ZHAO L, RUZICKA JA, TAYLOR EW; Interscience Conference on Antimicrobial Agents and Chemotherapy.

Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2000 Sep 17-20; 40: 285.

The Univ. of Georgia, Athens, GA

BACKGROUND: Taylor et al. (J AIDS Hum. Retrovirol. 15:393-94, 1997) reported previously that HIV-1 encodes a sequence in an overlapping reading frame of env gp41 with highly significant sequence similarity to the mammalian selenoprotein glutathione peroxidase (GPx). We have now demonstrated that in mammalian cells transfected with a plasmid designed to express the putative HIV-1 GPx as a selenoprotein, there is a significant increase in GPx activity in cell lysates, ranging from 30% to 99% relative to controls, depending on the cell type (Zhao et al., Proc. Natl. Acad. Sci. USA, in press). We have further assessed the hypothesis that HIV-1 encodes a functional GPx gene by bacterial expression of the Cys mutant of this hypothetical protein, and studies of its biological activity.METHODS: The putative HIV-1 GPx sequence was cloned into the pTRC-HIS expression vector as a Cys mutant (UGA to UGC mutation), expressed in E. coli as a 9 kDa protein, isolated from inclusion bodies, purified to homogeneity, renatured, and then assayed for GPx activity, using a standard enzyme assay.RESULTS: The purified mutant GPx (mGPx) protein had GPx-like activity of 4.2 +/- 0.6 units/mg protein (n=5), which is a significant level of activity for a Cys mutant of a Se-dependent GPx enzyme. The Km value with t-butyl hydroperoxide as substrate was 3.5x10[-5 ]M. The mGPx enzyme activity was pH dependent, and was found to be maximal at pH 6.5 in 20 mM phosphate buffer.CONCLUSIONS: HIV-1 encodes functional GPx activity in the region overlapping env gp41, as assessed by mammalian cell transfection studies and by assay of the purified mGPx protein. Because cellular GPx is a regulator of transcription factor NF-k B, this novel viral gene may play a role in the regulation of HIV-1 replication.KEYWORDS: Glutathione peroxidase; HIV-1; Selenium

Publication Types:

• Meeting Abstracts

Keywords:

• Base Sequence
• Cysteine
• Genes, env
• Glutathione Peroxidase
• HIV-1
• In Vitro
• Reading Frames
• Selenium
• Transcription, Genetic
• enzymology
• genetics

Other ID:

• GWAIDS0010932

UI: 102248430

From Meeting Abstracts