Small Angle X-ray Scattering Provides Insight into the Mode of RNA-Mediated Regulation of APOBEC3G Anti-HIV Activity

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Joseph E. Wedekind^*1, Richard Gillilan², Alena Janda¹, Jolanta Krucinska¹, Jason D. Salter¹, Ryan P. Bennett¹, Jay Raina³, and Harold C. Smith^1
1Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
²Macromolecular Structure Facility at the Cornell High Energy Synchrotron Source (MacCHESS), Cornell University, Ithaca, NY 14853
³Immunodiagnostics Inc., Woburn, MA 01801

^*correspondence to: joseph.wedekind@rochester.edu
 

Abstract:
Human APOBEC3G (hA3G) is an innate deoxycytidine-to-deoxyuridine deaminase that restricts HIV-1 infectivity by introducing catastrophic mutations into the viral genome in the absence of the HIV-1 factor vif.  hA3G from HIV-permissive, activated CD4+ T-cells exists as an inactive, high-molecular-mass (HMM) complex that can be transformed in vitro into an enzymatically active, low-molecular-mass (LMM) variant comparable to that identified in HIV-non-permissive CD4+ T-cells.  Similarly, hA3G has been detected in viral capsids as an inactivated ribonucleoprotein whose activity restoration depends upon the RNase H function of reverse transcriptase.  Collectively, these data support a model that suggests inactivation of hA3G depends on RNA. To elucidate how RNA influences the structural organization of hA3G, we determined low resolution structures of recombinantly expressed HMM- and LMM-variants by small angle X-ray scattering. The results indicated that LMM particles have an extended shape, dissimilar to known cytidine deaminase crystal structures.  Structure-based biochemical experiments corroborated these observations and suggested that each LMM particle comprises a tail-to-tail dimer of identical subunits.  Additional studies indicated that recombinant LMM-hA3G binds with avidity to nucleic acids and that this interaction blocks hA3G deamination.  Shape analysis of LMM- and HMM-particles led us to propose a hierarchical assembly model whereby symmetric association of LMM dimers produces inactive HMM aggregates.  These observations imply that the disruption of cellular HMM species will require regulation of protein-RNA, as well as protein-protein interactions, which has implications for therapeutic development.

abstract (pdf)