Immunoglobulin somatic hypermutation by APOBEC3/Rfv3 during retroviral infection - PubMed (original) (raw)

Immunoglobulin somatic hypermutation by APOBEC3/Rfv3 during retroviral infection

Kalani Halemano et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Somatic hypermutation (SHM) is an integral process in the development of high-affinity antibodies that are important for recovery from viral infections and vaccine-induced protection. Ig SHM occurs predominantly in germinal centers (GC) via the enzymatic activity of activation-induced deaminase (AID). In contrast, the evolutionarily related apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 3 (APOBEC3) proteins are known to restrict retroviruses, including HIV-1. We previously reported that mouse APOBEC3 encodes Recovery from Friend virus 3 (Rfv3), a classical resistance gene in mice that promotes the neutralizing antibody response against retrovirus infection. We now show that APOBEC3/Rfv3 complements AID in driving Ig SHM during retrovirus infection. Analysis of antibody sequences from retrovirus-specific hybridomas and GC B cells from infected mice revealed Ig heavy-chain V genes with significantly increased C-to-T and G-to-A transitions in wild-type as compared with APOBEC3-defective mice. The context of the mutations was consistent with APOBEC3 but not AID mutational activity. These findings help explain the role of APOBEC3/Rfv3 in promoting the neutralizing antibody responses essential for recovery from retroviral infection and highlight APOBEC3-mediated deamination as a previously unidentified mechanism for antibody diversification in vivo.

Keywords: Friend retrovirus; affinity maturation; antibody repertoire profiling; humoral immunity; restriction factor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Characterization of hybridomas from _Rfv3_-resistant versus -susceptible mice. (A) FV-specific mAbs from mA3+/s and _mA3_−/s mice. Hybridomas were derived from splenocytes of FV-infected mice at 21 and 28 dpi. (B) IgG subclass composition of FV-reactive mAbs. (C) Mutation frequency relative to germline. IgG cDNA was sequenced and compared against B6 germline V H sequences. (D) Relative binding of mAbs to native virions at 21 dpi, as evaluated by time-resolved fluorescence ELISA. (E and F) AID (E) and mA3 (F) hotspot mutations. The percentages of WR

C

or TY

C

mutations relative to the number of C or G mutations per mAb were calculated, respectively. (G) V H gene distribution. The number of hybridomas belonging to each V H gene family is shown. (H) Data are identical to D, except that mA3+/s mAbs encoding V H 1–19 were analyzed separately. In A, B, and G, the difference in percentages was evaluated using Fisher’s exact test. In C_–_F, median values are shown; differences between the two groups were evaluated using a two-tailed Mann–Whitney U test with P < 0.05 considered significant. NS, not significant.

Fig. 2.

Fig. 2.

NGS of GC B-cell V H genes from B6 WT versus mA3 KO mice. (A) Sorting strategy. RNA extracted from CD19+GL7+ cells were used for cDNA synthesis followed by IgG VH PCR with Illumina primers. (B) Sequence read counts from individual WT and KO mice. To minimize selection bias, identical sequences were collapsed into a single unique sequence. FV+ V H corresponds to the 16 V H genes in the FV-specific mAbs in Fig. 1_G_. (C) Validation of sorted GC B cells. The average mutation frequencies of paired GL7+ and GL7− populations from the three WT mice were computed for each of the 16 V H genes. Differences were evaluated using a two-tailed paired Student t test. (D) Total mutation frequency in WT (n = 3) and KO (n = 4) mice for V H 1–19 and V H 1–26 sequences. Differences were evaluated using a two-tailed unpaired Student t test with P < 0.05 considered as significant.

Fig. 3.

Fig. 3.

mA3 promotes TY

C

antibody mutations in specific V H genes. (A and B) The percentages of (A) AID hotspot and (B) mA3 hotspot mutations relative to the number of C or G mutations were evaluated in GC B-cell V H genes from WT (n = 3) and mA3 KO (n = 4) mice. Data are shown for V H 1–19 and V H 1–26, which are the major V H genes in the FV-specific mAb panel in Fig. 1_G_. The percentage of TC

C

mutations in V H 1–18 is shown in B. Differences were evaluated using a two-tailed unpaired Student t test with P < 0.05 considered as significant. (C) Prevalence of nonsynonymous TY

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mutations in WT versus mA3 KO IgH sequences. TY

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mutations from B6 WT (n = 16,680 noncollapsed sequence reads from three mice) and B6 mA3 KO (n = 51,096 noncollapsed sequence reads from four mice) sequences were combined, and those sequence reads that resulted in amino acid changes were evaluated in 2 × 2 contingency analyses. (Left) Five nonsynonymous V H 1–19 TY

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substitutions found in >0.4% of sequence reads in WT mice were compared with sequence reads in KO mice by Fisher’s exact test. Percentages from the entire V H 1–19 sequence dataset are shown in parentheses. (Right) IMGT collier-de-perles plot (27) showing the relative positions of the predicted amino acid mutations. (D) Ontogeny of nonsynonymous V H 1–19 TY

C

mutations in individual B6 WT mice. Phylogenetic trees were constructed using the neighbor-joining method, and clades supported with >70% bootstrap are indicated. Each dot within each line corresponds to a unique sequence. Note that M39I was detected in all three mice and that lineages with two TY

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mutations could emerge.

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