Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA-editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here, we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA-binding-defective mutants demonstrate clear separation of function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H while, at the same time, positively regulating its antiviral activity. Shaban et al. define positive and negative roles for RNA in regulating the activities of human APOBEC3H. RNA is essential for APOBEC3H localization, packaging into HIV-1 particles, and virus restriction. RNA also blocks DNA deaminase activity. Crystal structures demonstrate duplex RNA as a central feature of the underlying molecular mechanism.
Bibliographical noteFunding Information:
We thank D. Ebrahimi for providing helpful comments and M. Jarvis and J. Zhang for technical assistance. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pLAI.2 from K. Peden, courtesy of the MRC AIDS Directed Program, and anti-HIV-1 p24/CA mAb courtesy of B. Chesebro and K. Wehrly. This work was supported by grants from the US National Institutes of Health ( NIGMS R01-GM118000 to R.S.H. and H.A., NIGMS R35-GM118047 to H.A., and NCI R21-CA206309 to R.S.H.), the Prospect Creek Foundation (R.S.H), and the University of Minnesota (College of Biological Sciences, Academic Health Center, and Masonic Cancer Center to R.S.H.). Partial salary support for R.L.-K., C.M.R., and D.S. was provided by NIH training grants ( T32-HL007062 , T32-AI83196 , and T90-DE022732 , respectively). This work is based in part upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health ( NIGMS P41-GM103403 ). The Pilatus 6M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant ( S10 RR029205 ). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished University McKnight Professor, and an Investigator of the Howard Hughes Medical Institute.
© 2017 Elsevier Inc.
- DNA cytosine deaminase
- RNA duplex
- retrovirus restriction factor
- ribonucleoprotein complex
- structural virology