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. 19)

Landau N^1,2, Connor R¹, Paxton W¹, Ebright M¹, Choe S^1
1Aaron Diamond AIDS Research Center, New York, NY; ²New York University Medical School, New York, NY

We were interested to understand the molecular interactions that lead to the incorporation of HIV-1 Vpr into virions. To determine which virion component and which regions of Vpr itself mediate incorporation into the viral particle during assembly in an infected cell, we expressed Vpr linked to a nine amino acid “epitope tag” derived from the influenza hemagglutinin. Virions containing tagged Vpr were prepared by cotransfecting COS cells with a HIV vpr provirus and tagged-Vpr expression vector. The epitope tag did not interfere with Vpr incorporation and allowed for recognition of mutated forms of Vpr without regard to their amino acid sequence. The high levels of Vpr in the virions that were produced resulted from a specific interaction of Vpr with an HIV-1 component, since cotransfection of cells with the tagged Vpr expression vector and distantly related proviruses (murine leukemia virus and human T cell leukemia virus) resulted in no detectable virion-associated tagged Vpr. By transfecting cells with mutated HIV-1 proviruses defective for one or more of the virion structural genes, we showed that Vpr incorporation depends on the carboxy-terminal gag product, p6 (not on pol or env gene products or genomic RNA). In addition, using truncated and mutated forms of tagged Vpr, we showed that a 10 amino acid sequence near the carboxy-terminus is required for incorporation into virions. We are currently using this system to target larger polypeptide virions.

We have also localized the tagged-Vpr cells by immunofluorescence. The results showed that Vpr is a nuclear protein. We are currently localizing the amino acids that determine nuclear localization using the mutated tagged Vpr vectors.

Keywords: Amino Acid Sequence, Animals, Gene Products, env, Gene Products, gag, Genes, gag, Genes, pol, Genes, vpr, Genetic Vectors, HIV, HIV Protease, HIV-1, Humans, Nuclear Proteins, Proviruses, S100 Proteins, S100A12 protein, human, Virion, genetics

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1993-12-12
19

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