RAPID AND SENSITIVE DETECTION OF CELL-ASSOCIATED HIV-1 IN LATENTLY INFECTED CELL-LINES AND IN PATIENT CELLS USING SODIUM-N-BUTYRATE INDUCTION AND RT-PCR

To develop a rapid and sensitive means of detecting cell-associated hu man immunodeficiency virus (HIV), donor cells from HIV seropositive pa tients were treated with the potent viral activator sodium-n-butyrate (NaB) and subsequently assayed by both in situ RNA hybridization and a reverse transcriptase polymerase chain reaction (RT-PCR). The sensiti vity of RT-PCR was estimated to be equivalent to 1 x 10(-16) grams (0. 1 fg) or approximately 64 copies of the input standard viral RNA per r eaction. The present study takes advantage of the ability of NaB to in troduce changes in chromatin structure of latently infected cells, lea ding to increased HIV gene expression. Human ACH-2 and U1 cell lines w ere used as representatives of T-lymphocytic and monocytoid cells harb oring latent inducible proviruses. HIV gene expression was readily det ected when these cells were treated with NaB. Viral gag RNA was detect ed by both in situ and RT-PCR assays. When peripheral blood mononuclea r cells (PBMCs) from acquired immunodeficiency syndrome (AIDS) patient s, who were all negative for in situ hybridization and serum/ plasma p 24 assays, were used for detection of viral gene expression, four cate gories with distinct patterns of induction were observed. The first se t of patients showed HIV-positive PBMCs by RT-PCR without any added Na B, and suppression by added NaB or PHA. The second set of samples show ed induction of viral RNA by NaB alone. The third set could be induced with PHA, but not NaB, and the fourth set required both NaB and PHA f or induction of HIV gene expression. Our results suggest that direct t reatment of the cells with HIV activators may be useful in increasing sensitivity of the RT-PCR intended to be used for detection of cell-as sociated viral RNAs. This approach may be used to confirm true status of the HIV infection when p24 results are negative or HIV RNAs in seru m/plasma are below the threshold of detection. Moreover, this method m ay identify the presence of latent proviral genomes possibly reflectin g the true rate of cell-associated viral load in vivo and without poss ible mutations brought about by long-term co-cultivation assays with c ells from seronegative donors. (C) 1997 Wiley-Liss, Inc.