HIV restriction factor APOBEC3G binds in multiple steps and conformations to search and deaminate single-stranded DNA.

Michael Morse,Yuqing Feng,Mark C Williams,M Nabuan Naufer,Ioulia Rouzina,Linda Chelico

doi:10.7554/elife.52649

Michael Morse, Yuqing Feng + Show 4 more

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https://doi.org/10.7554/elife.52649

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Abstract

APOBEC3G (A3G), an enzyme expressed in primates with the potential to inhibit human immunodeﬁciency virus type 1 (HIV-1) infectivity, is a single-stranded DNA (ssDNA) deoxycytidine deaminase with two domains, a catalytically active, weakly ssDNA binding C-terminal domain (CTD) and a catalytically inactive, strongly ssDNA binding N-terminal domain (NTD). Using optical tweezers, we measure A3G binding a single, long ssDNA substrate under various applied forces to characterize the binding interaction. A3G binds ssDNA in multiple steps and in two distinct conformations, distinguished by degree of ssDNA contraction. A3G stabilizes formation of ssDNA loops, an ability inhibited by A3G oligomerization. Our data suggests A3G securely binds ssDNA through the NTD, while the CTD samples and potentially deaminates the substrate. Oligomerization of A3G stabilizes ssDNA binding but inhibits the CTD's search function. These processes explain A3G's ability to efficiently deaminate numerous sites across a 10,000 base viral genome during the reverse transcription process.

Highlights

The APOBEC3 family of proteins provides humans and other primates with innate immune antiviral resistance
APOBEC3G (A3G) is known for its ability to inhibit the infectivity of human immunodeficiency virus type 1 (HIV-1) (Harris and Liddament, 2004; Malim, 2009) when it is not targeted for degradation by the accessory protein viral infectivity factor (Vif) (Desimmie et al, 2014; Fisher et al, 1987; Goila-Gaur and Strebel, 2008; Sheehy et al, 2002; Strebel et al, 1987)
We find that A3G binds single-stranded DNA (ssDNA) in two distinct conformations, consistent with the N-terminal domain (NTD) securely binding the substrate over the timescale of 100 s and the C-terminal domain (CTD) transiently interacting with the substrate, physically contracting the ssDNA and allowing the catalytically active domain to search and deaminate target sequences

Summary

Introduction

The APOBEC3 family of proteins provides humans and other primates with innate immune antiviral resistance. Other members of the APOBEC3 family have either one or two domains, though for two domain proteins only one domain is enzymatically active (LaRue et al, 2008). For A3G, the C-terminal domain (CTD) is enzymatically active (Hacheet al., 2005; Navarro et al, 2005), though when isolated it binds ssDNA with low affinity (Chelico et al, 2010; Harjes et al, 2009). The N-terminal domain (NTD), though lacking deaminase activity, binds ssDNA and RNA with high affinity (Chelico et al, 2010; Huthoff et al, 2009). The NTD is critical in A3G’s packaging in the virion and contains the binding site for Vif

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