Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
202 Enzymatic Activity of CEM15 is Essential for its Function to Suppress the Infectivity of deltaVif Virion A. Takaori-Kondo*, K. Shindo, M. kobayashi, A. Arukin, T. Uchiyama Grad Sch of Med, Kyoto Univ, Kyoto, Japan
Background: HIV-1 Vif protein plays a crucial role in regulating virus infectivity. Recently, a cellular factor responsible for this function, named CEM15, has been identified by subtraction cloning. The amino acid sequence of CEM15 is homologous to that of apobec-1, the cytidine deaminase that is the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme, suggesting that CEM15 acts as an RNA editing enzyme to regulate virion infectivity. However, its precise mechanism is still unclear. In this study, we investigated whether the enzymatic activity of CEM15 affects the virus infectivity regulated by HIV-1 Vif protein.
Methods: 1) We made expression vectors for human CEM15, EGFP-CEM15, and CEM15 mutant (H257R), in which we mutated a putative zinc-coordinating residue that is conserved in other cytidine deaminases; 2) we transfected with EGFP-CEM15 with Vif and whole molecular clone into 293T cells and looked at the cellular localization of EGFP-CEM15 using fluorescence microscopy; and 3) we examined the infectivity of wild-type and deltaVif virus produced from 293T cells transfected with CEM15, EGFP-CEM15, or H257R using luciferase reporter system (pNL43/Luc and pNL43/deltaVif/Luc).
Results: 1) Expression of CEM15, EGFP-CEM15, and H257R were confirmed by Western blot; 2) transient transfection of EGFP-CEM15 suppressed the infectivity of deltaVif virus produced from 293T cells, indicating EGFP-fused CEM15 retained its function; 3) EGFP-CEM15 was localized in cytoplasm and co-expression of Vif protein or viral RNA did not change its localization; and 4) transient transfection of CEM15 introduced non-permissive phenotype into 293T cells, but H257R did not.
Conclusions: 1) Enzymatic activity of CEM15 is necessary for its function to suppress the infectivity of deltaVif virus; and 2) CEM15 is localized in cytoplasm, which is not affected by expression of Vif protein or viral RNA.
