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


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.
BouHamdan
M
M
Center for Human Virology, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Kulkosky
J
J
Duan
L X
LX
Pomerantz
R J
RJ
D019380:Anti-HIV Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000494:pharmacology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D015694:Gene Products, rev / Q000235:genetics / Q000494:pharmacology*; D016336:Gene Products, vpr / Q000235:genetics / Q000494:pharmacology*; D005822:Genetic Vectors; D015488:HIV Antigens / Q000032:analysis; D016655:HIV Core Protein p24 / Q000032:analysis; D015658:HIV Infections / Q000517:prevention & control*; D015497:HIV-1 / Q000187:drug effects* / Q000472:pathogenicity / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D007135:Immunoglobulin Variable Region; D019429:Integrase Inhibitors / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014779:Virus Replication / Q000187:drug effects*; D054321:rev Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus
D019380:Anti-HIV Agents; D015694:Gene Products, rev; D016336:Gene Products, vpr; D015488:HIV Antigens; D016655:HIV Core Protein p24; D007135:Immunoglobulin Variable Region; D019429:Integrase Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C092667:anti-HIV-1 Rev SFv; D054321:rev Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus; D015500:Chloramphenicol O-Acetyltransferase