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Aims: Primary head/neck mucosal melanomas (MMs) are rare and exhibit aggressive biologic behaviour and elevated mutational loads. The molecular mechanisms responsible for high genomic instability observed in head/neck MMs remain elusive. The DNA cytosine deaminase APOBEC3B (A3B) constitutes a major endogenous source of mutation in human cancer. A3B‐related mutations are identified through C‐to‐T/−G base substitutions in 5′‐TCA/T motifs. Herein, we present immunohistochemical and genomic data supportive of a role for A3B in head/neck MMs. Methods and results: A3B protein levels were assessed in oral (n = 13) and sinonasal (n = 13) melanomas, and oral melanocytic nevi (n = 13) by immunohistochemistry using a custom rabbit α‐A3B mAb (5210‐87‐13). Heterogeneous, selective‐to‐diffuse, nuclear only, A3B immunopositivity was observed in 12 of 13 (92.3%) oral melanomas (H‐score range = 9–72, median = 40) and 8 of 13 (62%) sinonasal melanomas (H‐score range = 1–110, median = 24). Two cases negative for A3B showed prominent cytoplasmic staining consistent with A3G. A3B protein levels were significantly higher in oral and sinonasal MMs than intraoral melanocytic nevi (P < 0.0001 and P = 0.0022, respectively), which were A3B‐negative (H‐score range = 1–8, median = 4). A3B levels, however, did not differ significantly between oral and sinonasal tumours (P > 0.99). NGS performed in 10 sinonasal MMs revealed missense NRAS mutations in 50% of the studied cases and one each KIT and HRAS mutations. Publicly available whole‐genome sequencing (WGS) data disclosed that the number of C‐to‐T mutations and APOBEC3 enrichment score were markedly elevated in head/neck MMs (n = 2). Conclusion: The above data strongly indicate a possible role for the mutagenic enzyme A3B in head/neck melanomagenesis, but not benign melanocytic neoplasms. [ABSTRACT FROM AUTHOR]

Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CLpro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CLpro, and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CLpro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains. Assessing antiviral activity: Nirmatrelvir, ensitrelvir, and GC376 are SARS-CoV-2 3CLpro inhibitors that provide a promising strategy for treating individuals with COVID-19. However, as antivirals become more widely used, SARS-CoV-2 may develop mutations that confer resistance against these antivirals. Here, Heilmann et al. developed a vesicular stomatitis virus (VSV)–based system, where the SARS-CoV-23CLpro was essential for replication of VSV. The authors used this system to select for nirmaltrelvir resistance mutations, identifying several. Some of the mutations were specific to nirmaltrelvir, whereas others conferred resistance to additional 3CLpro inhibitors. The authors validated the identified resistance-conferring mutations using multiple other strategies, suggesting that this VSV-based system could be used to further understand SARS-CoV-2 antiviral resistance. —CM [ABSTRACT FROM AUTHOR]

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions (1-9). Most of these [...]

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication (1-3). The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host's antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation (4-10). Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-b is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β-Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins., Mammals have evolved cellular proteins termed restriction factors that function to prevent the spread of mobile genetic elements including retroviruses (1-3). As a counter-defence, most retroviruses, including the human pathogen [...]

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons (1-6). [...]

Ig class switch recombination (CSR) and somatic hypermutation serve to diversify antibody responses and are orchestrated by the activity of activation-induced cytidine deaminase and many proteins involved in DNA repair and genome surveillance. Msh5, a gene encoded in the central MHC class III region, and its obligate heterodimerization partner Msh4 have a critical role in regulating meiotic homologous recombination and have not been implicated in CSR. Here, we show that MRL/Ipr mice carrying a congenic [H-2.sup.b/b] MHC interval exhibit several abnormalities regarding CSR, including a profound deficiency of IgG3 in most mice and long micro-homologies at Ig switch (S) joints. We found that Msh5 is expressed at low levels on the [H-2.sup.b] haplotype and, importantly, a similar long S joint microhomology phenotype was observed in both Msh5 and Msh4-null mice. We also present evidence that genetic variation in MSH5 is associated with IgA deficiency and common variable immune deficiency (CVID) in humans. One of the human MSH5 alleles identified contains two nonsynonymous polymorphisms, and the variant protein encoded by this allele shows impaired binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA deficiency patients carrying disease-associated MSH5 alleles show increased donor/acceptor microhomology, involving pentameric DNA repeat sequences and lower mutation rates than controls. Our findings suggest that Msh4/5 heterodimers contribute to CSR and support a model whereby Msh4/5 promotes the resolution of DNA breaks with low or no terminal microhomology by a classical nonhomologous end-joining mechanism while possibly suppressing an alternative microhomology-mediated pathway. immunoglobulin subclass deficiency | mismatch repair | Msh4

Functional antibody genes assembled by V(D)J joining are subsequently diversified by somatic hypermutation, gene conversion and class-switch recombination. Recent evidence indicates that all three processes are caused by the deamination of cytosine to uracil at sites within the immunoglobulin (Ig) loci, with the pattern of diversification depending on the pathway used for resolving the initiating dU-dG lesion. Whereas DNA deamination targeted to the endogenous Ig locus triggers a program of somatic gene diversification that underpins adaptive immunity, deamination targeted to foreign DNA might have arisen initially as a form of innate immunity. Furthermore, the observation that members of the DNA deaminase family can target inappropriate genes suggests they might also contribute to mutations during genome evolution, as well as in cancer.

Author(s): Svend K. Petersen-Mahrt [1]; Reuben S. Harris [1]; Michael S. Neuberger (corresponding author) After gene rearrangement, immunoglobulin variable genes are diversified by somatic hypermutation or gene conversion, whereas the [...]