Functional analysis of the two cytidine deaminase domains in APOBEC3G

Abstract

Human APOBEC3G (hA3G), a cytidine deaminase with two cytidine deaminase domains (CDs), has been identified as an anti-HIV-1 host factor. Although the two CDs of hA3G have been extensively characterized, there is still debate on the role of the CDs in the biological function of hA3G. In this work, we constructed three hA3G mutants CD1-1, CD2-2 and CD2-1, which contain duplicate CD1 domain, duplicate CD2 domain and position switched CD domain respectively, and investigated the effect of CD domain replacement or switch upon virion encapsidation, Vif-mediated degradation, deamination and antiviral activity of hA3G. The results showed that the two CD domains were functionally equivalent in virion encapsidation and the interaction with HIV-1 Vif of hA3G, whereas CD domain switch or replacement greatly affected the sensitivity to Vif induced degradation, editing and antiviral activity of hA3G. Although the CD2 domain was shown to possess the deamination activity, CD2-2 incorporated efficiently into HIV-1 was unable to mutate viral cDNA, suggesting that CD1 also involved in the enzymatic function. Interestingly, CD2-1 retained considerable deamination activity with a different sequence preference. Taken together, our results suggest that CD domain may play a structural role in virion encapsidation and Vif-mediated degradation of hA3G, and coordination of the two CD domains is required for its editing and antiviral activity.

The in vitro Biochemical Characterization of an HIV-1 Restriction Factor APOBEC3F: Importance of Loop 7 on Both CD1 and CD2 for DNA Binding and Deamination

“…The incorporation/encapsidation into HIV virions, which is mainly mediated through RNA binding by the A3 proteins, requires both CD1 and CD2 domains of A3F, whereas only CD1 of A3G is needed 32; 34; 35; 36; 37; 38; 39 . On the other hand, while HIV Vif targets A3G for E3 ubiquitin ligase mediated degradation through binding to the CD1 of A3G, Vif targets A3F by binding to its CD2 domain 25; 26; 40; 41; 42; 43.…”

DNA mutagenic activity and capacity for HIV-1 restriction of the cytidine deaminase APOBEC3G depend on whether DNA or RNA binds to tyrosine 315

“…1A) and aa 314 -326, sequence IYDDQGRCQEGLR (one missed trypsin cleavage) ( Fig. 1B), suggesting that these peptides contain cross-linked 79 Br/ 81 Br-modified amino acid residues (see details under "Experimental procedures" and in Fig. 1, A and B, legend).…”

“…Tyr-181 in tryptic peptide aa 181-194 and Tyr-315 in tryptic peptide aa 314 -320 were identified as BrdU-modified residues in cross-linked samples (MS/MS Figure 1. A, initial identification of 79 Br-and 81 Br-containing BrdU-cross-linked A3G peptides aa 181-213 that matched predicted MS1 values. Tryptic peptides of A3G/BrdU cross-linked sample were analyzed on Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer and searched for BrdU modifications, including 79 Br and 81 Br isotope difference with Compass software (Compass IsotopePattern, Bruker Daltonics).…”

“…Gel-shifted and control samples were analyzed using Q Exactive (Thermo Fisher Scientific) mass spectrometer, and the MS data were initially searched for differences in peptides identified in the ssDNA cross-linked in A3G sample due to altered m/z compared with those identified in control A3G lacking ssDNA cross-linking. Peptides corresponding to these differences were analyzed for 79 Br and 81 Br BrdU-modified double peak peptides with Compass Software (Isotope-Pattern, Bruker Daltonics) and the chemistry of modification set as corresponding to BrdU, C 9 H 11 N 2 O 8 P 1 Br. Two sets of peaks, with m/z values in the range of 1111-1113 (charge ϩ4) and 663-665 (charge ϩ3), were identified that showed MS1 peaks matching the predicted m/z values of the A3G-BrdUmodified peptides, aa 181-213, sequence YYILLHIMLGEILR-HSMDPPTFTFNFNNEPWVR (one missed trypsin cleavage) ( Fig.…”

Human Immunodeficiency Virus Reverse Transcriptase

“…Between these, only the C-terminus is capable of deaminating ssDNA cytosine to uracil (Haché et al 2005 ;Iwatani et al 2006 ;Langlois et al 2005 ;Navarro et al 2005 ;Newman et al 2005 ). The catalytically inactive N-terminus, in contrast, is more important for binding to single-stranded nucleic acid substrates, an activity that mediates RNA-dependent oligomerization and encapsidation and that may also help orient A3G for processive deamination ... (Huthoff et al 2009Malim 2007 #1965;Friew et al 2009Gooch andCullen 2008 #2129;Li et al 2011Navarro et al 2005 #528;Chelico et al 2010. Although it has been proposed that monomeric A3G may also be capable of encapsidation, deamination, and restriction (Opi et al 2006 ), recent evidence suggests the particular mutant employed in this study, while monomeric, may not actually encapsidate, raising the question of whether its apparent ability to restrict represents a specifi c effect on HIV or a nonspecifi c poisoning of producer cells [(Friew et al 2009 ;Shlyakhtenko et al 2011 ) and Mueller and Harris Labs unpublished data].…”