Role of envelope/GP120 in disease progression: Studies in the SIV-Macaque model and the impact of HIV-1 replicative fitness on neuropathogenesis

The AIDS pandemic continues to expand and has become a major public health challenge of modern times. Following human immunodeficiency virus (HIV-1) infection all individuals do not progress to full-blown AIDS equally. It is crucial to differentiate between rapid and slow progressors so that the rapid progressors could benefit from aggressive antiretroviral therapy (ART) early on, and the slow progressors could avoid its undue side effects. The differences in rate of disease progression may be linked to biological characteristics of the evolving viruses. One such characteristic that may influence disease progression is the replicative fitness of the dominant HIV-1 variants. Conducting longitudinal studies to investigate the rate of HIV-1 disease progression among humans poses serious ethical issues mainly due to the potential of withholding ART that could have been beneficial to an infected participant enrolled in the study.; Experimental simian immunodeficiency virus (SIV) infection in Macaca mulatta (macaques) is an excellent model to study dynamics of disease progression and analyze the characteristics of evolving variants that may contribute to their replicative fitness. To investigate these biological properties in SIV-macaque model, we evaluated viral growth kinetics of 30 SIV isolates obtained from 7 rapid- and 8 slow progressors, at two different time points during disease progression after inoculation with a reference SIV strain (SIVmac239). This enabled us to initially estimate the replication fitness of the macaque derived SIV isolates and classify the animals as slow- or rapid progressors (Chapter V). In the next phase, we analyzed complete env sequences, from different stages of the disease, for mutations which could have contributed to the replicative fitness of evolving SIV variants (Chapter VI).; To examine the possible phenotypes that could have resulted from the evolutionary changes in the envelope region of slow- and rapid-progressors, and to determine accurate viral fitness values, two SIV-Deltaenv recombinant viruses tagged with EGFP or DsRed2 were used to clone the complete env gene from macaque-derived SIV isolates (Chapter III & VII). Viruses generated by the recombinant clones were used in dual growth competition experiments between the reference SIVmac239 and envelope-recombinant viruses, each tagged with a different fluorescence marker (Chapter VII).; Compartmental evolution is another feature of SIV-macaque model and HIV-1 infection in humans. The central nervous system (CNS) provides an ideal niche for independent evolution of HIV-1 variants that may contribute to neuropathogenesis of HIV-1-associated dementia (HAD)/NeuroAIDS. To extend and apply our animal study to humans, we recovered HIV-1 gp120 sequences from different regions of the brain, and blood of autopsied patients. In addition to sequence analysis, recombinant viruses constructed from these sequences were evaluated for their replicative fitness in CD4.CCR5-expressing cell line, and human peripheral blood mononuclear cells (PBMC). We also explored the potential mechanisms that may underlie the variation in replicative fitness of the viruses isolated from different compartments.; In summary, we have shown that in the SIV-macaque model, viruses isolated early on, differ in their replicative fitness from those isolated at the late stages of the disease. In brief, viruses in rapid progressor macaques gain fitness and there is loss of fitness among the isolates from slow progressors. These findings correlate with envelope evolution, being more evident in slow progressors than in rapid progressors. Further corroboration of these findings became evident in dual growth competition experiments conducted with env-recombinant viruses (Chapter VII). In preparation of the groundwork for these experiments we report the successful engineering of novel multi-use fluorescent-tagged SIV (Chapter III). These constructs express all of their genes along...