Limitation of Tat-Associated Transcriptional Processivity in HIV-Infected PBMC

Abstract

The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage depends on the function of the viral transcriptional regulatory protein, Tat, which increases the efficiency of RNA elongation (transcriptional processivity) in response to cellular activation. To quantify levels of Tat function in vivo, a quantitative competitive RT-PCR assay was developed that reflects levels of TAR leader fragments (nonprocessive transcripts) and viral mRNA (processive transcripts), indicating low or high levels of Tat function, respectively. The abundance of these RNA species was measured in peripheral blood mononuclear cells (PBMC) of 22 HIV-1-positive individuals (CD4(+) T cell counts 63-934/mm3) and in established cell line models of HIV constitutive replication (H9IIIB) and reversible latency (U1 and ACH-2). In PBMC, the level of total viral transcripts ranged over four orders of magnitude; however, nonprocessive transcription predominated: 70% of PBMC samples had a ratio of processive to total transcripts of <0.3 and none of the samples had 100% processivity. The cell line studies revealed that, even in activated H9IIIB cells, nonprocessive transcription dominates and that latently infected cells can have different transcriptional responses to activation. This is the first study that enumerates degrees of transcriptional processivity in the circulating mononuclear cell compartment and the results suggest that limitation of Tat function may be a common phenotype throughout the course of the disease.

Analysis of Human Immunodeficiency Virus Type 1 Transcriptional Elongation in Resting CD4+T Cells In Vivo

“…Our results are consistent with previous findings from other groups who analyzed RNA in unfractionated PBMCs from viremic patients (1,2). The preponderance of short or nonprocessive transcripts in resting CD4 ϩ T cells compared to full-length transcripts supports the notion that suboptimal conditions for transcription elongation contribute to latency in resting CD4…”

“…Studies in cell lines and unfractionated peripheral blood mononuclear cells (PBMCs) from infected individuals have shown that the HIV-1 long terminal repeat produces two distinct populations of transcripts, a set of short, prematurely terminated transcripts of about 60 nucleotides and a set of full-length mRNAs (1,2,42,48,58,59,70). In the absence of Tat, the short abortive transcripts predominate, whereas in the presence of Tat, there is a dramatic increase in the levels of long transcripts.…”

Loss of the Brm-Type SWI/SNF Chromatin Remodeling Complex Is a Strong Barrier to the Tat-Independent Transcriptional Elongation of Human Immunodeficiency Virus Type 1 Transcripts

“…Hence, latent infection would need to have been established in resting memory T cells originating from activated T cells that were infected with HIV-1 during the process of their inactivation. Whereas latently infected cells produce little HIV-1 proviral mRNA in patients with HAART (2,3,7,12), it was previously reported that abortive transcripts of about 60 nt are detectable in resting T cells from AIDS patients undergoing HAART (14). Interestingly, we show from our present data that Brm and some other components of the SWI/SNF complex, which are marginal in resting CD4 ϩ T cells, are transiently induced when they are activated by CD3 and CD28 costimulation (Fig.…”

Assays for precise quantification of total (including short) and elongated HIV-1 transcripts

“…1). Because of their extensive internal base pairing, these abortive transcripts are thought to be partially protected from degradation when released from the transcriptional machinery, and their accumulation has been reported in cell line models (Kao et al, 1987) as well as in peripheral blood lymphocytes of HIV-1 infected individuals on ART (Adams et al, 1994; Adams et al, 1999; Lassen et al, 2004). In the context of HIV cure research and the quest for HIV latency-reversing drugs, there is a compelling need to develop techniques that can accurately quantify both short and processive transcripts in clinical samples, since their relative levels can be used to infer the levels of HIV transcriptional initiation and inhibition of elongation.…”

“…Semi-quantitative detection by end-point reverse transcription (RT) polymerase chain reaction (RT-PCR) was used in the seminal papers (Adams et al, 1994; Adams et al, 1999). The subsequent development of more accurate PCR-based quantification techniques, such as real-time PCR (qPCR) and droplet digital PCR (ddPCR), may allow for more reliable quantification of HIV short abortive transcripts.…”