The long term goal of this proposal is to better understand the non-coding region of HIV so as to shed light on the contribution of this region to reverse transcription, viral gene expression, protein synthesis and viral replication. Deletion mutagenesis in this region may also help in the development of an attenuated HIV vaccine, and extensive research using the SIV model will be conducted to determine whether this may be the case. This research will provide important new information on a relatively poorly understood area of the HIV genome. Among the questions to answer are the following: 1. Whether a deletion of the 54 nt segment downstream of the primer binding site will permanently attenuate HIV replication in tissue culture, and to understand the molecular basis for reversions that involve a series of smaller deletions within the 54 nt region. To determine whether a deletion mutation at the 3' end of the 54 nt region may also compromise HIV replication capacity and interfere with the production of viral DNA, mRNA, and proteins to the same extent as the 54 nt deletion. 2. To determine whether a similar deletion in simian immunodeficiency virus (SIV), to that of the 54 nt deletion in HIV, will also impact severely on viral replication, generation of viral DNA, synthesis of viral mRNA, and production of viral protein. 3. Whether combining the 54 nt deletion with the 184V substitution in RT, previously shown to increase RT fidelity, may diminish the likelihood of reversion taking place. Also, what will the effect be of combining the 184V substitution in RT with deletion of the A-rich loop on the rapidity of reversion? 4. To determine whether combination of the large and small deletion mutations in SIV, together with the 184V substitution in RT that increases fidelity, will likewise result in a lesser likelihood of reversion to wild-type virus over extended periods. 5. To determine whether SIV variants, containing large deletions in the non-coding region, with or without the 184V substitution in RT, may serve as attenuated vaccines for purposes of stimulating anti-SIV humoral and cellular immune responsiveness in macaques. 6. To determine whether these attenuated SIV constructs may protect against subsequent challenge with virulent forms of SIV and SIV/HIV hybrids.
