Genotype analysis of HIV-1 isolated from an individual with long term nonprogressive infection.

The goal of this MQP was to determine whether genetic alterations in HIV-1 sequences from a Long-Term Non-Progressor (LTNP) HIV-1 Patient (LTNP-4) are unique and potentially responsible for the interesting nonprogressive phenotype. Candidate genes and DNA sequences were initially identified by sequence comparisons of HIV-1 between progressors and LTNP that were available in the literature. PCR conditions were developed to successfully amplify portions of vpu, vif, env, pol, and gag genes. Sequences were obtained from cloned amplicons. Interesting point mutations were identified in all genes sequenced. Two positive candidates were tested in rescue experiments. In the first rescue, a plasmid containing a wild type vif fragment was transfected into cell cultures with LTNP-4 virions. This transfection re-established wild type growth of the culture, providing evidence that this vif mutation may represent a key feature of the LTNP-4 phenotype. In the second rescue, a wild type vpu gene was mutated by site-directed mutagenesis to mimic an intriguing LTNP-4 mutation. The results illustrated that this vpu mutation most likely does not contribute to the LTNP-4 status of the patient. Also, an immunofluorescence experiment was performed to determine whether LTNP-4 infected cells contain reduced levels of vif protein as hypothesized. There was no vif staining in the LTNP-4 cells, which suggests that this protein is not being expressed. Ultimately, discovering the mutations responsible for LTNP-4 phenotype is a step in the direction of uncovering new treatment methods for HIV-1