Abstract | OBJECTIVES: To deliver antiretroviral agents or other foreign proteins into progeny virions and evaluate their inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication. STUDY DESIGN/METHODS: HIV-1 encodes proteins in addition to gag, pol, and env, some of which are packaged into virus particles. One essential retroviral enzyme is integrase (IN), which has been used as a target for developing agents that inhibit virus replication. In previous studies, we demonstrated that intracellular expression of single-chain variable antibody fragments (SFvs), which bind to IN, results in resistance to productive HIV-1 infection in T-lymphocytic cells. Because the highly conserved accessory HIV-1 Vpr protein can be packaged within virions in quantities similar to those of the major structural proteins, this primate lentiviral protein may be used as a fusion partner to deliver antiviral agents or other foreign proteins into progeny virions. In these studies, the fusion proteins Vpr- chloramphenicol acetyl transferase (CAT) and Vpr-SFv-IN have been developed. Stable transfectants expressing these fusion proteins were generated from PA317 cells and SupT1 T-lymphocytic cells and analyzed using immunofluorescence microscopy. After challenge of SupT1 cells with HIV-1, p24 antigen expression was evaluated. The incorporation of these fusion proteins were evaluated by immunoprecipitation of virions using a Vpr antibody. RESULTS: Expression of the fusion proteins was confirmed by immunofluorescent staining in PA317 cells transfected with the plasmids expressing Vpr-CAT and Vpr-SFv-IN proteins. Stable transfectants expressing these fusion proteins were generated from SupT1 T-lymphocytic cells. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression, was inhibited in cells expressing Vpr-SFv-IN. It was demonstrated that Vpr- chloramphenicol acetyl transferase (Vpr-CAT and Vpr-SFv-IN proteins can be efficiently packaged into the virions and that Vpr-SFv-IN also decreases the infectivity of virions into which it is encapsidated. CONCLUSIONS: An anti- integrase single-chain variable fragment moiety can be delivered into HIV-1 virions by fusing it to Vpr. Vpr-SFv-IN decreases HIV-1 production in human T-lymphocytic cells. The benefits of "intravirion" gene therapy include immunization of target cells as well as decreasing infectivity of HIV-1 virions harboring the fusion construct. Thus, this approach to anti-HIV-1 molecular therapies has the potential to increase inhibitory effects against HIV-1 replication and virion spread.
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Authors | M BouHamdan, J Kulkosky, L X Duan, R J Pomerantz |
Journal | Journal of human virology
(J Hum Virol)
2000 Jan-Feb
Vol. 3
Issue 1
Pg. 6-15
ISSN: 1090-9508 [Print] United States |
PMID | 10774802
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- Anti-HIV Agents
- Gene Products, rev
- Gene Products, vpr
- HIV Antigens
- HIV Core Protein p24
- Immunoglobulin Variable Region
- Integrase Inhibitors
- Recombinant Fusion Proteins
- Recombinant Proteins
- Single-Chain Antibodies
- anti-HIV-1 Rev SFv
- rev Gene Products, Human Immunodeficiency Virus
- vpr Gene Products, Human Immunodeficiency Virus
- Chloramphenicol O-Acetyltransferase
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Topics |
- Anti-HIV Agents
(pharmacology)
- Blotting, Western
- Cell Line
- Chloramphenicol O-Acetyltransferase
(pharmacology)
- Cloning, Molecular
- Enzyme-Linked Immunosorbent Assay
- Fluorescent Antibody Technique
- Gene Products, rev
(genetics, pharmacology)
- Gene Products, vpr
(genetics, pharmacology)
- Genetic Vectors
- HIV Antigens
(analysis)
- HIV Core Protein p24
(analysis)
- HIV Infections
(prevention & control)
- HIV-1
(drug effects, pathogenicity, physiology)
- HeLa Cells
- Humans
- Immunoglobulin Variable Region
- Integrase Inhibitors
(pharmacology)
- Recombinant Fusion Proteins
(biosynthesis, genetics, pharmacology)
- Recombinant Proteins
- Single-Chain Antibodies
- Virus Replication
(drug effects)
- rev Gene Products, Human Immunodeficiency Virus
- vpr Gene Products, Human Immunodeficiency Virus
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