Inhibition of HIV-1 replication and infectivity by expression of a fusion protein, VPR-anti-integrase single-chain variable fragment (SFv): intravirion molecular therapies.

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.

• 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.)

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

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