Early transcription from nonintegrated DNA in human immunodeficiency virus infection - PubMed (original) (raw)

Early transcription from nonintegrated DNA in human immunodeficiency virus infection

Yuntao Wu et al. J Virol. 2003 Oct.

Abstract

Replication of human immunodeficiency virus (HIV) involves obligatory sequential processes. Following viral entry and reverse transcription, the newly synthesized viral DNA integrates into the host chromatin. Integration is mandatory for viral production, yet HIV infection of CD4 T cells in vivo results in high levels of nonintegrated DNA. The biological potential of nonintegrated HIV DNA is unclear; however, prior work has demonstrated a limited transcription of the nef gene by nonintegrated HIV in infected quiescent T-cell populations. In a kinetic analysis of HIV infection of metabolically active transformed and primary CD4 T cells, we find an unexpected transient expression of both early and late message by nonintegrated HIV DNA. However, only the early multiply spliced transcript was measurably translated. This restriction of protein expression was due in part to inadequate Rev function, since expression of Rev in trans resulted in the expression of the late structural gene gag by nonintegrated HIV DNA.

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Figures

FIG. 1.

FIG. 1.

Viral reverse transcription and integration in wild-type- and D116N mutant-infected cells. CEM-50 cells (106) were infected with equivalent levels of wild-type (Wt) HIV-1NL4-3 or an integrase mutant, HIV-1IN/D116N (D116N). Cells were harvested at different times postinfection, and nuclear DNA was purified. (A) Total cellular DNA was amplified by PCR as previously described (34). The sample immediately following the addition of virus was defined as the time zero sample. (B) The 1-LTR circle was also measured by PCR amplification as described in Materials and Methods. Both the 1-LTR circle and the 2-LTR circle were amplified by the primer pair as indicated. (C) Real-time PCR. The same DNA used in panels A and B was subjected to real-time PCR quantification using primers targeting the late RT product (filled triangles and circles) and 2-LTR circles (open triangles and circles). (D) Expansion of the 2-LTR circle data from panel C. (E) Viral DNA integration in cells infected with wild-type or D116N mutant HIV. Total cellular DNAs from D116N mutant- or wild-type-infected cells, harvested at different times postinfection (lanes 1 to 8), were subjected to _Alu_-PCR amplification (+Alu) as described in Materials and Methods. To ensure that the amplification was specific to integrated viral DNA, amplification was also carried out in the absence of the Alu primer (−Alu). The PCR products were analyzed by gel electrophoresis, transferred to a nylon membrane, and hybridized with a digoxigenin-labeled probe. (F) Plot of amplification signals as captured on a cooled CCD camera. Each experiment was reproduced three or more times.

FIG. 2.

FIG. 2.

Early HIV transcription is similar for CEM cells infected by wild-type (wt) or nonintegrating D116N mutant HIV. (A) Comparison of viral transcription at 12 h postinfection. mRNAs from 104 infected cells were harvested. For each sample, mRNA was purified, treated with DNase I, serially diluted (1:5), and reverse transcribed using random decamers. One-fourth of the cDNA was subjected to PCR amplification using the primer pair F2-B4 as described in Materials and Methods. To ensure that comparisons were quantitative, samples were normalized by the content of the coamplified cellular β-actin transcript. One-fifth of the product was applied to a 2% agarose gel, transferred to a nylon membrane, and hybridized with digoxigenin-labeled probes specific for tat, tat-rev (rev panel), tat-rev-nef (nef panel), or human β-actin. The same cDNA was further amplified by the primer pair F2-B3, F2-B2, or F2-B1 to detect singly spliced env and vif viral transcripts or unspliced gag-pol transcripts. Amplified products were resolved on a 6% polyacrylamide gel and visualized by ethidium bromide staining. (B) Time course analysis of viral transcription in wild-type and D116N mutant infection. Samples taken at each time point following infection were analyzed as for panel A and normalized by the β-actin transcript. To ensure that the amplified unspliced transcripts were not derived from viral DNA, mRNA was also directly subjected to amplification using F2-B1 without reverse transcription (panel −RT).

FIG. 3.

FIG. 3.

Integrase-defective D116N HIV does not replicate in CEM cells. Cells were infected with either wild-type (Wt) or D116N mutant HIV at equal viral loads. Viral replication was monitored by measuring extracellular p24 levels. Data are means of triplicate determinations. Error bars, standard deviations.

FIG. 4.

FIG. 4.

Early viral transcription is resistant to the integrase inhibitor L-708,906. (A) Effect of the integrase inhibitor on viral replication. CEM cells were treated with various amounts of L-708,906 1 h before infection with wild-type (Wt) or D116N mutant HIV. Virus replication was monitored by measuring p24 production in three independent determinations. Values are means and standard deviations. (B) Viral mRNA from the infected cells, harvested at 12 or 72 h, was analyzed by quantitative RT-PCR as in Fig. 2.

FIG. 5.

FIG. 5.

Viral protein synthesis is limited to early products in the absence of integration. CEM cells were infected as in Fig. 1 and were harvested at the indicated time points. A total of 5 × 105 cells were loaded onto an SDS-14% polyacrylamide gel, resolved, and electroblotted onto a nitrocellulose membrane for Western blot analysis (lanes 4 to 14). (A) A human anti-HIV antiserum was incubated with the membrane, followed by a goat anti-human secondary antiserum conjugated with peroxidase. The right panel is a shorter exposure of lanes 12 to 14. (B) Following signal detection, the blot was stripped and reblotted with a sheep anti-Nef antiserum, followed by a rabbit anti-sheep secondary antibody conjugated to peroxidase. (C) As a control, the same blot was also reblotted with a monoclonal antibody against human actin and a goat anti-mouse secondary antibody conjugated to peroxidase. To serve as Nef controls, 0.5 ng of purified HIV-1 Nef protein (lane 1), 5 × 105 Jurkat cells expressing the nef gene of HIV-1NL4-3 (lane 2), and 5 × 105 uninfected CEM cells (lane 3) were applied to the gel. Wt, wild type.

FIG. 6.

FIG. 6.

In the absence of integration, induced Rev expression permits late structural protein synthesis. ΔKS Jurkat cells, which possess a Rev-inducible system based on the tetracycline operon, were infected with wild-type (Wt) virus (lanes 1 and 2) or the D116N mutant (lanes 3 and 4) in the absence (lanes 2 and 3) or presence (lanes 1 and 4) of Rev induction by the addition of tetracycline at the time of infection. Uninfected Jurkat cells were used as a control (lane 5). For Western blot analysis, 5 × 105 cells were loaded onto an SDS-4 to 20% polyacrylamide gel. The blot was probed with a human anti-HIV serum (top panel), as described in the legend to Fig. 5A, and with a rabbit anti-Rev serum reacted with a goat anti-rabbit secondary antibody (bottom panel). p55_gag_ synthesis was detected by Western blotting in D116N mutant infection with Rev induction (lane 4).

FIG. 7.

FIG. 7.

Early HIV transcription from nonintegrated DNA in primary human T cells. Primary human CD4 T cells from peripheral blood of healthy donors were purified, stimulated with anti-CD3/CD28-conjugated beads at a ratio of 5 beads per cell, and then infected with wild-type (Wt) or D116N mutant virus as described in the legend to Fig. 1. mRNA molecules from infected cells were subjected to RT-PCR analyses as described in the legend to Fig. 2. (A) Multiply spliced nef, singly spliced env, and unspliced transcripts. (B) The presence of multiple transcripts in infected cells did not lead to replication of the D116N mutant in stimulated primary T cells, as measured by extracellular p24 production. Data are means for triplicate determinations plus standard deviations. (C) For comparison of transcriptional activity in cells infected with wild-type versus D116N mutant HIV, the ratio of chemiluminescent signals between nef and β-actin was plotted for each time point. These results were reproduced using primary CD4 T cells from multiple donors (data not shown).

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