HIV-1 quasispecies heterogeneity mediates Tat function, molecular epistasis, and neuropathogenesis
Authors:   , Spector Cassandra , Nonnemacher Michael R. , Wigdahl Brian 
Published: August 2022

Over 38 million people worldwide continue to live with human immunodeficiency virus type 1 (HIV-1). HIV-1 remains a major public health concern due to chronic infection that cannot be cured by conventional antiretroviral therapies (ART). Infected cells continue to produce and secrete viral proteins, including the transactivator of transcription (Tat), even in viral load suppressed individuals. This predisposes people with HIV (PWH) to increased risk for developing HIV-1-associated comorbidities, such as HIV-1-associated neurocognitive disorder (HAND). The HIV-1 genome is susceptible to genetic diversification due to the error prone viral reverse transcriptase, as well as from adaptation in response to host restriction factors and immune selection pressures. This results in the formation of unique viral quasispecies (vQS) populations in individuals. Genetic variation within vQS may manifest in Tat as functional amino acid (AA) mutations. Tat's canonical function is to bind host factors and the HIV-1 transcription response element (TAR) RNA to enhance viral transcription efficiency, but also participates in pathogenic activities that contribute to overall HIV-1 pathogenesis and comorbidities. Previous studies have sought to characterize functional genetic variation between both Tat exons, which are separated by about 3 kilobases in the HIV-1 genome. However, prior sequencing technologies could not associate observed variants within and between exons from the same molecule of DNA. Therefore, the Pacific Biosciences (PacBio) Sequel platform was utilized to obtain long read sequences encompassing both Tat exons. PBMC gDNA from therapy-controlled participants of the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort was used to isolate tat-containing 4 kilobase amplicons to be sequenced. This sequencing dataset allowed for studies on viral diversity, co-occurring AAs, and functional conservation in Tat. Though deviations from expected occurrence composed a small proportion of observed co-variants, these presented potential variants of interest for future studies. AA functional sequence analyses displayed two patterns of conserved activity split across annotated Tat functions and demonstrated heterogeneity of Tat functional ability between vQS populations. Sequencing data from this study was applied to questions involving Tat's interactions with other viral proteins and its role in neuropathogenesis. Given the observed genetic diversity within tat, further studies investigated selection pressure between tat and rev, another HIV-1 gene that encodes a transcriptional regulatory protein and shares an overlapping reading frame with tat. Selection pressures observed on each overlapping gene segment as well as on individual overlapping codons supported the presence of segregated functional domain organization between the two genes. AA sequence logos of each gene further suggested adaptation of AAs that overlapped with functionally essential AAs in the opposite protein. To address the contribution of Tat AA variation to occurrence of HAND, Tat vQS sequences were associated with paired neuropsychological assessment test data administered to CARES Cohort participants. Neurocognitive phenotypes emerged that were led by dominant deficits in neurocognitive domains. Vectorization and dimensional reduction of Tat sample-specific sequences overlayed with domain deficit scores showed that sequence similarity between participants also associated with neurocognitive impairment in certain domains. Overall, these studies have led to advances in understanding Tat vQS composition and protein function in PWH. The data and results were applied to address questions on pathogenic mechanisms and the development of HIV-1 comorbidities involving Tat.