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. 12)

Friborg J, Daniel N, Yao XJ, Cohen EA
Laboratoíre de Rétrovírologíe Humaine, Université de Montreal, Montréal, CP 6128 Station A, Montréal, Canada H3C 3JY

The HIV-1 vpu gene encodes a 16 kDa class I integral membrane phosphoprotein which facilitates the release of viral particles from infected cells and delays the rate of syncytium formation in CD4 cells. The predicted 81 amino acid (a.a.) sequence of vpu suggests an amphipathic structure of the protein. The C-terminal hydrophilic region projecting from the cytoplasmic membrane face contains a 12 a.a. sequence which is highly conserved among vpu proteins from diverse HIV-1 isolates. We have previously demonstrated that this well conserved region represents the phosphorylation dependent domain of vpu that modulates its ability to reduce the rate of syncytium formation. The vpu-facilitated virion release and delayed cytopathic affect were shown to be the consequence of two functional sites. A recent report also revealed that sequences within the C-terminal region of vpu are critical for its degradative activity on CD4. The strongly hydrophobic N-terminal region of 27 a.a. presumably constitutes the anchor domain of vpu. In this study we present a mutational analysis of this region in order to ascertain its structural and functional role in the vpu phenotype.

In the current experiments, vpu mutants spanning the hydrophobic region were generated by a site-specific mutagenesis using a PCR based protocol. These mutants were tested for membrane association in an in vitro transcription- coupled translation and translocation system in presence of canine pancreatic microsomal membranes. We have defined two regions with predicted alpha-helix structures (sequences De 7-Ala 14 and Val 20-Ser 23) required for membrane association. These vpu mutants were introduced into an infectious molecular clone of HIV-1 and assessed for vpu biological activity. The effect of these mutations on vpu-facilitated virion release and delayed cytopathic effect will be discussed.

Keywords: Antigens, CD4, Base Sequence, CD4-Positive T-Lymphocytes, Cell Membrane, DNA Primers, Gene Products, vpu Genes, vpu, HIV-1, Humans, In Vitro Mutagenesis, Site-Directed, Polymerase Chain Reaction, Protein Biosynthesis, Transcription, Genetic, Virion, genetics, immunology

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1993-12-12
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