Unperturbed posttranscriptional regulatory Rev protein function and HIV-1 replication in astrocytes

Abstract

Astrocytes protect neurons, but also evoke proinflammatory responses to injury and viral infections, including HIV. There is a prevailing notion that HIV-1 Rev protein function in astrocytes is perturbed, leading to restricted viral replication. In earlier studies, our finding of restricted viral entry into astrocytes led us to investigate whether there are any intracellular restrictions, including crippled Rev function, in astrocytes. Despite barely detectable levels of DDX3 (Rev-supporting RNA helicase) and TRBP (anti-PKR) in primary astrocytes compared to astrocytic cells, Rev function was unperturbed in wild-type, but not DDX3-ablated astrocytes. As in permissive cells, after HIV-1 entry bypass in astrocytes, viral-encoded Tat and Rev proteins had robust regulatory activities, leading to efficient viral replication. Productive HIV-1 infection in astrocytes persisted for several weeks. Our findings on HIV-1 entry bypass in astrocytes demonstrated that the intracellular environment is conducive to viral replication and that Tat and Rev functions are unperturbed.

Figure 1. HIV-1 infection in astrocytes.

HIV-1 NL4-3 or NLENY1 infection was done in lymphocytes (Jurkat), primary human fetal astrocytes (HFA), and SVGA cells. (A) Diagrammatic view of HIV-1 viruses, NL4-3 (T-tropic) wild type, and recombinant NLENY1 genetically engineered NL4-3 showing YFP gene insertion. (B) SVGA or Jurkat cells in 6-well culture plates were infected with NLENY1 or NL4-3 virus. After 6–18 days of infection, cells were fixed and directly examined for green fluorescence or immunostained for viral p24 (Fig. 1B). Mock-infected SVGA, HFA and lymphocytes served as negative controls for p24 antibody. (C) HFA in 60-mm dishes were infected with HIV-1 NL4-3 or NLENY1 for 8–28 days, then immunostained for p24 and GFAP, then examined by UV microscope. Upper panels show NL4-3-infected GFAP-positive astrocytes 8 days after infection; middle panels show NLENY1 infection in HFA on 28 days; bottom panels show mock-infected GFAP-positive HFA as control.

Figure 2. HIV-1 Tat protein expression and its biological activity in astrocytes.

(A) Jurkat or SVGA were cultured in 6-well culture plates. In one set, cultures were infected with NL4-3 and assessed for Tat expression. HIV-1 NL4-3-infected Jurkat cells at 6 days after infection, pNL4-3 or CMV-Tat expression vector transfected SVGA were immunostained for Tat. In immunostaining, uninfected Jurkat or empty-vector transfected SVGA served as controls for Tat antibody. HFA after 48 h of transfection with Tat-GFP expression vector, were observed by fluorescent microscopy. (B) For Tat-mediated HIV-1 LTR transactivation in astrocytes, SVGA reporter cells (LTR-GFP or LTR-RFP) were transfected with Tat expression vector or HFA were cotransfected with Tat-expressing and LTR-GFP vectors. At 48 h after transfection, HFA were immunostained for GFAP (red); fluorescence was observed by UV microscope.

Figure 3. Tat-mediated HIV-1 LTR transcriptional regulation in astrocytes.

(A) HIV-1 wild-type or mutant infectious molecular clones and Tat expression vector are shown (Fig. 3A inset). SVGA-LTR-GFP (stable) cells were transfected with 1.0 µg of HIV-1 YU-2 molecular clone, pMtat(-), Tat vector, or pcDNA empty vector. LTR-GFP fluorescence was monitored 48 h later by UV microscope or read in a fluorimeter. (B) Stable HeLa or SVGA cells expressing minimal levels of Tat (undetectable by immunostaining) were transfected with 0.5 µg of LTRGFP plasmid. SVGA-pcDNA (stable) cells transfected with LTR-GFP vector served as controls. HIV-LTR activity in SVGA-Tat was comparable to that in HeLa-Tat cells (p≤0.31; n = 2). (C) Stable SVGA reporter cells (LTR-GFP or LTR-RFP) were infected with VSV-NLENY1 or transfected with HIV-1 YU-2 infectious molecular clone or HIV-1 infectious molecular clone mutated for tat gene pMtat(-). Red (Tat-mediated) and green fluorescence (HIV-1 infection) was observed 48 h later by UV microscope.

Figure 4. HIV-1 Rev protein expression and its biological activity in astrocytes.

(A) SVGA cells were seeded overnight in 6-well culture plates, then transfected with Rev-BFP expression vector or empty vector (control). After 24 h, cultures were fixed and their nuclei stained with propidium iodide. Fluorescence was observed by UV microscope. (B) Rev- and Tat-responsive reporters and their regulation by Rev and Tat. Stable single and dual reporter expression vectors are shown. SVGA-LTR-gag-GFPRRE/LTR-RFP cells were seeded overnight in 6-well culture plates, then transfected with Tat, Rev, dTat (control), or Tat+Rev expression vector (pCV1); 24 h later, Tat- and Rev-mediated green and red fluorescence signals were observed by UV microscope.

Figure 5. Posttranscriptional regulatory function of HIV-1 Rev in astrocytes.

(A) Diagram of Tat- and Rev-responsive or Tat-independent but Rev-dependent reporter vectors and their functioning. (B) SVGA-LTR-gag-GFPRRE cells seeded overnight in 6-well culture plates were transfected in duplicate with either Tat, Rev, Tat+Rev, dTat, pcDNA (control), or dTat +Rev vectors. GFP fluorescence was captured 48 h later using a digital camera. (C) HFA were seeded overnight in 6-well culture plates. The next day, HFA were co-transfected in duplicate with HIV-1 LTR gagGFPRRE and either Tat, Rev, Tat+Rev, dTat+Rev, dTat, or pcDNA (control). Astrocytes were fixed 48 h later and immunostained for GFAP. (D) SVGA or (E) HFA seeded in 6-well culture plates were co-transfected in duplicate with CMV-gagGFPRRE and either Tat, Rev, Tat+Rev, dTat, pcDNA, or dTat+Rev. GFP fluorescence was captured 48 h later.

Figure 6. HIV Rev predominantly regulates the posttranscriptional viral life gene expression in astrocytes.

SVGA seeded in 6-well culture plates were transfected in duplicate with full length HIV-1 infectious DNA clone, HIV DNA mutated for Rev pMrev(-), HIV DNA mutated for Tat pMtat(-) or pcDNA vector as control. Cells were fixed 48 h later and immunostained for HIV gag (p24) and nuclei. The immunostained cells were photographed (A) and counted (B) for p24 positive cells in ten random fields under low power field (LPF) in duplicate sets. Means were plotted and p values calculated using t test (p<0.004) (n = 3). (C) HIV-1 LTR-gagGFPRRE activity in SVGA and Hela cells: Stable SVGA-LTRgagGFPRRE and Hela-LTRgagGFPRRE cells were seeded overnight in 12-well culture plates. On the next day, reporter cells were transfected with Tat−Rev, Rev, or control pcDNA vector. Leptomycin B (10 nM) was used as a positive control to inhibit Rev function. At 72 h after transfection, GFP-positive cells were counted in tests and controls in duplicate sets, then plotted (p<0.0007; p<0.0005) (n = 2).

Figure 7. RNA helicase DDX3 regulates HIV-1 Rev function in astrocytes.

(A–B) Astrocytes (HFA and SVGA) and HIV-1-permissive cells (Jurkat, HEK, and HeLa) were cultured in 6-well culture plates, and 24 h later, lysed and processed for Western blotting. Natural expression levels of RNA helicases (DDX1, DDX3, and RHA), HAX-1, Sam68, PKR, and TRBP in SVGA and HFA are shown for comparison with HIV-1-permissive cells. (C) SVGA were cultured in 6-well culture plates and, 24 h later, transfected with DDX3-HA expression vector or control vector. After 48 h, immunostaining for DDX3 was done in transiently transfected astrocytes using HA tag antibody (inset). In another set, SVGA-LTR-gagGFP cells were seeded in 12-well culture plates and transfected with Tat+Rev, Tat+Rev+antisense-DDX3 (AS), or pcDNA vector (control). Leptomycin-B was used a positive control for inhibition of Rev function. (D) Ectopic DDX3 expression partially reinstated AS-ablated Rev function. Cultured SVGA-LTR-gagGFP reporter cells in 12-well plates were transfected with Tat+Rev expression vector alone or in combination with DDX3, AS, or mutant DDX3 (d-DDX3, functionally dead mutant). In parallel, reporter cells were transfected with control empty vector. Leptomycin B was used as a positive control for inhibition of Rev function. At 30 h after transfection, GFP-positive cells were counted in 10 random fields in duplicate sets and plotted. (E) HeLa and SVGA cells in duplicate were transfected with HIV-1 NL4-3 infectious molecular clone (pNL4-3) alone or in combination with AS or d-DDX3 expression vectors. In parallel, cells were transfected with empty vector (control). At 48 h after transfection, p24 in supernatants was monitored in triplicate by ELISA (p<0.004 and p<0.009) (n = 2).

Figure 8. HIV-1 entry bypass in astrocytes and Rev function.

(A) SVGA-LTR-GFP and SVGA-LTR-gagGFP reporter cells were seeded in 6-well culture plates. Reporter cells were infected with VSV-NL4-3. At 48 h, infected cells were fixed and immunostained for p24. Uninfected cells served as controls for p24 antibody. Immunostained specimens were observed by UV microscope and photographed. (B) HFA in flaskets were infected with VSV-NL4-3 and 48 h later immunostained for p24 and GFAP. Uninfected HFA served as a control for p24 antibody. After immunostaining, nuclei were stained with DAPI and examined by UV microscope. (C) HFA cultured overnight in 6-well culture plates were left untreated or treated with Dynasore. HFA cultures were infected with VSV-NL4-3 or NL4-3 virus. In parallel, uninfected cultures were used as controls. HFA cultures were followed for 20 days, after which the supernatants were analyzed for p24 (ELISA), normalized with normal controls, and plotted (p<0.05; p<0.05) (n = 3).

Figure 9. HIV-1 entry bypass in astrocytes leads to persistent infection.

HFA seeded in T-25 flasks were infected with VSV-pseudotyped NLENY1 virus, and, 2 h later, washed and cultured at 37°C. Cultures were followed for 111 days. Every 10 days, cultures were examined for green fluorescence (YFP) in cells; culture supernatants were collected for p24 analysis. (A) YFP (green) expression at 111 days in HIV-1-infected HFA is shown. (B) Viral p24 levels were analyzed after normalization with uninfected controls and plotted (n = 2).