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.humoral immunity | restriction factor | antibody repertoire profiling | affinity maturation | Friend retrovirus N atural recovery from viral infections and vaccine-induced protection are typically associated with potent neutralizing antibodies (1), so it is of great importance to understand fully the mechanisms that generate high-affinity antibodies. Part of the process of affinity maturation to improve the recognition of foreign antigens involves somatic hypermutation (SHM), which occurs predominantly in germinal centers (GCs) via the enzymatic activity of activation-induced deaminase (AID) (2, 3). AID catalyzes the deamination of deoxycytidines to deoxyuridines in single-stranded Ig DNA. In vitro reconstitution and analysis of in vivo Ig mutations demonstrated that AID preferentially deaminates in the 5′-WRC-3′ context (W = A or T; R = purine = A or G; the underlined C corresponds to the deaminated deoxycytidine) (4-6). On the other hand, a deoxycytidine preceded by a pyrimidine, e.g., in the dinucleotide context YC (Y = C or T) is referred to as an AID "coldspot." It is well established that AID is the critical enzyme mediating antibody class-switching and SHM. However, it remains unknown if other deaminases can complement AID in driving SHM.The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 3 (APOBEC3) proteins are deoxycytidine deaminases expressed in lymphocytes, including T and B cells, that can restrict retroviruses, including HIV-1 (7). APOBEC3 becomes packaged into virions and inhibits reverse transcription in the next target cell. Retrovirus inhibition could occur through the enzymatic action of APOBEC3, which involves deaminating deoxyc...

Knowledge of the processes that underlie IgG subclass switching could inform strategies designed to counteract infections and autoimmunity. Here we show that TLR7 ligands induce subsets of memory CD4 and CD8 T cells to secrete interferon γ (IFNγ) in the absence of antigen receptor stimulation. In turn, TLR ligation and IFNγ cause B cells to express the transcription factor, T-bet, and to switch immunoglobulin production to IgG2a/c. Absence of TLR7 in T cells leads to the impaired T-bet expression in B cells and subsequent inefficient IgG2a isotype switching both in vitro and during the infection with Friend virus in vivo. Our results reveal a surprising mechanism of antiviral IgG subclass switching through T-cell intrinsic TLR7/IL-12 signaling.

Major conceptual roadblocks impede the development of an HIV-1 vaccine that can stimulate a potent neutralizing antibody response. Animal models that support HIV-1 replication and allow for host genetic manipulation would be an ideal platform for testing various immunological hypotheses, but progress on this research front has been slow and disappointing. In contrast, many valuable concepts emerged from more than 50 years of studying the Friend retrovirus model. This was recently exemplified by the identification of an innate restriction gene, Apobec3, that could promote the retrovirus-specific neutralizing antibody response. Here we review both classical and recent data on humoral immunity against Friend retrovirus infection, and highlight the potential of this model for unraveling novel aspects of the retrovirus-specific antibody response that may guide HIV-1 vaccine development efforts.

B cell functional defects are associated with delayed neutralizing antibody development in pathogenic lentivirus infections. However, the timeframe for alterations in the antibody repertoire and somatic hypermutation (SHM) remains unclear. Here, we utilized the SIV/rhesus macaque (RM) model to investigate the dynamics of immunoglobulin VH gene diversity and SHM following infection. Three RMs were infected with SIVmac239 and VH1, VH3 and VH4 genes were amplified from peripheral blood at 0, 2, 6, 24 and 36 weeks post-infection for next-generation sequencing. Analysis of over 3.8 million sequences against currently available RM germline VH genes revealed a highly biased VH gene repertoire in outbred RMs. SIV infection did not significantly perturb the predominant IgG1 response, but overall immunoglobulin SHM declined during the course of SIV infection. Moreover, SHM at the AID deamination hotspot, WRC, rapidly decreased and was suppressed throughout SIV infection. In contrast, a transient increase in mutations at the APOBEC3G deamination hotspot, CCC, coincided with a spike in APOBEC3G expression during acute SIV infection. The results outline a timetable for altered VH gene repertoire and IgG SHM in the SIV/RM model and suggest a burst of APOBEC3G-mediated antibody SHM during acute SIV infection.

Ribonuclease L (RNase L) is a Type I Interferon regulated factor that can significantly inhibit retroviruses in vitro and may activate cytoplasmic sensing pathways to augment adaptive immunity. However, the antiretroviral activity of RNase L remains to be validated in vivo. We investigated the role of RNaseL in counteracting Friend retrovirus (FV) infection relative to a well-described restriction factor, Apobec3. C57BL/6 wild-type (WT) and RNaseL knock-out (KO) mice exhibited similar acute FV infection levels despite significant transcriptional induction of Oligoadenylate Synthetase 1, which produces activators of RNase L. Apobec3 KO mice showed higher FV infection levels relative to WT mice, but deletion of RNaseL in Apobec3 KO mice did not augment FV infection. Moreover, RNaseL did not influence FV-specific IgG responses and recovery from viremia by 28 days post-infection. The results suggest that RNase L is not an evolutionarily-conserved host defense mechanism to counteract retroviruses in vivo.