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 human immunodeficiency virus (HIV), donor cells from HIV seropositive patients 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 sensitivity 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 reaction. The present study takes advantage of the ability of NaB to introduce changes in chromatin structure of latently infected cells, leading to increased HIV gene expression. Human ACH-2 and U1 cell lines were used as representatives of T-lymphocytic and monocytoid cells harboring latent inducible proviruses. HIV gene expression was readily detected when these cells were treated with NaB. Viral gag RNA was detected by both in situ and RT-PCR assays. When peripheral blood mononuclear cells (PBMCs) from acquired immunodeficiency syndrome (AIDS) patients, who were all negative for in situ hybridization and serum/plasma p24 assays, were used for detection of viral gene expression, four categories with distinct patterns of induction were observed. The first set of patients showed HIV-positive PBMCs by RT-PCR without any added NaB, and suppression by added NaB or PHA. The second set of samples showed 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 for induction of HIV gene expression. Our results suggest that direct treatment of the cells with HIV activators may be useful in increasing sensitivity of the RT-PCR intended to be used for detection of cell-associated viral RNAs. This approach may be used to confirm true status of the HIV infection when p24 results are negative or HIV RNAs in serum/plasma are below the threshold of detection. Moreover, this method may identify the presence of latent proviral genomes possibly reflecting the true rate of cell-associated viral load in vivo and without possible mutations brought about by long-term co-cultivation assays with cells from seronegative donors.
Kashanchi
F
F
Laboratory of Molecular Virology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Melpolder
J C
JC
Epstein
J S
JS
Sadaie
M R
MR
D002087:Butyrates / Q000494:pharmacology*; D020148:Butyric Acid; D004396:Coloring Agents; D004996:Ethidium / Q000737:chemistry; D015683:Gene Products, gag / Q000235:genetics; D006679:HIV Seropositivity / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000821:virology; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014775:Virus Activation / Q000187:drug effects*; D017735:Virus Latency
D002087:Butyrates; D004396:Coloring Agents; D015683:Gene Products, gag; D012367:RNA, Viral; D020148:Butyric Acid; D004996:Ethidium