Novel, live attenuated simian immunodeficiency virus constructs containingmajor deletions in leader RNA sequences

We have constructed a series of simian immunodeficiency virus (SIV) mutants containing deletions within a 97-nucleotide (nt) region of the leader sequ ence. Deletions in this region markedly decreased the replication capacity in tissue culture, i.e., in both the C8166 and CEMx174 cell lines, as well as in rhesus macaque peripheral blood mononuclear cells. In addition, these deletions adversely affected the packaging of viral gnomic RNA into virion s, the processing of Gag precursor proteins, and patterns of viral proteins in virions, as assessed by biochemical labeling and polyacrylamide gel ele ctrophoresis. Different levels of attenuation were achieved by varying the size and position of deletions within this 97-nt region, and among a series of constructs that were generated, it was possible to rank in vitro virule nce relative to that of wild-type virus. In all of these cases, the most se vere impact on viral replication was observed when the deletions that were made were located at the 3' rather than 5' end of the leader region. The po tential of viral reversion over protracted periods was investigated by repe ated viral passage in CEMx174 cells. The results showed that several of the se constructs showed no signs of reversion after more than 6 months in tiss ue culture. Thus, a series of novel, attenuated SIV constructs have been de veloped that are significantly impaired in replication capacity yet retain all viral genes. One of these viruses, termed SD4, may be appropriate for s tudy with rhesus macaques, in order to determine whether reversions will oc cur in vivo and to further study this virus as a candidate for attenuated v accination.