HIV-1 exhibits numerous strategies to evade host immunity. These strategies include persistence in sanctuary sites, defined as anatomical or privileged cellular sites in which viral replication may continue despite highly active antiretroviral therapy (HAART). The central nervous system (CNS) is an important sanctuary site for HIV-1. Penetration of the CNS by HIV-1 occurs early in infection, and can result in a wide range of pathological and clinical manifestations - all of which contribute significantly to morbidity and mortality. It is therefore important to develop an improved understanding of the adaptive behavior of HIV-1 in the CNS. The overriding goal of the proposed research is to use HIV-1 variants sequenced from cerebrospinal fluid (CSF), to elucidate HIV-1 CNS evolutionary pathways and explore genetic determinants of neuroadaptation through an in-depth, integrated, phylogenetic and functional approach. In order to perform the proposed research, we will use patient samples from the well-characterized CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study. Using standard analysis of clonal sequences and heteroduplex tracking assays (HTA), phylogenetic compartmentalization of contemporaneous intra-patient CNS- and blood-derived viruses has been observed. For this research, we will utilize the single genome amplification (SGA) technique to generate HIV-1 variant sequences. When compared to more conventional sequencing methods, SGA has been shown to decrease taq- induced recombination, template resampling, nucleotide misincorporation and cloning bias and produces a more accurate representation of in-vivo HIV-1 quasispecies. We hypothesize that CNS-specific patterns of evolution observed through longitudinal characterization of HIV-1 envelope (env) and polymerase (pol) reflect attempts by the virus to adapt and persist within the CNS. Furthermore, the use of SGA to generate these variants will provide a more accurate representation of viral quasispecies than currently reported in the literature. Phylogenetic analysis and in-vitro characterization of these variants is expected to reveal novel patterns of CNS-specific evolution and functional consequences of the observed genetic changes.
The specific aims of this proposal are therefore: 1) To determine patterns of CNS compartmentalization of HIV-1 env in a cohort of HAART naive individuals 2) To determine patterns of CNS compartmentalization of antiretroviral drug resistance mutations and effects on the HIV-1 pol and env genes in a cohort of HAART experienced individuals, and 3) To determine the functional consequences of genetic variation in SGA-derived HIV-1 env sequences. Understanding adaptive measures undertaken by HIV-1 to persist in viral reservoirs and sanctuary sites are of paramount importance in defining strategies to improve viral suppression and obtain eradication of HIV-1 in the infected host. To our knowledge, current scientific literature contains no application of the novel SGA method to the characterization of HIV-1 variants in the CNS. Importantly, the successful execution of this proposal will allow me to acquire the technical skill and experience necessary to become an innovative and productive independent investigator in translational HIV-1 pathogenesis research.
Infection of the central nervous system (CNS) by the human immunodeficiency virus-1 (HIV-1) can result in a wide range of pathological and clinical manifestations. The widespread use of highly active antiretroviral therapy (HAART) has led to a clear reduction in the incidence of HIV-associated dementia (HAD), one of the most severe manifestations of HIV-1 CNS infection. Despite this decrease, the prevalence of minor HIV-1 associated cognitive impairment appears to be on the rise. Although more subtle, these impairments can be disabling and have been demonstrated to be independently associated with an increased risk for mortality in those with HIV-1. An improved understanding of HIV-1 neuroadaptation will be important for promoting the development of targeted therapies against HIV-1 in this unique sanctuary site.
