Class-switched memory B cells remodel BCRs within secondary germinal centers.

Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.

Pseudorabies Virus DNA Polymerase Processivity Factor UL42 Inhibits Type I IFN Response by Preventing ISGF3-ISRE Interaction.

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.

Tet DNA demethylase is required for plasma cell differentiation by controlling expression levels of IRF4.

Antibodies produced by plasma cells are critical for protection from infection. It has been demonstrated that global epigenetic modification, such as changes in DNA methylation, occurs during differentiation of plasma cells from B cells. However, the precise mechanisms by which DNA methylation controls plasma cell differentiation are not fully understood. We examined the effect of deficiency of DNA demethylases, Tet2 and Tet3, on B-cell activation and plasma cell differentiation, by generating conditional Tet2/3 double-KO (Tet dKO) B cells. We found that Tet dKO B cells failed to differentiate into plasma cells upon immunization with antigens. Tet dKO B cells proliferated normally and were capable of generating cells with IRF4int, but not with IRF4hi, the majority of which were CD138+ plasma cells. IRF4 overexpression rescued the defect of Tet dKO B cells in plasma cell differentiation, suggesting that Tet2/3-dependent high IRF4 expression is required for plasma cell differentiation. We identified CpG sites in the Irf4 locus that were demethylated specifically in plasma cells and in a Tet2/3-dependent manner. Our results suggest that Tet2/3-dependent demethylation of these CpG sites is dispensable for initial IRF4 expression but is essential for high IRF4 expression which is prerequisite for plasma cell differentiation.

Vitamin C Facilitates Demethylation of the Foxp3 Enhancer in a Tet-Dependent Manner.

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-ß (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.

TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBV-persistent mice.

OBJECTIVE: To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB). METHODS: TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status. RESULTS: In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (i.e., capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range. CONCLUSIONS: Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB.

Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: a new perspective for vaccine development.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX-specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the NmX CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.

The intracellular DNA sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication.

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between -54 and -43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor.

Immune-induced evolutionary selection focused on a single reading frame in overlapping hepatitis B virus proteins.

Viruses employ various means to evade immune detection. Reduction of CD8(+) T cell epitopes is one of the common strategies used for this purpose. Hepatitis B virus (HBV), a member of the Hepadnaviridae family, has four open reading frames, with about 50% overlap between the genes they encode. We computed the CD8(+) T cell epitope density within HBV proteins and the mutations within the epitopes. Our results suggest that HBV accumulates escape mutations that reduce the number of epitopes. These mutations are not equally distributed among genes and reading frames. While the highly expressed core and X proteins are selected to have low epitope density, polymerase, which is expressed at low levels, does not undergo the same selection. In overlapping regions, mutations in one protein-coding sequence also affect the other protein-coding sequence. We show that mutations lead to the removal of epitopes in X and surface proteins even at the expense of the addition of epitopes in polymerase. The total escape mutation rate for overlapping regions is lower than that for nonoverlapping regions. The lower epitope replacement rate for overlapping regions slows the evolutionary escape rate of these regions but leads to the accumulation of mutations more robust in the transfer between hosts, such as mutations preventing proteasomal cleavage into epitopes.

Sindbis virus replicase-based DNA vaccine construct encoding FMDV-specific multivalent epitope gene: studies on its immune responses in guinea pigs.

Foot-and-mouth disease (FMD) is still a perennial global menace affecting livestock health and production. It is imperative to figure out new ways to curb this disease. In this study, a sindbis virus replicase-based DNA vaccine, pSinCMV-Vac-MEG990, encoding a multivalent epitope gene (representing tandemly linked VP1 C-terminal halves of three foot-and-mouth disease virus (FMDV) serotypes) was constructed. In vitro transfection studies in BHK-21 cells revealed that the construct was able to express FMDV-specific antigen but does not overproduce the antigen. Immunization of guinea pigs with the construct at dose rate of 10, 5, 2 and 1 µg per animal through intramuscular route showed significant neutralizing antibody induction at all doses against all serotype tested as compared to non-immunized controls. On viral challenge of guinea pigs 4 week post-immunization with 1000 GPID(50) of FMDV serotype A, it was observed that the immunization not only delayed the appearance and reduced the severity of FMD lesions significantly (P < 0.05) but also provided complete protection in several guinea pigs. In fact, two of six and one of six guinea pigs were completely protected in 10 and 5 µg immunized groups, respectively. These results suggest that the development of the replicase-based DNA vaccine may provide a promising approach as an alternative vaccine strategy for controlling FMD.

A backup role of DNA polymerase kappa in Ig gene hypermutation only takes place in the complete absence of DNA polymerase eta.

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.

The Functions of Hepatitis B Virus Encoding Proteins: Viral Persistence and Liver Pathogenesis.

About 250 million people worldwide are chronically infected with Hepatitis B virus (HBV), contributing to a large burden on public health. Despite the existence of vaccines and antiviral drugs to prevent infection and suppress viral replication respectively, chronic hepatitis B (CHB) cure remains a remote treatment goal. The viral persistence caused by HBV is account for the chronic infection which increases the risk for developing liver cirrhosis and hepatocellular carcinoma (HCC). HBV virion utilizes various strategies to escape surveillance of host immune system therefore enhancing its replication, while the precise mechanisms involved remain elusive. Accumulating evidence suggests that the proteins encoded by HBV (hepatitis B surface antigen, hepatitis B core antigen, hepatitis B envelope antigen, HBx and polymerase) play an important role in viral persistence and liver pathogenesis. This review summarizes the major findings in functions of HBV encoding proteins, illustrating how these proteins affect hepatocytes and the immune system, which may open new venues for CHB therapies.

Preparation and functional evaluation of monoclonal antibodies targeting Hepatitis B Virus Polymerase.

HBV pol plays a critical role in the replication of hepatitis B virus (HBV). Previous studies conducted on HBV pol have produced limited evidence on HBV pol expression due to the lack of effective detection methods. The present study used the HBV pol (159-406 aa) protein as a target to screen for specific monoclonal antibodies that recognize HBV pol and subsequently evaluate their diagnostic and therapeutic value. Four antibodies (P3, P5, P12, P20) against HBV pol were obtained. Among them, the P20 antibody indicated optimal binding with HBV pol as demonstrated by Western blotting (WB) in a cell model transfected with the HBV genome. We also expressed P5 and P12 antibodies in mouse liver cells by transfection and the results indicated significant antiviral effects caused by these two antibodies especially P12. In summary, the present study established an antibody which was denoted P20. This antibody can be used to detect HBV pol expression by four HBV genomes via WB analysis. In addition, the antibody denoted P12 could exert antiviral effects via intracellular expression, which may provide a promising approach for the treatment of chronic hepatitis B.

Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.

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.

Immune cell infiltrate-associated dysregulation of DNA repair machinery may predispose to papillary thyroid carcinogenesis.

BACKGROUND: An altered immune microenvironment may contribute to papillary thyroid cancer development, as immune infiltrates are identified postoperatively in many papillary thyroid cancer cases with or without diagnosed thyroiditis. Oxygen radicals, endogenous or inflammation-induced, can generate DNA damage, which causes mutations when repaired incorrectly. We hypothesized that infiltrating immune cells might promote aberrant DNA repair, predisposing thyrocytes to papillary thyroid cancer. METHODS: Quantitative reverse-transcriptase polymerase chain reaction assays measured gene expression in fresh-frozen samples (n = 55). RNA-seq data was obtained for papillary thyroid cancer and normal thyroid samples from the Cancer Genome Atlas (n = 564), and Hashimoto's-affected and normal thyroids from the Genotype-Tissue Expression project (n = 279). Immune cell marker expression levels were compared to histological estimates and to selected DNA repair genes. Immunohistochemistry localized gene expression to specific cell types. RESULTS: DNA polymerase theta expression by quantitative reverse-transcriptase Polymerase chain reaction was higher in papillary thyroid cancer and papillary thyroid cancer-adjacent samples than in benign normal thyroid (P < .001). Immune markers including CD4 correlated with DNA polymerase theta expression (r = 0.50) but not other DNA repair genes examined. Benign tissue with Hashimoto's exhibited increased DNA polymerase theta (P < .0001) and CD3E (P < .0001) expression. DNA polymerase theta localized to thyrocytes, not lymphocytes. CONCLUSION: We identified a strong correlation between immune cell infiltrate and dysregulated thyrocyte DNA repair genes, likely reflecting a pathway to papillary thyroid cancer development.

The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques.

BACKGROUND: ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. RESULTS: We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses. CONCLUSION: The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV.

DNA polymerase with characteristics of reverse transcriptase purified from human milk.

A DNA polymerase purified from a particulate fraction of human milk has biochemical and biophysical properties similar to those of viral reverse transcriptases. This enzyme is immunologically distinct from cellular DNA polymerases obtained from a variety of human sources.

DNA polymerase kappa counteracts inflammation-induced mutagenesis in multiple organs of mice.

In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)-induced N2 -deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock-in (Polk-/- ) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk-/- and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk-/- mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk-/- mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk-/- than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP-induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation-induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320-330, 2019. © 2019 Wiley Periodicals, Inc.

Infection of human B cells with Epstein-Barr virus results in the expression of somatic hypermutation-inducing molecules and in the accrual of oncogene mutations.

Infection of human B cells with Epstein-Barr virus (EBV) was seen to result in activation-induced cytidine deaminase (AID) and polymerase-eta (pol-eta) gene expression. AID and pol-eta are cellular gene products that play central roles in the DNA-modifying processes involved in immunoglobulin gene class switch recombination and somatic hypermutation. Errors in these processes can result in oncogene mutation/translocation, thereby contributing to lymphomagenesis. It was seen that EBV infected, AID, and pol-eta expressing B cells accumulated mutations in cellular proto-oncogenes (BCL-6 and p53) that are known to be involved in the genesis of B cell lymphoma. The nature of the mutations seen in these oncogenes was consistent with the known activity of AID and pol-eta. These findings indicate that EBV induced AID and pol-eta expression, and that this was associated with oncogene mutation, providing a novel means by which EBV infection of B cells may contribute to lymphomagenesis.

Potential inhibition of somatic hypermutation by nucleoside analogues.

Somatic hypermutation, which occurs in antigen-activated germinal centre B lymphocytes, diversifies the genes that encode immunoglobulin variable regions and leads to the 'affinity maturation' of the humoral immune response. Hypermutation affects dC/dG and dA/dT pairs with approximately equal frequency in vivo. DNA polymerase-theta contributes to hypermutagenesis at dC/dG pairs and DNA polymerase-eta is substantially involved in the generation of hypermutations at dA/dT pairs. The biochemical properties of polymerases-theta and -eta indicate that their DNA synthetic activities are potentially susceptible to inhibition by nucleoside analogues, so it is feasible that nucleoside analogues reduce the accumulation of dC/dG- and dA/dT-targeted hypermutations in vivo. Nucleoside analogues could hence impair the humoral adaptive immune response of HIV-infected patients who are prescribed these chemotherapeutic agents.

Cell cycle expression of two replicative DNA polymerases alpha and delta from Schizosaccharomyces pombe.

We have investigated the expression of two Schizosaccharomyces pombe replicative DNA polymerases alpha and delta during the cell cycle. The pol alpha+ and pol delta+ genes encoding DNA polymerases alpha and delta were isolated from S. pombe. Both pol alpha+ and pol delta+ genes are single copy genes in haploid cells and are essential for cell viability. In contrast to Saccharomyces cerevisiae homologs, the steady-state transcripts of both S. pombe pol alpha+ and pol delta+ genes were present throughout the cell cycle. Sequence analysis of the pol alpha+ and pol delta+ genes did not reveal the Mlu I motifs in their upstream sequences that are involved in cell cycle-dependent transcription of S. cerevisiae DNA synthesis genes as well as the S. pombe cdc22+ gene at the G1/S boundary. However, five near-match Mlu I motifs were found in the upstream region of the pol alpha+ gene. S. pombe DNA polymerases alpha and delta proteins were also expressed constantly throughout the cell cycle. In addition, the enzymatic activity of the S. pombe DNA polymerase alpha measured by in vitro assay was detected at all stages of the cell cycle. Thus, these S. pombe replicative DNA polymerases, like that of S. pombe cdc17+ gene, are expressed throughout the cell cycle at the transcriptional and protein level. These results indicate that S. pombe has at least two regulatory modes for the expression of genes involved in DNA replication and DNA precursor synthesis.