Background: Short, unique, persistent, in-frame deletions have been previously detected within p6gag sequences obtained from 3 HIV-1-infected long-term nonprogressors (LTNPs). These deleted residues are not known to be involved in late domain activity of p6gag or in vpr binding.

Methods: Using PCR mutagenesis, we created 11 mutants with serial, 2 amino acid deletions within p6gag of SIVmac239 downstream of the late domain, to investigate whether the loss of any 2 amino acids can result in viral attenuation. The first 8 mutants had deletions within residues 19–28 and the last 3 were within residues 47–51. The late domain, PTAP, is located upstream of these deletions within residues 11–14. Replication was evaluated by measurement of p27gag by standard antigen capture following DEAE transfection or infection of various cell lines. Viral protein was obtained from virus pelleted from transfected 293T cell supernatants and analyzed by Western blot.

Results: Nine (9) of the 11 mutants were replication competent with only a slight initial delay or slightly lower peak height than the wild type (WT) virus. Two (2) of the mutants, 1 with a VD deletion at residues 20 and 21 (dVD) and 1 with a DL deletion at residues 21 and 22 (dDL), did not replicate. Capsid antigen was just above the level of detection within the supernatants of 293T cells transfected with dVD. Western blot analysis demonstrated a barely discernible band migrating at 27kD. We were unable to detect any capsid antigen within supernatants of 293T cells transfected with dDL by either antigen capture or Western blot. We substituted the D for an A within the WT background at residue 21 to evaluate the need for a negative charge. This substitution resulted in a fully replication competent virus. We have also created 8 serial, overlapping, 2 amino acid deletion mutants beginning 3 residues downstream of PTAP within HIV-1, including 2 of the LTNP deletions. Only 1 of 8 HIV-1 mutants (dES with a deletion at residues19 and 20) exhibited a marked delay in replication kinetics.

Conclusions: Our findings indicate that SIV and HIV are remarkably tolerant to deletions within p6gag downstream of the late domain. The replication defect in dVD is likely a result of increased gag instability. Studies in monkeys will be needed to determine whether such deletions in p6gag can result in an attenuated phenotype in the absence of an obvious growth defect in standard cell culture systems.