% median infective doses of the 3/92 stock of SIVmacJ5 on day 20 (12,36).…”

Genomic Analysis of the Viral Population in Genital Secretions Early after Infection of Simian Immunodeficiency Viruses in Macaque Monkeys

“…The nucleotide sequences of the analyzed clones and SIV clones reported previously were aligned using the Clustal W computer program (25), and a phylogenetic tree was constructed using the Neighbor-Joining method (23) in the Tree View program (19). The sequence data of SW strains (MM251, MM239, MM3l6 and 32H Pj5) were obtained from GenBank (7)(8)(9)22).…”

Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum.

“…To date, the most potent protection has been obtained with live attenuated vaccines. The genetic disruption of the nef gene significantly restricts the replication of SIV in macaques and prevents the induction of disease (Kestler et al, 1991 ;Rud et al, 1994). Chronic infection with attenuated isolates of SIVmac can confer complete protection against superinfection with pathogenic SIVmac (Daniel et al, 1992).…”