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. 2017 Aug 25;7(1):9487.
doi: 10.1038/s41598-017-10272-7.

HIV-1 envelope glycoprotein stimulates viral transcription and increases the infectivity of the progeny virus through the manipulation of cellular machinery

Xiaozhuo Ran  1 , Zhujun Ao  1   2   3 , Adriana Trajtman  2 , Wayne Xu  4 , Gary Kobinger  5 , Yoav Keynan  2 , Xiaojian Yao  6   7   8
Affiliations

HIV-1 envelope glycoprotein stimulates viral transcription and increases the infectivity of the progeny virus through the manipulation of cellular machinery

Xiaozhuo Ran et al. Sci Rep. .

Abstract

During HIV infection, large amounts of progeny viral particles, including infectious virus and a large proportion of defective viral particles, are produced. Despite of the critical role of the infectious viruses in infection and pathogenesis in vivo, whether and how those defective viral particles, especially the virus-associated envelope glycoprotein (vEnv), would impact viral infection remains elusive. In this study, we investigated the effect of vEnv on HIV-infected T cells and demonstrated that the vEnv was able to stimulate HIV transcription in HIV-infected cells, including peripheral blood mononuclear cells (PBMCs) isolated from HIV patients. This vEnv-mediated HIV transcription activation is mediated primarily through the interaction between vEnv and CD4/coreceptors (CCR5 or CXCR4). Through transcriptome analysis, we found that numerous cellular gene products involved in various signaling pathways were modulated by vEnv. Among them, we have further identified a cellular microRNA miR181A2, which is downregulated upon vEnv treatment, resulting in increased HIV LTR histone H3 acetylation and HIV transcription. Furthermore, we also found a vEnv-modulated cellular histone deacetylase, HDAC10, whose downregulation is associated with the increased infectivity of progeny viruses. Altogether, these findings provide evidence of the important role vEnv plays in modulating cellular environments and facilitating HIV expression and infection.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
HIV-1 virus-associated Env (vEnv) activates LTR-driven gene expression. (A) Upper panel: Detection of infectivity of AT-2-treated HIV. Wild-type HIV virus was treated with the indicated concentrations of AT-2 for 1 hour at 37 °C and used to infect C8166 cells for three days. The p24 level in the supernatant was detected by p24 ELISA (n = 2). Lower panel: Luciferase expression in TZMb1 cells infected with AT-2-treated virus for 24 hours (n = 2). (B) Schematic for producing HIV Env-VLP. HIV Gag-pol (Δ8.2) and Env (X4/R5-tropic) plasmids were co-transfected into 293 T cells; after 48 hours, Env-VLP in the supernatant was collected and concentrated by ultracentrifugation. Purified Env-VLPs were used to treat various cells. (C) Upper panel: Western blot confirming the presence of gp120 and p24 of Env-VLP. Lower panel: Luciferase expression in TZMb1 cells treated with Env(X4)-VLP, Env(R5)-VLP or VLP or untreated for 24 hours (n = 3). (D) Upper panel: Luciferase expression in TZMb1 cells treated with varying amounts (0-10 ng) of Env(X4)-VLP (n = 3). Lower panel: Luciferase expression in TZMb1 cells treated with Env(X4)-VLP for different periods (n = 3). (E) Effect of various viral glycoproteins on HIV transcription. Upper panel: Detection of the presence of different viral glycoprotein in HIV VLP by western blotting. Each VLP stock was lysed, and glycoproteins were detected using corresponding antibodies (data in the right panel and the left panel are from two experiments). Lower panel: Luciferase expression was detected in TZMb1 cells treated with Env-VLP, VSVG-VLP, HA-NA-M2, EBOLA-VLP or VLP (without Env) or untreated for 24 hours, and luciferase activity was measured (n = 3). (F) Luciferase comparative transcription (luciferase/GAPDH, n = 3) in TZMb1 cells treated with Env-VLP, VSVG-VLP, VLP (without Env) or untreated (upper panel). Luciferase activity was measured in TZMb1 cells treated with Env-VLP, VSVG-VLP or VLP or untreated cells (n = 2) (Lower panel). Data are the mean and sd. Ns, not significant p > 0.05; *p < 0.05. (two-tailed unpaired t-test; multiple-t test; correction for multiple comparison using the Holm-Sidak method)
Figure 2
Figure 2
Interaction between HIV virus-associated envelope glycoprotein (vEnv) and CD4/coreceptors is essential for HIV transcription activation. (A) Expression of luciferase in TZMb1 cells treated with Env-VLP in presence of different concentrations of an anti-gp120 neutralizing antibody, which targets the interaction region between CD4 and gp120. After 24 hrs, luciferase activity in the TZMb1 cells was detected (n = 3). TZMb1 cells were first cultured in the presence of CCR5 inhibitor TAK779 or CXCR4 inhibitor Bicyclam JM-2987 for 2 hr. Then cells were treated with Env(R5)-VLP (B) or Env(X4)-VLP (C) in the presence of CCR5 inhibitor TAK779 or CXCR4 inhibitor Bicyclam JM-2987. After 24 hours, cells were collected, lysed and used to measure luciferase activity (n = 4). TZMb1 cells treated with Env(X4 or R5)-VLP, shed Env proteins (D), recombinant gp120 (CM) (E), or the recombinant gp120 (Bal) (F). After 24 hours, cells were collected, lysed and used to measure luciferase activity (n = 3). Data are the mean and sd. Ns, not significant p > 0.05 (two-tailed unpaired t-test).
Figure 3
Figure 3
HIV vEnv induced viral transcription in HIV-infected CD4 T cell line (J-Lat 6.3), HIV-infected non-stimulated PBMCs, and latent-infected PBMCs in ART-treated patients. (A) Increased transcription of HIV gag in J-Lat 6.3 T cells treated with Env(X4)-VLP. J-Lat 6.3 T cells were treated or non-treated with Env(X4)-VLP for 24 hrs. HIV gag mRNA was detected by RT-PCR, normalized against the housekeeping gene GAPDH (n = 3), and expressed as comparative transcription level. (B) J-Lat 6.3 T cells were treated with Env(X4)-VLP for 0, 6, and 24 hrs (as indicated) and after 24 hrs, cells were lysed and HIV comparative transcription levels (gag/GAPDH) and the reporter gene GFP comparative transcription levels (GFP/GAPDH) were detected by RT-PCR (left panel). Also, the Env(X4)-VLP treated or untreated J-Lat 6.3 T cells were lysed and the expression of GFP was detected by western blot with corresponding antibody (right panel). (C) HIV comparative transcription levels in HIV-infected resting PBMCs treated with Env(X4)-VLP or untreated (n = 3). PBMCs were isolated from five donors and infected with HIV virus for 24 hrs without stimulation and the infected PBMCs were washed and kept in culture medium for 2 days. Then, cells were treated with Env(X4)-VLP or untreated. Meanwhile, the untreated HIV-infected PBMCs, untreated and uninfected PBMCs or Env(X4)-VLP-treated uninfected PBMCs were used as controls. After 24 hours of treatment, HIV comparative transcription levels (gag/GAPDH) were detected by RT-PCR (left panel). Meanwhile, the infected resting PBMCs treated or untreated with Env-VLP, (as described in left panel) were co-cultured with C8166 T cells for three days. Then, the HIVp24 levels in the supernatant of co-cultures were measured by anti-HIVp24 ELISA (right panel). (D) HIV comparative transcription levels were detected in PHA or anti-CD3/CD28 stimulated HIV-infected PBMCs followed by the treatment with Env-VLP or not (n = 3). (E) HIV comparative transcription levels in non-stimulated PBMCs isolated from seven HIV latent patients, that were treated or non-treated with Env(X4)-VLP for 24 hrs, and then the HIV gag mRNA was detected by RT-PCR (n = 3). Data are the mean and sd. Ns, not significant p > 0.05. (two-tailed unpaired t-test)
Figure 4
Figure 4
Treatment of vEnv(X4) of J-Lat 6.3 T cells induces the transcriptional changes of multiple cellular genes involved in different signaling pathways. (A) Heatmap of genes (p < 0.05) in J-Lat 6.3 T cells untreated (n = 2) or treated with Env(X4)-VLP (n = 3) in technical replicates showing hierarchical clustering. (Lifescope v2.5.1 software from Life Technologies with the 2-mismatch setting). (B) Enrichment of genes modulated by vEnv. The fold enrichment was calculated based on the frequency of genes annotated to the term compared to their frequency in the genome in the DAVID Bioinformatics database.
Figure 5
Figure 5
vEnv regulates the miR181A2/P300/CBP-associated factor (PCAF) expression, and increases HIV LTR histone H3 Acetylation. (A) The miR181A2 mRNA level was reduced in J-Lat 6.3 T cells treated with Env(X4)-VLP for 24 hrs, as compared to the untreated cells (n = 3) (left panel). The reduced miR181A2 mRNA levels in PBMCs from 3 donors treated with Env-VLP, as compared to the untreated cells (n = 2) (right panel). (B) The PCAF mRNA (left panel) and protein levels (right panel) in J-Lat 6.3 T cells treated with Env-VLP or untreated (n = 3). (C) The HIV gag transcription levels in J-Lat 6.3 T cells overexpressed with miR181A2 or miR181A2-3p (inhibitor), as compared to the cells transduced with empty lentiviral vectors (n = 3). J-Lat 6.3 T cells were transduced with lentiviral vectors encoding miR181A2, miR181A2-3p or transduced with empty vector for 24 hrs and kept on culture for another 72 hrs. Then, cells were collected and the HIV comparative transcription (gag/GAPDH) was measured by RT-PCR. (D) The PCAF mRNA (left panel) and protein levels (right panel) in miR181A2 or miR181A2-3p overexpressing J-Lat 6.3 T cells (n = 3). (E) The schematic diagram of the positions of the nucleosomes bound to the HIV-1 LTR and the location of the primers used for the real-time PCR in the ChIP assay. (F) The detection of histone H3 acetylation ratio in HIV LTR -109- + 82 by CHIP (n = 3). J-Lat 6.3 T cells were treated with Env(X4)-VLPs for 24 hrs. In parallel, cells treated with histone deacetylase inhibitor VOR or VPA were included as positive controls and the untreated cells act as a negative control. After treatment, cells were lysed and analyzed by CHIP assay (left panel). Also, the global histone H3 acetylation in J-Lat 6.3 T cells treated with Env(X4)-VLP, VOR, VPA or untreated were detected by western blot with anti-histone H3 acetylated antibody. Data are mean and sd. Ns, not significant p > 0.05. (Two-tailed unpaired t-test).
Figure 6
Figure 6
vEnv downregulates HDAC10 and enhances the infectivity of progeny viruses. (A) The HDAC10 levels in J-Lat 6.3 T cells (left panel) (n = 3) and PBMCs from 3 donors treated with Env(X4)-VLP (right panel) (n = 3) were measured by RT-PCR. Also, HDAC10 protein expressing levels in J-Lat 6.3 T cells treated with Env(X4)-VLP were also detected by western blot with anti-HDAC10 antibody (middle panel). (B) HDAC10 knock-down (KD) enhances HIV viral infection and/or replication. First, the shRNA-mediated HDAC10-KD in Jurkat T cells was checked by detecting HDAC10 expression with antiHDAC10 antibody (left panel). Then, both HDAC10-KD and the control Jurkat T cells were infected by HIV virus (N119) for 12 hrs. Then the infected cells were cultured in the presence or absence of AZT. After 72 hrs, the HIV integrated DNA was quantified by Alu-LTR- nested PCR procedure as described in Methods Section (middle panel), and HIVp24 level in the supernatant was detected by anti-p24 ELISA (right panel). (C) The progeny viruses from HIV infected HDAC10-KD Jurkat cells were more infectious than that from the control Jurkat cells. Equal amounts of progeny viruses (normalized by amounts of p24 levels) produced either from HDAC10-KD or the control Jurkat T cells were used to infect C8166 T cells, CEM-SS T cells, or TZMB1 cells. At different time intervals, the HIV Gag p24 levels in supernatant from infected C8166 T cells (left panel) and CEM-SS T cells (middle panel) were qualified by anti-p24 ELISA. Also, the HIV LTR-driving luciferase activity in TZMb1 cells infected with progeny virus from HDAC10-KD or control cells (n = 2) were detected by luciferase assay. Data are mean and sd. Ns, not significant, p > 0.05; *p < 0.05.(Two-tailed unpaired t-test; Multiple-t test; Correction for multiple comparison used the Holm-sidak method).
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