Background:  HIV-1 utilizes a network of host vacuolar sorting proteins to bud from the infected cell.  Viral budding requires interactions between the gag protein p6 and several cellular proteins, Tsg101 and AIP1, mediated by specific amino acid motifs in p6, PT/SAP and LYP, LRSL, respectively.  The Replication Capacity Assay  measures the ability of recombinant HIV-1 containing the C-terminal 83 amino acids of gag, all of protease, and the first 305 amino acids of reverse transcriptase of virus isolated from the plasma of HIV-infected patients, to replicate in the absence of antiretroviral inhibitors, and has been shown to be predictive of CD4 decline.  Wild type  populations of HIV-1 have broad distributions of replication capacity values.  The source of this variation is not well understood.  We examined the relationship between mutations in gag and replication capacity in subtype B, wild type HIV-1.

Methods:  Utilizing the virologic phenotype-genotype database (about 17,000), we selected wild type viruses for which replication capacity data were available (1063). We further selected viruses that had replication capacity values less than the 10th percentile (n = 64), greater than the 90th percentile (n = 80), and close to the median (97.4% +1.4%, n = 24) of the replication capacity distribution for those wild type viruses. We determined the Gag sequences and performed univariate analyses (Fisher’s exact test) to look for specific mutations in gag or protease that were associated with either high or low replication capacity. 

Results:  Mutations in gag that were significantly associated with high replication capacity values included insertions at 458 and mutations at 418, 470, and 473. Gag mutations significantly associated with low replication capacity values included those at positions 429, 483, and 484.  Of note, insertions at 458 represent duplications or partial duplications of the PT/SAP motif, effectively lengthening the Tsg101 binding site. Mutations at 483 and 484 occur at the LYP motif that is required for p6 binding to AIP1.  Additional analyses revealed correlations between the appearance of these mutations and clusters of mutations elsewhere in gag.

Conclusions:  The observed variability in replication capacity in wild type  populations of HIV can be explained in part by gag mutations that occur at specific sites that serve critical roles in binding to cellular proteins required for viral budding. Mutations in gag that are associated with low replication capacity may suggest other potential sites for interaction with cellular machinery. Clustered gag mutations may represent compensatory changes that enhance viral fitness.

Keywords: replication capacity; p6 gag; TSG101