Affinity maturation of the humoral response is mediated by somatic hypermutation of the immunoglobulin (Ig) genes and selection of higher-affinity B cell clones. Activation-induced cytidine deaminase (AID) is the first of a complex series of proteins that introduce these point mutations into variable regions of the Ig genes. AID deaminates deoxycytidine residues in single-stranded DNA to deoxyuridines, which are then processed by DNA replication, base excision repair (BER), or mismatch repair (MMR). In germinal center B cells, MMR, BER, and other factors are diverted from their normal roles in preserving genomic integrity to increase diversity within the Ig locus. Both AID and these components of an emerging error-prone mutasome are regulated on many levels by complex mechanisms that are only beginning to be elucidated.

Background: Exosomes are extracellular vesicles that have been implicated in intercellular communication.Results: Exosomes that originate from human cells infected with HIV-1 contain virus-derived small noncoding RNA. Conclusion: Virus-derived small RNA present in exosomes exert functional consequences in naive recipient cells. Significance: Viral RNA molecules present in exosomes may be critical mediators of intercellular viral spread in infected hosts.

No abstract

Various epigenetic marks at the HIV-1 5′LTR suppress proviral expression and promote latency. Cellular antisense transcripts known as long noncoding RNAs (lncRNAs) recruit the polycomb repressor complex 2 (PRC2) to gene promoters, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), thus promoting nucleosome assembly and suppressing gene expression. We found that an HIV-1 antisense transcript expressed from the 3′LTR and encoding the antisense protein ASP promotes proviral latency. Expression of ASP RNA reduced HIV-1 replication in Jurkat cells. Moreover, ASP RNA expression promoted the establishment and maintenance of HIV-1 latency in Jurkat E4 cells. We show that this transcript interacts with and recruits PRC2 to the HIV-1 5′LTR, increasing accumulation of the suppressive epigenetic mark H3K27me3, while reducing RNA Polymerase II and thus proviral transcription. Altogether, our results suggest that the HIV-1 ASP transcript promotes epigenetic silencing of the HIV-1 5′LTR and proviral latency through the PRC2 pathway.

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase–mediated cytidine deamination of immunoglobulin genes. MutS homologue (Msh) 2−/− mice have reduced A-T mutations and CSR. This suggests that Msh2 may play a role in repairing activation-induced cytidine deaminase–generated G-U mismatches. However, because Msh2 not only initiates mismatch repair but also has other functions, such as signaling for apoptosis, it is not known which activity of Msh2 is responsible for the effects observed, and consequently, many models have been proposed. To further dissect the role of Msh2 in SHM and CSR, mice with a “knockin” mutation in the Msh2 gene that inactivates the adenosine triphosphatase domain were examined. This mutation (i.e., Msh2G674A), which does not affect apoptosis signaling, allows mismatches to be recognized but prevents Msh2 from initiating mismatch repair. Here, we show that, similar to Msh2−/− mice, SHM in Msh2G674A mice is biased toward G-C mutations. However, CSR is partially reduced, and switch junctions are more similar to those of postmeiotic segregation 2−/− mice than to Msh2−/− mice. These results indicate that Msh2 adenosine triphosphatase activity is required for A-T mutations, and suggest that Msh2 has more than one role in CSR.

After encounter with antigen, the antibody repertoire is shaped by somatic hypermutation (SHM), which leads to an increase in the affinity of antibodies for the antigen, and class-switch recombination (CSR), which results in a change in the effector function of antibodies. Both SHM and CSR are initiated by activation-induced cytidine deaminase (AID), which deaminates deoxycytidine to deoxyuridine in single-stranded DNA (ssDNA). The precise mechanism responsible for the formation of ssDNA in V regions undergoing SHM has yet to be experimentally established. In this study, we searched for ssDNA in mutating V regions in which DNA–protein complexes were preserved in the context of chromatin in human B cell lines and in primary mouse B cells. We found that V regions that undergo SHM were enriched in short patches of ssDNA, rather than R loops, on both the coding and noncoding strands. Detection of these patches depended on the presence of DNA-associated proteins and required active transcription. Consistent with this, we found that both DNA strands in the V region were transcribed. We conclude that regions of DNA that are targets of SHM assemble protein–DNA complexes in which ssDNA is exposed, making it accessible to AID.

c Resting memory CD4؉ T cells are the largest reservoir of persistent infection in HIV-1-positive subjects. They harbor dormant, stably integrated virus despite suppressive antiretroviral therapy, posing an obstacle to a cure. Surface markers that identify latently infected cells remain unknown. Microarray analyses comparing resting latently infected and uninfected CD4 ؉ T cells generated in vitro showed profound differences in the expression of gene programs related to transcriptional and posttranscriptional regulation, cell proliferation, survival, cycle progression, and basic metabolism, suggesting that multiple biochemical and metabolic blocks contribute to preventing viral production in latently infected cells. We identified 33 transcripts encoding cell surface markers that are differentially expressed between latently infected and uninfected cells. Quantitative reverse transcriptase PCR (RT-QPCR) and flow cytometry analyses confirmed that the surface marker CD2 was expressed at higher levels on latently infected cells. To validate this result in vivo, we sorted resting memory CD4 ؉ T cells expressing high and low surface levels of CD2 from six HIV-1-infected subjects successfully treated with antiretroviral drugs for at least 3 years. Resting memory CD4 ؉ CD2high T cells from all subjects harbored higher HIV-1 DNA copy numbers than all other CD4 ؉ T cell subsets. Moreover, after ex vivo viral reactivation, robust viral RNA production was detected only from resting memory CD4 ؉ CD2 high T cells but not from other cell subsets. Altogether, these results show that a high CD2 expression level is a hallmark of latently infected resting memory CD4 ؉ T cells in vivo.

Somatic hypermutation and class switch recombination (CSR) contribute to the somatic diversification of antibodies. It has been shown that MutS homologue (Msh)6 (in conjunction with Msh2) but not Msh3 is involved in generating A/T base substitutions in somatic hypermutation. However, their roles in CSR have not yet been reported. Here we show that Msh6 − / − mice have a decrease in CSR, whereas Msh3 − / − mice do not. When switch regions were analyzed for mutations, deficiency in Msh6 was associated with an increase in transition mutations at G/C basepairs, mutations at RGYW/WRCY hotspots, and a small increase in the targeting of G/C bases. In addition, Msh6 − / − mice exhibited an increase in the targeting of recombination sites to GAGCT/GGGGT consensus repeats and hotspots in Sγ3 but not in Sμ. In contrast to Msh2 − / − mice, deficiency in Msh6 surprisingly did not change the characteristics of Sμ-Sγ3 switch junctions. However, Msh6 − / − mice exhibited a change in the positioning of Sμ and Sγ3 junctions. Although none of these changes were seen in Msh3 − / − mice, they had a higher percentage of large inserts in their switch junctions. Together, our data suggest that MutS homologues Msh2, Msh3, and Msh6 play overlapping and distinct roles during antibody diversification processes.