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. 2016 Jan;97(1):220-224.
doi: 10.1099/jgv.0.000326. Epub 2015 Oct 23.

The CD8+ Cell Non-Cytotoxic Antiviral Response Affects RNA Polymerase II-mediated Human Immunodeficiency Virus Transcription in Infected CD4+ Cells

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The CD8+ Cell Non-Cytotoxic Antiviral Response Affects RNA Polymerase II-mediated Human Immunodeficiency Virus Transcription in Infected CD4+ Cells

Dalibor Blazek et al. J Gen Virol. .
Free PMC article

Abstract

A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.

Figures

Fig. 1.
Fig. 1.
CAF inhibits transcription from the HIV G6LTRCAT reporter gene. (a) Depiction of the HIV G6LTRCAT reporter assay. Six Gal-binding sites (dark green rectangles) are bound by six molecules of Gal-CyclinT1 (Gal-CycT1; light green ovals). RNAPII (yellow oval) binds the HIV-LTR promoter (black rectangle). Active transcription results in CAT reporter gene (pink rectangle) expression. Gal-CycT1/Cdk9 phosphorylates the C-terminal domain of RNAPII (light green arrow) which results in the productive transcription of the CAT reporter gene (yellow arrow). (b) Jurkat T-cells described in (a) were transfected with either the Gal-empty vector or Gal-CycT1. The cells were also incubated for 48 h with CAF or control fluids from CNAR-positive and negative subjects, respectively. The grey bars correspond to relative CAT activity. The line bars represent mean and sd for two independent studies.
Fig. 2.
Fig. 2.
CD4+ cells infected with a single-round vesicular stomatitis virus (VSV)-HIV-GFP virus have less RNAPII on the HIV promoter, and the viral Gag and Tat coding regions, upon treatment with CD8+ cells from HIV-infected subjects with CNAR. (a) Location of primers used in ChIP assay on the HIV genome. Positions of the ChIP primers are indicated by red squares. The numbers above red squares represent the nucleotide positions of individual ChIP primers. Green rectangles represent individual HIV genes and numbers 1 and 9719 represent the first and final nucleotides of the HIV genome, respectively. (b) CD4+ cells in a CNAR assay result in lower recruitment of RNAPII to the HIV-LTR promoter and the HIV genome. ChIP analyses for the presence of RNAPII on the regions of the HIV genome (LTR, gag, tat) are shown. The amount of RNAPII on the LTR promoter and gag and tat genes in the control experiment was set at 100 %. The IgG corresponds to the empty bead control. The results of the study come from two independent experiments and quantitative PCR (qPCR) was performed in triplicate for each experiment. CNAR denotes results with CD8+ cells from CNAR-positive subjects; CTRL denotes results with CD8+ cells from normal uninfected subjects. The line bars represent mean and SD for two independent studies.

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