HIV-1 proprotein processing as a target for gene therapy.
The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.
Cordelier
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Department of Pathology, Jefferson Medical College, Philadelphia, PA 19107, USA.
Zern
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Strayer
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D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D015683:Gene Products, gag / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D019302:HIV Envelope Protein gp160 / Q000378:metabolism; D015658:HIV Infections / Q000276:immunology / Q000628:therapy*; D006801:Humans; D008214:Lymphocytes / Q000276:immunology; D011498:Protein Precursors / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods; D014779:Virus Replication; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D019302:HIV Envelope Protein gp160; D011498:Protein Precursors; D015842:Serine Proteinase Inhibitors; D000515:alpha 1-Antitrypsin; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; C091950:pr160 gag-pol precursor protein, Human immunodeficiency virus 1