Quantification of Deaminase Activity-Dependent and -Independent Restriction of HIV-1 Replication Mediated by APOBEC3F and APOBEC3G through Experimental-Mathematical Investigation

Tomoko Kobayashi; Yoshiki Koizumi; Junko S. Takeuchi; Naoko Misawa; Yuichi Kimura; Satoru Morita; Kazuyuki Aihara; Yoshio Koyanagi; Shingo Iwami; Kei Sato

doi:10.1128/JVI.00062-14

Quantification of Deaminase Activity-Dependent and -Independent Restriction of HIV-1 Replication Mediated by APOBEC3F and APOBEC3G through Experimental-Mathematical Investigation

^aLaboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, Kyoto, Japan
^bSchool of Medicine, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japan
^cDepartment of Mathematical and Systems Engineering, Shizuoka University, Shizuoka, Japan
^dInstitute of Industrial Science, The University of Tokyo, Tokyo, Japan
^eGraduate School of Information Science and Technology, The University of Tokyo, Tokyo, Japan
^fDepartment of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan

S. R. Ross, Editor

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FIG 1
Schematic diagram of the HIV-1 life cycle and the antiviral effect of APOBEC3 proteins. (Left) Incoming WT APOBEC3 proteins (purple) cause (i) the inhibition of the viral RT process independent of their deaminase activity (blue arrow) and (ii) deaminase activity-dependent C-to-U mutations in minus-strand viral DNA (vDNA) resulting in G-to-A mutations in plus-strand vDNA (red arrow), both of which lead to the abrogation of viral replication; (Right) on the other hand, incoming CI APOBEC3 proteins (blue) cause only the inhibition of the viral RT process independent of their deaminase activity (blue arrow).
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FIG 2
Antiviral ability of APOBEC3F and APOBEC3G proteins. (A) Western blotting of the released HIV-1 particles. The level of HIV-1 particles released into the culture supernatant was analyzed by Western blotting, and representative results are shown. (B) Quantification of antiviral activity of APOBEC3F and APOBEC3G by experimental and mathematical analyses. The infectivity of released HIV-1 particles was quantified by TZM-bl assay. The assay was performed in triplicate. The averages of experimental data are shown as dots with standard deviations represented by bars, and a graph of the best-fit model is superimposed.
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FIG 3
Quantification of mutation-dependent and -independent antiviral effects by APOBEC3F and APOBEC3G. (A) A Venn diagram of the relative contribution rates of deaminase activity-dependent G-to-A mutations [Mutation, RC_Mu(x)] and deaminase activity-independent RT inhibition [RT inhibition, RC_RT(x)] to the total antiviral effect of APOBEC3 proteins. (B) Relative contribution rate of the different modes of antiviral activities with APOBEC3F and APOBEC3G. RC_Mu(x) (red area) and RC_RT(x) (blue area) of APOBEC3F and APOBEC3G are estimated as described in the text. RC_RT(x) in APOBEC3F, 11 ng, 26.5% ± 36.7%; 33 ng, 27.9% ± 30.6%; 100 ng, 30.6% ± 20.2%; 300 ng, 36.0% ± 12.9%. RC_RT(x) in APOBEC3G, 11 ng, 1.64% ± 1.02%; 33 ng, 0.64% ± 0.41%; 100 ng, 0.26% ± 0.17%; 300 ng, 0.11% ± 0.08%.
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FIG 4
Mutation signatures and in silico analysis. (A and B) Summary of viral DNA sequence analyzed in this study. (A) Raw sequence data (the sequences are available upon request). (B) Summarized table. ^a, The number in parenthesis represents the amount of transfected APOBEC3-expressing plasmid; ^b, the numbers of analyzed nucleotides and clones (in parenthesis) are shown; ^c, hypermutation index (38, 43) is calculated as follows: (no. of G-to-A mutations − no. of A-to-G mutations)/sequence length (bp) × 1,000; ^d, the number and the percentage (in parenthesis) of dinucleotide contexts of G-to-A mutation sites in total G-to-A mutations are shown. ND, not determined, probably due to a high level of G-to-A hypermutations mediated by APOBEC3G in the primer-binding region of template DNA that resulted in the failure of the PCR process under this condition (53, 54). (C) In silico analyses. The effects of G-to-A mutations mediated by APOBEC3F (GA to AA, red line) or APOBEC3G (GG to AG, blue line) on the proportion of proviral DNA with stop codons (S, y axis) were estimated with equation 9 as described in the text.