ADAM10 – A CELLULAR METALLOPROTEASE REQUIRED FOR HIV REPLICATION

B Friedrich¹, J Murray Ph.D.², D Rubin M.D.³, T Hodge Ph.D.², WA O’Brien M.D.^1,2, and MR Ferguson Ph.D.¹

¹Department of Experimental Pathology, UTMB, ²Zirus, Inc. Buford, GA, ³VATVHS and Vanderbilt University, Nashville, TN

Background: HIV uses cellular factors to successfully complete replication. ADAM10 was identified by gene trap insertional mutagenesis. This is based on integration of an MMLV-derived shuttle vector carrying a promoterless neomycin resistant gene. Cells surviving both gene knockout and lethal virus infection carry an interrupted gene required by the pathogen. Objective: To identify mechanism of action of ADAM10 during HIV replication. Methods: Potential clinical relevance of ADAM10 was assessed by siRNA treatment of primary macrophages. U373-MAGI-CCR5 cells express β-galactosidase under control of the HIV LTR promoter. Virus production was assessed by p24 production 7d post-infection. HIV protein production and nuclear entry were detected by Western blot and real time PCR. New viral DNA formation (reverse transcription) and integration was quantitated by Real Time PCR. Results: In primary macrophages, transfection of ADAM10-siRNA 1-2d prior to HIV infection, or 12h after infection, resulted in 85% reduction in HIV p24 production at 7d compared to mock treatment. In primary macrophages, formation of full length HIV DNA was not inhibited in ADAM10-siRNA transfected cells. β-gal production was inhibited after infection in ADAM10 siRNA-treated U373-MAGI cells. Transfection of ADAM10 siRNA affected virus production in virally infected cells, but not full length HIV provirus transfected cells. Conclusion: The site of ADAM10 activity is after reverse transcription and before HIV protein production, likely at the level of integration or nuclear entry. The absence of cytotoxic effects associated with downregulation in primary macrophages suggests that ADAM10 may not be essential for the cell, and may provide a unique target for therapeutic intervention. This project is supported by Zirus, Inc.