DEVELOPMENT OF A CELL CULTURE, SCREENING, AND SEQUENCING METHOD FOR HIGH-THROUGHPUT ISOLATION OF HIV-POSITIVE CD4+ T-CELLS FROM HIV-INFECTED PATIENTS

Abstract

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that targets immune cells critical to innate and adaptive immune responses in infected patients. Despite viremia suppression by antiretroviral drug treatments, chronic immune activation persists in HIV-1-infected patients, increasing their risk of HIV-associated chronic comorbidities. Defective HIV-1 proviruses harboring genetic mutations may contribute to persistent immune activation in suppressed HIV-1-infected patients through expression of canonical or novel viral proteins. In this study, we presented the steps taken to develop optimal culture conditions for CD4+ T cells from suppressed HIV-infected patients, and an unbiased nested polymerase chain reaction (PCR) approach for screening of HIV-1 positive cells. Of the 507 potential HIV-1 positive clones detected, we chose three for further bioinformatic analysis that resulted in observation of two novel open reading frames within HIV-1 integrase and reverse transcriptase genes.