1st National Conference Human Retroviruses and Related Infections Washington, DC - December 12-16, 1993

Natl Conf Hum Retrovir Relat Infect 1993 Dec 12-16;1: (abstract no. 16)

Cohen EA, Yao XJ, Lavallee C, Jolicoeur P, Subramanian R, Ladha A
Laboratoire de Rétrovirologìe Humaine, Université de Montréal, CP 6128, Montréal, Canada, H3C 3J7

The assembly and maturation of HIV-1 virus particles is a complex process in which the structural gag, pol, and env gene products are expressed in the form of polyprotein precursors. The 96 amino acids Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors and its incorporation must occur via an interaction with a component normally found in the virus particle. To investigate the mechanism of incorporation of vpr into HIV-1 virion, proviral DNA constructs (vpr-) harboring mutations or deletions in specific virion-associated gene products were cotransfected with a vpr expressor in COS cells. Virus released from the transfected cells was tested for the presence of vpr by immunoprecipitation using vpr specific antibodies. The results of these experiments show that vpr is trans-incorporated into virions but with a lower efficiency than when vpr is expressed from a proviral construct. The minimal viral genetic information necessary for vpr incorporation was a deleted provirus encoding only the p55 gag precursor protein. Incorporation of vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of vpr with the p55 gag precursor protein was demonstrated by coprecipitation experiments using vpr specific antibodies. To delineate the domain(s) of vpr that are critical for its incorporation, a mutational analysis of vpr was undertaken. Deletion analysis indicate that a region encompassing amino-acids 78 to 84 at the C-terminal is essential for vpr incorporation. In addition, individual substitution in a predicted alpha helix motif located between amino acid 15 and 35 affected significantly vpr incorporation. Overall, these results demonstrate a novel mechanism by which viral protein can be incorporated into viral particles. Identification of specific virion association motif(s) in the vpr protein may permit the development of chimeric molecules that can be specifically targeted into the mature HIV-1 virion to affect its structural organization or functional integrity.

Keywords: Animals, Gene Products, env, Gene Products, gag, Gene Products, vpr, Genes, env, Genes, gag, Genes, pol, Genes, vpr, HIV-1, Humans, Protein Precursors, Proviruses, Viral Proteins, Virion, genetics

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1993-12-12
16

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