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Alphaherpesviruses are large dsDNA viruses with an ability to establish persistent infection in hosts, which rely partly on their ability to evade host innate immune responses, notably the type I IFN response. However, the relevant molecular mechanisms are not well understood. In this study, we report the UL42 proteins of alphaherpesvirus pseudorabies virus (PRV) and HSV type 1 (HSV1) as a potent antagonist of the IFN-I-induced JAK-STAT signaling pathway. We found that ectopic expression of UL42 in porcine macrophage CRL and human HeLa cells significantly suppresses IFN-α-mediated activation of the IFN-stimulated response element (ISRE), leading to a decreased transcription and expression of IFN-stimulated genes (ISGs). Mechanistically, UL42 directly interacts with ISRE and interferes with ISG factor 3 (ISGF3) from binding to ISRE for efficient gene transcription, and four conserved DNA-binding sites of UL42 are required for this interaction. The substitution of these DNA-binding sites with alanines results in reduced ISRE-binding ability of UL42 and impairs for PRV to evade the IFN response. Knockdown of UL42 in PRV remarkably attenuates the antagonism of virus to IFN in porcine kidney PK15 cells. Our results indicate that the UL42 protein of alphaherpesviruses possesses the ability to suppress IFN-I signaling by preventing the association of ISGF3 and ISRE, thereby contributing to immune evasion. This finding reveals UL42 as a potential antiviral target., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Demethylation of CpG motifs in the Foxp3 intronic element, conserved noncoding sequence 2 (CNS2), is indispensable for the stable expression of Foxp3 in regulatory T cells (Tregs). In this study, we found that vitamin C induces CNS2 demethylation in Tregs in a ten-eleven-translocation 2 (Tet2)-dependent manner. The CpG motifs of CNS2 in Tregs generated in vitro by TGF-β (iTregs), which were methylated originally, became demethylated after vitamin C treatment. The conversion of 5-methylcytosin into 5-hydroxymethylcytosin was more efficient, and the methyl group from the CpG motifs of Foxp3 CNS2 was erased rapidly in iTregs treated with vitamin C. The effect of vitamin C disappeared in Tet2(-/-) iTregs. Furthermore, CNS2 in peripheral Tregs in vivo, which were demethylated originally, became methylated after treatment with a sodium-dependent vitamin C transporter inhibitor, sulfinpyrazone. Finally, CNS2 demethylation in thymic Tregs was also impaired in Tet2(-/-) mice, but not in wild type mice, when they were treated with sulfinpyrazone. Collectively, vitamin C was required for the CNS2 demethylation mediated by Tet proteins, which was essential for Foxp3 expression. Our findings indicate that environmental factors, such as nutrients, could bring about changes in immune homeostasis through epigenetic mechanisms., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Patients with the variant form of xeroderma pigmentosum (XPV) syndrome have a genetic deficiency in DNA polymerase (Pol) eta, and display accordingly an increased skin sensitivity to UV light, as well as an altered mutation pattern of their Ig V genes in memory B cells, alteration that consists in a reduced mutagenesis at A/T bases. We previously suggested that another polymerase with a different mutation signature, Pol kappa, is used as backup for Ig gene hypermutation in both humans and mice in cases of complete Pol eta deficiency, a proposition supported in this study by the analysis of Pol eta x Pol kappa double-deficient mice. We also describe a new XPV case, in which a splice site mutation of the first noncoding exon results in a decreased mRNA expression, a mRNA that otherwise encodes a normal Pol eta protein. Whereas the Pol eta mRNA level observed in patient's fibroblasts is one-twentieth the value of healthy controls, it is only reduced to one-fourth of the normal level in activated B cells. Memory B cells from this patient showed a 50% reduction in A/T mutations, with a spectrum that still displays a strict Pol eta signature. Pol eta thus appears as a dominant enzyme in hypermutation, its presence precluding the use of a substitute enzyme even in conditions of reduced availability. Such a dominant behavior may explain the lack of Pol kappa signature in Ig gene mutations of some XPV patients previously described, for whom residual Pol eta activity might exist.

Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous V(H) and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in V(H) genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the V(H) gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.

Mutations arising in Ig V genes during an immune response are most likely introduced by one or several error-prone DNA polymerases. Many of the recently described nonreplicative DNA polymerases have an intrinsic fidelity compatible with such an activity, the strongest candidates being polymerase (pol) eta, pol iota, pol zeta, and pol mu. We report in this work that mice inactivated for either of the two polymerases related to pol beta (i.e., pol mu and pol lambda) are viable and fertile and display a normal hypermutation pattern.

Autoantibodies to nuclear and smooth muscle are common in hepatitis B virus (HBV) infection. To understand their origin, we scanned protein databases and found that HBV-DNA polymerase (HBV-pol) shares 7-9 amino acid sequences with nuclear (MHC II trans-activator, nuclear pore core protein, nuclear mitotic apparatus, and polymyositis sclerosis Ag) and smooth muscle proteins (caldesmon and myosin). Twenty-mer peptides with relevant homologues and an irrelevant control peptide were constructed and ELISAs were established. Sixty-five children with chronic HBV infection, 104 patients with other chronic liver diseases (CLD), 36 patients with extrahepatic autoimmune diseases, and 24 healthy controls were investigated. Double reactivity to HBV-pol peptides and corresponding self homologues was observed in 40% of HBV-positive patients as compared with four (4%) with other chronic liver diseases, two (6%) with extrahepatic autoimmune diseases, and in none of the healthy controls (p < 0.001 for all). Double reactivity to myosin or caldesmon peptides and their HBV-pol homologues was associated with anti-smooth muscle Ab positivity by immunofluorescence (p < 0.05 for both). HBV-positive sera double reactive for myosin or caldesmon and their homologous HBV-pol peptides also reacted with the native proteins on immunoblot. Fifty to ninety percent Ab inhibition to individual HBV-pol and HBV-pol99-118 peptides was noted by preincubation with individual HBV-pol/self homologue peptide and native proteins, respectively, but not with control peptide. Our results show that cross-reactive immunity targeting homologous sequences of viral and self proteins may partly account for autoantibody production in HBV infection.

S-Antigen (S-Ag) is a well characterized 45,000 m.w. photoreceptor cell protein. When injected into susceptible animal species, including primates, it induces an experimental autoimmune uveitis, a predominantly T cell-mediated autoimmune disease of the retina and uveal tract of the eye, and of the pineal gland. In this study we found an amino acid sequence homology between a uveitopathogenic site of S-Ag, several viral proteins and one additional nonviral protein. An experimental autoimmune uveitis and pinealitis was induced in Lewis rats with these different synthetic peptides, corresponding to the amino sequence of hepatitis B virus DNA polymerase, gag-pol polyprotein of Baboon endogenous virus and gag-pol polyprotein of AKV murine leukemia virus and potato proteinase inhibitor IIa, which contain three or more consecutive amino acids identical to peptide M in S-Ag. Lymph node cells from rats immunized with either peptide M or the different synthetic peptides showed a significant degree of cross-reaction. Mononuclear cells from monkeys (Macaca fascicularis) immunized with peptide M also showed significant proliferation when incubated with either peptide M or synthetic peptides as measured by in vitro lymphocyte mitogenesis assay using [3H]TdR. Based on our findings we conclude that a viral infection may sensitize the mononuclear cells that can cross-react with self proteins by a mechanism termed molecular mimicry. Tissue injury from the resultant autoantigenic event can take place in the absence of the infectious virus that initiated the immune response.