Somatic Diversification of Rearranged Antibody Gene Segments by Intra- and Interchromosomal Templated Mutagenesis.

The ability of the humoral immune system to generate Abs capable of specifically binding a myriad of Ags is critically dependent on the somatic hypermutation program. This program induces both templated mutations (i.e., gene conversion) and untemplated mutations. In humans, somatic hypermutation is widely believed to result in untemplated point mutations. In this study, we demonstrate detection of large-scale templated events that occur in human memory B cells and circulating plasmablasts. We find that such mutations are templated intrachromosomally from IGHV genes and interchromosomally from IGHV pseudogenes as well as other homologous regions unrelated to IGHV genes. These same donor regions are used in multiple individuals, and they predominantly originate from chromosomes 14, 15, and 16. In addition, we find that exogenous sequences placed at the IgH locus, such as LAIR1, undergo templated mutagenesis and that homology appears to be the major determinant for donor choice. Furthermore, we find that donor tracts originate from areas in proximity with open chromatin, which are transcriptionally active, and are found in spatial proximity with the IgH locus during the germinal center reaction. These donor sequences are inserted into the Ig gene segment in association with overlapping activation-induced cytidine deaminase hotspots. Taken together, these studies suggest that diversity generated during the germinal center response is driven by untemplated point mutations as well as templated mutagenesis using local and distant regions of the genome.

HIV-1 variants are archived throughout infection and persist in the reservoir.

The HIV-1 reservoir consists of latently infected cells that persist despite antiretroviral therapy (ART). Elucidating the proviral genetic composition of the reservoir, particularly in the context of pre-therapy viral diversity, is therefore important to understanding reservoir formation and the persistence of latently infected cells. Here we investigate reservoir proviral variants from 13 Zambian acutely-infected individuals with additional pre-therapy sampling for a unique comparison to the ART-naïve quasispecies. We identified complete transmitted/founder (TF) viruses from seroconversion plasma samples, and additionally amplified and sequenced HIV-1 from plasma obtained one year post-infection and just prior to ART initiation. While the majority of proviral variants in the reservoir were most closely related to viral variants from the latest pre-therapy time point, we also identified reservoir proviral variants dating to or near the time of infection, and to intermediate time points between infection and treatment initiation. Reservoir proviral variants differing by five or fewer nucleotide changes from the TF virus persisted during treatment in five individuals, including proviral variants that exactly matched the TF in two individuals, one of whom had remained ART-naïve for more than six years. Proviral variants during treatment were significantly less divergent from the TF virus than plasma variants present at the last ART-naïve time point. These findings indicate that reservoir proviral variants are archived throughout infection, recapitulating much of the viral diversity that arises throughout untreated HIV-1 infection, and strategies to target and reduce the reservoir must therefore permit for the clearance of proviruses encompassing this extensive diversity.

Clustered Mutations at the Murine and Human IgH Locus Exhibit Significant Linkage Consistent with Templated Mutagenesis.

Somatic hypermutation generates a myriad of Ab mutants in Ag-specific B cells, from which high-affinity mutants are selected. Chickens, sheep, and rabbits use nontemplated point mutations and templated mutations via gene conversion to diversify their expressed Ig loci, whereas mice and humans rely solely on untemplated somatic point mutations. In this study, we demonstrate that, in addition to untemplated point mutations, templated mutagenesis readily occurs at the murine and human Ig loci. We provide two distinct lines of evidence that are not explained by the Neuberger model of somatic hypermutation: 1) across multiple data sets there is significant linkage disequilibrium between individual mutations, especially among close mutations, and 2) among those mutations, those <8 bp apart are significantly more likely to match microhomologous regions in the IgHV repertoire than predicted by the mutation profiles of somatic hypermutation. Together, this supports the role of templated mutagenesis during somatic diversification of Ag-activated B cells.