Envelope glycoprotein of HIV induces interference and cytolysis resistance in CD4+ cells: mechanism for persistence in AIDS - PubMed (original) (raw)
Envelope glycoprotein of HIV induces interference and cytolysis resistance in CD4+ cells: mechanism for persistence in AIDS
M Stevenson et al. Cell. 1988.
Abstract
Masking of host cell receptors following retroviral infection is the basis for the phenomenon of virus interference. Amphotropic retrovirus vectors were used to express the HIV envelope glycoprotein in a human CD4+ cell line. Envelope expression is accompanied by a reduction in the level of surface CD4 receptor molecules and correlates with the presence of intracellular envelope-CD4 receptor complexes. Cells expressing the HIV envelope acquire a cytolysis-resistant phenotype such that infection with HIV leads to a non-cytopathic persistent virus infection. Furthermore, phorbol ester-mediated stimulation of viral replication in persistently infected cells results in renewed cytolytic effects which, due to the absence of CD4 in the cell population, are absolutely independent of syncytium formation. This study elucidates the mechanism by which viral persistence is initiated and maintained in the course of AIDS.
Figures
Figure 1.
Construction of Retroviral Vector Containing HIV Envelope Coding Sequences The complete HIV envelope gene from HIV clone N1G-F was inserted into the Sall site of the retrovirus expression vector pDOL, and the cytomegalovirus immediate early promoter was inserted at the BamHI site. Respective regions of the vector are shown to scale (sizes in base pairs in brackets). Boxed areas represent promoter/enhancer sequences and arrows indicate direction of transcription. Symbols: MLV, murine leukemia virus; CMV, cytomegalovirus; B, BamHI; Sm, Smal; Sa, Sall; K, Kpnl; X, Xhol; Xb, Xbal; H, HindIII; Ps, Pstl; ψ, retroviral packaging sequences; p, Pvull; Bg, BgIII; S, Sacl.
Figure 2.
Southern Blot Analysis of Integrated HIV Envelope Sequences in CEM Cells CEM cells were infected with retroviral env and tat expression vectors by cocultivation with DNA transfected amphotrophic packaging cell line PA317 in the presence of polybrene. Infected cells were selected in G418 and analyzed for envelope sequences. a, CEM cells infected with pDOL CMV·env; b, uninfected CEM cells; c, CEM cells infected with pDOL·tat. Genomic DNA was prepared by guanidine thiocyanate lysis, restriction endonuclease digested, electrophoresed, and transferred to nylon membranes. Blots were hybridized under high stringency conditions to a 32P-labeled probe prepared by nick translation of a Sall–Xhol fragment of λ-N1G-F. Molecular weight markers are provided by 32P end-labeled HindIII digests of λDNA.
Figure 3.
Expression of HIV Envelope–Specific RNA and Antigen in CEM Cells (A) Poly (A)+ mRNA from guanidine thiocyanate cell lysates was selected by passage over oligo d-T cellulose columns, spotted onto nylon membranes (2, 1, and 0.5 μg) and hybridized with a β-tubulin probe (Hall et al., 1983) or HIV probe as in Figure 2: a, uninfected CEM cells; b, CEM cells infected with pDOL CMV·env; c, CEM cells infected with pDOL·tat. Blots were autoradiographed for either 3 hr (β-tubulin probe) or 18 hr (HIV probe). (B) Indirect immunofluorescence visualization of HIV envelope in G418 selected cells. Acetone fixed cells were reacted with HIV-positive serum from an HIV seropositive hemophiliac (antibody titer, 1:5160). Bound HIV-specific IgG was detected with FITC conjugated goat anti-human IgG. Uninfected CEM cells were used as a negative control. Photomicrographs were taken on Kodak Pan film (20 sec exposure at 400 ASA). Note syncytium in CEM pDOL·env photomicrograph.
Figure 4.
Reduced Surface CD4 Expression in Envelope-Expressing CEM Cells Immunofluorescence flow cytometry analysis of CD4 expression with OKT4 and OKT4A antibodies. G418 selected cells were incubated with the respective monoclonal antibody (4°C, 30 min) and analyzed on an Ortho model 50H cytofluorograph. The graph displays relative fluorescence intensity versus cell number. Horizontal bar indicates channels analyzed, and mean channel number of cells in this area are indicated on each plot. Background fluorescence (using FITC conjugated second antibody alone) was less than 7%. Plots are representative results of three separate analyses.
Figure 5.
Envelope-Expressing CEM Cells Display Expected T Cell Receptor Gene Rearrangements and Have Abundant CD4 mRNA (A) A, uninfected CEM cells; B, CEM cells infected with pDOL CMV·env; C, CEM cells infected with pDOL·tat. Cellular DNA was blotted and hybridized to a T cell receptor β-chain probe (Yanagi et al., 1984) or CD4 cDNA (Maddon et al., 1985) as described in Figure 3A. (B) Northern blot analysis of relative T4 levels was performed by electrophoresis of total cellular RNA (10 μg/lane) on 1% agarose gels followed by transfer to nylon membranes. Blots were hybridized to 32P-labeled CD4 specific cDNA or β-tubulin. Autoradiography times were 36 hr and 4 hr for CD4 and β-tubulin probes, respectively.
Figure 6.
CD4/HIV Envelope Complexes in G418 Selected CEM Cells CEM cells infected with pDOL CMV·env (A and B) or uninfected CEM cells (C and D) were metabolically labeled with 35S-methionine and 35S-cysteine for 6 hr and immunoprecipitated with OKT4A (A and C) or OKT4 (B and D) monoclonal antibodies as described in Experimental Procedures. Cell lysates were preabsorbed with protein A-Sepharose beads overnight at 4°C and then incubated with monoclonal antibody–treated beads with gentle rotation at 4°C for 12 hr. Typically, 100 μg Sepharose was adsorbed with 200 μl monoclonal antibody and conjugated with extract of 107 cells. Conjugated beads were washed, and immunoprecipitates eluted in sample buffer at 65°C for 30 min. Electrophoresis was performed on a 15% polyacrylamide gel with a 3.5% stacking gel. Molecular weights were provided by prestained protein markers (Bethesda Research Laboratories).
Figure 7.
Accumulation of Unintegrated Viral DNA in Infected CEM Cells At the indicated times following infection, unintegrated viral DNA was analyzed in Hirt extracts from aliquots of 5 × 106 cells. Samples were resolved on 0.8% agarose gels and hybridized with an HIV probe. All Hirt samples were run undigested, while Xbal endonuclease digested genomic DNA from persistently infected CEM·env cells 44 days postinfection is shown for comparison.
Figure 8.
Effect of TPA on HIV Transcription in Persistently Infected CD4 Cells Persistently infected CEM cells (expressing pDOL · env CEM) were seeded in 25 cm3 flasks at 5–10 × 105 cells per ml in a total volume of 5 ml. Treatment was initiated by addition of TPA (in DMSO) to a final concentration of 1 ng/ml and/or α-amanitin (in methanol) to a final concentration of 1 μg/ml. After 24 hr at 37°C, total cellular RNA was extracted from the cells by guanidine thiocyanate lysis, and blotted in doubling dilutions onto nitrocellulose. Blots were hybridized with an HIV probe as indicated in Figure 2, or a β-actin probe (Gunning et al., 1983) and autoradiographed for 3 hr at −80°C. Hybridization intensity was determined by scanning densitometry; the induction index is the hybridization intensity relative to the signal obtained with untreated control cells.
Figure 9.
Transmission Electron Micrographs of TPA-Induced Cells Persistently Infected with HIV pDOL CMV·env (Figure 1) expressing CEM cells persistently infected with HIV before (A) and after (B and C) a 24 hr incubation with TPA as detailed in Figure 8. Cell pellets (approximately 5 × 105 cells) were fixed in 400 μl of picric acid-paraformaldehyde-glutaraldehyde (PPG) postfixed with osmium tetroxide, dehydrated, and embedded in Araldite 6005. Thin sections were stained with uranyl acetate and lead citrate, and examined with a Philips EM 300 electron microscope. Magnifications: 1.5× K (A and B); 15× K (C).
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