Abstract
The apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) family proteins bind RNA and single-stranded DNA, and create C-to-U base modifications through cytidine deaminase activity. APOBEC3G restricts human immunodeficiency virus 1 (HIV-1) infection by creating hypermutations in proviral DNA, while HIV-1-encoded vif protein antagonizes such restriction by targeting APOBEC3G for degradation. APOBEC3G also inhibits hepatitis B virus (HBV): APOBEC3G co-expression inhibits HBV replication and evidences exist indicating APOBEC3G-mediated HBV hypermutations in patients. HBV encodes a small non-structural X protein (HBx) with a recognized activating effect on HBV life cycle. In this work, we report the discovery that HBx selectively and dose-dependently decreases the protein level of co-expressed APOBEC3G in transfected Huh-7 cells. The effect was shown to take place post-translationally, but does not rely on protein degradation via proteasome or lysosome. Further work demonstrated that intracellular APOBEC3G is normally exported via exosome secretion and inhibition of exosome biogenesis causes retention of intracellular APOBEC3G. Finally, HBx co-expression specifically enhanced externalization of APOBEC3G via exosomes, resulting in decrease of intracellular APOBEC3G protein level. These data suggest the possibility that in addition to other mechanisms, HBx-mediated activation of HBV might also involve antagonizing of intracellular restriction factor APOBEC3G through promotion of its export.
Highlights
The APOBEC family consists of evolutionarily and structurally related proteins that bind RNA and single-stranded DNA and, through a conserved zinc-dependent cytidine deaminase activity, create C to U base modifications on substrate ssDNA and, for some members, RNA12
The most significant example is the discovery that APOBEC3G (A3G) and some other APOBEC3 subfamily proteins restrict human immunodeficiency virus 1 (HIV-1) infection by heavily editing viral ssDNA during reverse transcription resulting in hypermutated proviral DNA14
Immunoblot using Flag and HA antibodies revealed that protein levels of A3A, A3C, A3F and A3H displayed minimal differences with or without HBx co-expression, whereas A3G protein level in cells co-transfected with HBx was markedly lower compared to cells co-transfected with vector control (Fig. 1B)
Summary
The APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family consists of evolutionarily and structurally related proteins that bind RNA and single-stranded DNA (ssDNA) and, through a conserved zinc-dependent cytidine deaminase activity, create C to U base modifications on substrate ssDNA and, for some members, RNA12. The most significant example is the discovery that APOBEC3G (A3G) and some other APOBEC3 subfamily proteins restrict human immunodeficiency virus 1 (HIV-1) infection by heavily editing viral ssDNA during reverse transcription resulting in hypermutated proviral DNA14. The vif protein encoded by HIV-1 binds multiple A3 proteins including A3G, and targets the later for ubiquitination and proteasome degradation, antagonizing A3-mediated restriction[15]. Unlike HIV-1, no HBV-encoded protein has been shown to regulate A3G. We report the discovery that HBx is capable of selectively and down-regulating intracellular protein level of co-expressed A3G. Our data indicate that such down-regulation of A3G by HBx is most likely effected on already synthesized A3G proteins at the post-translational level, but does not seem to involve A3G protein degradation through proteasome or lysosome. We provide evidences showing that intracellular A3G is normally exported in exosomes and such externalization of A3G is enhanced by HBx, which correlates with HBx-mediated down-regulation of intracellular A3G
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