2nd National Conference Human Retroviruses and Related Infections Washington, DC - January 29 - February 2, 1995

Natl Conf Hum Retrovir Relat Infect 1995 Jan 29-Feb 2;2: (abstract no. 31)

Wu X¹, Liu H¹, Xiao H¹, Kim J¹, Seshaiah P², Natsoulis G³, Boeke JD², Hahn BH¹, Kappes JC^1
1University of Alabama at Birmingham, Birmingham, Alabama 35249; ²Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; ³Avigen Inc., Alameda, California 94501

The human immunodeficiency virus (HIV) Vpr and Vpx proteins are incorporated into virions through virus type-specific interactions with the Gag precursor polyprotein. To examine whether Vpr and Vpx can be utilized to target foreign proteins to virions, their coding regions were fused in-frame with the bacterial staphylococcal nuclease (SN) and chloramphenicol acetyltransferase (CAT) genes. In a T7-based expression system, both VprSN and VpxSN fusion proteins were expressed in mammalian cells. When coexpressed with Gag. VprSN and VpxSN were incorporated into virus-like particles (VLPs) released into the extracellular medium. Incorporation of VprSN and VpxSN into VLPs was shown to be mediated by viral specific signals and to occur even in the presence of wild-type Vpr or Vpx proteins. VLP associated Vpr- and Vpx- SN fusion proteins exhibited nuclease activity like purified SN protein, as determined by in vitro digestion of lambda phage DNA. Expression of HIV-1 and HIV-2 proviral DNAs in trans with HIV-regulated VprSN and VpxSN expression plasmids produced virus particles containing both fusion and wild type Vpr and Vpx proteins. The association of VpxSN with HIV-2 virions was confirmed by continuous gradient sucrose analysis. Vpr- and Vpx- CAT fusion proteins also associated with virions when expressed in trans with HIV proviral DNAs. These results demonstrate the feasibility of incorporating foreign proteins into HIV virions by expression as gene fusions with vpr and vpx. Such findings may suggest potential application of virion-associated accessory proteins to gene therapy based approaches for limiting the spread of HIV by specific delivery/targeting of virus-toxic molecules.

Keywords: Animals, Cats, Chloramphenicol O-Acetyltransferase, Gene Products, gag, Genes, gag, Genes, vpr, HIV, HIV Integrase, HIV Protease, HIV Protease Inhibitors, HIV-1, HIV-2, Humans, In Vitro, Recombinant Fusion Proteins, VPX protein, Human immunodeficiency virus 2, Viral Regulatory Proteins, Virion, genetics

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1995-01-29
31

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