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Somatic hypermutation has two phases: phase 1 affects cytosine-guanine (C/G) pairs and is triggered by the deamination of cytosine residues in DNA to uracil; phase 2 affects mostly adenine-thymine (A/T) pairs and is induced by the detection of uracil lesions in DNA. It is not known how, at V(D)J genes in mice, hypermutations accumulate at A/T pairs with strand bias without perturbing the strand unbiased accumulation of hypermutations at C/G pairs. Additionally, it is not known why, in contrast, at switch regions in mice, both C/G-targeted and A/T-targeted hypermutations accumulate in a strand unbiased manner. To explain the strand bias paradox, we propose that phase 1 and phase 2 hypermutations are generated at different stages of the cell cycle.

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.

The "affinity maturation" of the humoral immune response is driven by antigen-activated somatic hypermutation (SHM) of the genes that encode antibody variable regions and the subsequent antigenic selection of mutant clones. The molecular mechanism of SHM is yet to be completely elucidated. SHM affects cytosine-guanine (C/G) and adenine-thymine (A/T) pairs with approximately equal frequency in vivo. The proposition that error-prone DNA-dependent DNA synthesis explains A/T-targeted hypermutagenesis seems to have mainstream support within the hypermutation research community at present. A major feature of SHM in vivo is that C/G hypermutation is strand unbiased, whereas A/T hypermutation is strand biased. We show that the "DNA-based polymerase error" model of A/T-targeted hypermutagenesis does not explain this important aspect of SHM.

A major function of J-C intronic matrix attachment regions (MAR) during immune diversification via somatic hypermutation (SHM) at immunoglobulin loci may be to manipulate the topology of DNA within the upstream target domain. The suggestion that SHM induction requires MAR-induced torsional strain, in conjunction with DNA remodelling at the J-C intron, completes the definition of a cogent paradigm within which all extant molecular data on the issue may be interpreted. Moreover, the suggestion that a mutagenic mechanism relieves MAR-generated superhelicity could provide an indication as to the evolutionary basis of SHM.

Somatic hypermutation (SHM) diversifies the genes that encode immunoglobulin variable regions in antigen-activated germinal centre B lymphocytes. Available evidence strongly suggests that DNA deamination potentiates phase I SHM and subsequently triggers phase II SHM. A concise review of this evidence is followed by a detailed critique of two possible models which suggest that polymerase-eta potentiates phase II SHM via either its DNA-dependent or its RNA-dependent DNA synthetic activity. Quantitative analysis, in the context of extant data that define the features of SHM, favours the RNA-dependent mechanism.

The history and current development of the reverse transcriptase model of somatic hypermutation (RT-model) is reviewed with particular reference to the genesis of strand-biased mutation signatures in rearranged immunoglobulin variable genes (V(D)J). The recent disagreement in the field as to whether strand bias really exists or not has been critically analysed and the confusion traced to the putative presence, in some mutated V(D)J sequence collections, of polymerase chain reaction (PCR)-recombinant artefacts. Recent analysis of somatic hypermutation in xeroderma pigmentosum variant patients, by the group of PJ Gearhart and others, has established that the Y-family translesion DNA repair enzyme, DNA polymerase eta (eta), is responsible for the striking A-T targeted strand-bias mutation signature seen in all mouse and human collections of somatically mutated V(D)J sequences. This evidence, together with our own recent demonstration that human DNA polymerase eta is a reverse transcriptase, leads to the conclusion that the strand-biased A-T mutation signature is caused either by: (i) error-prone DNA-dependent DNA repair synthesis by pol-eta of single-strand nicks preferentially in the non-transcribed strand; and/or (ii) by error-prone cDNA synthesis of the transcribed strand by pol-eta using the pre-mRNA as the copying template, primed by the nicked transcribed DNA strand, followed by replacement of the original transcribed strand by cDNA. Analysis of the total mutation pattern also suggests that the major transitions observed in SHM (A-->G, C-->T and G-->A) can be explained by known RNA editing mechanisms active on pre-mRNA which are then written into cDNA during synthesis of the transcribed strand by error-prone cellular reverse transcriptases such as pol-eta.

Available evidence about the mechanisms and distribution of somatic hypermutation (SHM) of rearranged immunoglobulin (IgV) genes is reviewed with particular emphasis on the 5' boundary. In heavy (H) chain genes, the 5' boundary of SHM is the transcription start site; in contrast to kappa light (L) chain genes, it is located in the leader (L) intron. DNA-based models of SHM cannot account for this difference. However, an updated reverse transcriptase (RT)-based model invoking error-prone RT activity of DNA polymerase eta copying IgV pre-mRNA templates to produce cDNA of the transcribed strand (TS) of IgV DNA, which then replaces the corresponding section of the original TS, can explain the difference. This explanation incorporates recent knowledge of pre-mRNA processing, in particular, binding of the splicing-associated protein termed U2AF to a pyrimidine-rich tract in the L intron of pre-mRNA of kappa L chains that may block RT progression further upstream to the end of the pre-mRNA template (transcription start site). Reasons why this block may not occur in H chains and other aspects of the updated RT-model are discussed.

We have proposed previously that error-prone reverse transcription using pre-mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase-eta exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y-family polymerase is a reverse transcriptase (RT). This possibility was tested using a product-enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27-37 nucleotides. Human pol-eta and two other Y-family enzymes that are dispensable for SHM, human pols-iota and -kappa, copied a heteropolymeric DNA-primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol-eta was confirmed using a second sample from an independent source. Human DNA pols-beta and -mu, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol-eta was the most efficient RT of the Y-family enzymes assayed but was much less efficient than an HIV-RT standard in vitro. It is thus feasible that pol-eta acts as both a RNA- and a DNA-dependent DNA polymerase in SHM in vivo, and that Y-family RT activity participates in other mechanisms of physiological importance.

We have used the polymerase chain reaction (PCR) in an attempt to clone and sequence the exons and hitherto unavailable contiguous flanks of all members of the small V(H) 9 germline gene family from inbred mouse strains and sublines that have had a common ancestry within the last century, and to analyze the molecular evolution of these sequences. Fifteen genuine germline genes were isolated (designated V(H) 9.1 through V(H) 9.15) from strains and sublines of DBA, BALB, 129 and C57BL inbred mice. Of the 15 genuine isolates, nine are novel: seven sequences from DBA strains and sublines ( V(H) 9.3 to V(H) 9.9) and two sequences from C57BL strains ( V(H) 9.13 and V(H) 9.14). We have identified sequencing errors and PCR recombinant artefacts in previously published sequences. We detected no sequence divergence of individual genes shared by the strains and sublines studied. However, we isolated two genes from DBA strains and sublines, V(H) 9.1 and V(H) 9.3, that differ only by five nucleotides encoding three amino acid changes that are concentrated within a 33 nucleotide (11 codon) region. Of these 11 codons, eight encode a putative antigen binding site. There were no differences in the remaining 733 nucleotides sequenced (including both 5' and 3' flanking regions). Potential explanations for the generation of V(H) 9.1 and V(H) 9.3 are discussed.

The mechanism by which encephalitic flaviviruses enter the brain to inflict a life-threatening encephalomyelitis in a small percentage of infected individuals is obscure. We investigated this issue in a mouse model for flavivirus encephalitis in which the virus was administered to 6-week-old animals by the intravenous route, analogous to the portal of entry in natural infections, using a virus dose in the range experienced following the bite of an infectious mosquito. In this model, infection with 0.1 to 10(5) PFU of virus gave mortality in approximately 50% of animals despite low or undetectable virus growth in extraneural tissues. We show that the cytolytic effector functions play a crucial role in invasion of the encephalitic flavivirus into the brain. Mice deficient in either the granule exocytosis- or Fas-mediated pathway of cytotoxicity showed delayed and reduced mortality. Mice deficient in both cytotoxic effector functions were resistant to a low-dose peripheral infection with the neurotropic virus.

Cytolysis and interferon(IFN)-gamma production are two independent effector functions of activated cytotoxic T (Tc) cells. We have used the Tc-cell response against the flavivirus, Murray Valley encephalitis virus (MVE), to investigate the requirements for inducing these two functions with regard to antigen-concentration and CD8 coreceptor involvement. Cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo MVE-immune as well as in vitro peptide-restimulated splenocytes at lower peptide concentrations than IFNgamma-production (100-fold lower in the case of primary ex vivo effectors). Little difference was observed in CD8 dependency. Importantly, neither of the effector populations were triggered to produce IFN-gamma by virus-infected target cells, although cytolysis occurred. This result raises the possibility that the levels of presentation of cognate antigen on virus-infected cells in vivo may be below the threshold required for the IFN-gamma production.

Peptide presentation by MHC class II molecules plays a pivotal role in determining the peripheral T cell repertoire as a result of both positive and negative selection in the thymus. Homozygous I-A(g7) expression imparts susceptibility to autoimmune diabetes in the nonobese diabetic mouse, and recently, it has been proposed that this arises from ineffectual peptide binding. Following biosynthesis, class II molecules are complexed with class II-associated invariant chain peptides (CLIP), which remain associated until displaced by Ag-derived peptides. If I-A(g7) is a poor peptide binder, then this may result in continued occupation by CLIP to the point of translocation to the cell surface. To test this hypothesis we generated affinity-purified polyclonal antisera that recognized murine CLIP bound to class II molecules in an allele-independent fashion. We have found abnormally high natural levels of cell surface class II occupancy by CLIP on nonobese diabetic splenic B cells. Experiments using I-A-transfected M12.C3 cells showed that I-A(g7) alone was associated with elevated levels of CLIP, suggesting that this was determined solely by the amino acid sequence of the class II molecule. These results indicated that an intrinsic property of I-A(g7) would affect both the quantity and the repertoire of self-peptides presented during thymic selection.

We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H-2K(k)-restricted determinants from the FLAVIVIRUS: Murray Valley encephalitis virus (MVE), MVE(1785) (REHSGNEI) and MVE(1971) (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MVE(1785)- and MVE(1971)-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MVE(1785) Tc cells recognized a determinant (TDGEERVI) that shares with the determinant used for stimulation only the carboxyl-terminal amino acid residue, one of two H-2K(k) anchor residues. These reactivity patterns were also observed in peptide-dependent IFN-gamma production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few "immunodominant" viral determinants, are remarkably peptide cross-reactive.

The evidence supporting the reverse transcriptase model of somatic hypermutation is critically reviewed. The model provides a coherent explanation for many apparently unrelated findings. We also show that the somatic hypermutation pattern in the human BCL-6 gene can be interpreted in terms of the reverse transcriptase model and the notion of feedback of somatically mutated sequences to the germline over evolutionary time.

We used the CD8+ cytotoxic T (Tc) cell immune response against the flavivirus, Murray Valley encephalitis virus (MVE), restricted by the H-2Kk major histocompatibility complex (MHC) class I molecule, to investigate immunodominance. Split-clone limiting dilution analysis revealed almost exclusive recognition of two peptides, MVE1785 and MVE1971, derived from the viral NS3 protein. The precursor frequency of MVE-reactive Tc cells was determined by limiting dilution analysis for cytotoxic function and intracellular staining for interferon-gamma; the latter gave a 100-fold higher estimate of MVE-reactive Tc cell precursors. MHC class I cell surface stabilization assays revealed that affinity for H-2Kk as well as halflives of the peptide-H-2Kk-complexes were markedly different for the two peptides. However, a kinetic study of antigen presentation showed that both peptides are presented for recognition by Tc cells with a comparable kinetics during the latent period of virus infection. Nevertheless, the lower affinity peptide MVE1785 elicited roughly twofold more Tc cell clones than the high-affinity peptide MVE1971. While the cytolytic activity against both determinants was similar after in vitro restimulation at the peak of the primary response, the smaller pool of memory anti-MVE1971 Tc cells correlated with an impaired memory response against that determinant, suggesting that the available T-cell repertoire is a major factor influencing the establishment of T-cell memory.

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.

Cytotoxic T (Tc)-cell responses against influenza virus infection in BALB/c (H-2d) mice are dominated by Tc clones reactive to the viral nucleoprotein (NP). Here, we report investigations using recombinant vaccinia viruses (VV) encoding major histocompatibility complex (MHC) class I H-2Kd molecules differing by a single amino acid from glutamine (wild-type, Kdw) to histidine (mutant, Kdm) at position 114 located in the floor of the peptide-binding groove. Influenza-infected target cells expressing Kdw were strongly lysed by Kd-restricted Tc cells against A/WSN influenza virus or the immunodominant peptide of viral NP (NPP147-155), whereas infected Kdm-expressing targets gave little or no lysis, respectively, thus showing the immunodominance of NPP147-155. Kdm-expressing target cells saturated with synthetic NPP147-155 (10-5 M) were lysed similarly to Kdw-expressing targets by NPP147-155-specific Tc cells. Thus the defect in influenza-infected Kdm-expressing targets was quantitative; insufficient Kdm-peptide complexes were expressed. Tc-cell responses against four other viruses or alloantigens showed no effect of Kdm. When peptide transport-defective cells were infected with VV-Kdw or VV-Kdm and co-infected with a recombinant VV encoding an endoplasmic reticulum-targeted viral peptide, two influenza haemaglutinin peptides caused higher expression of Kdw than NPP147-155 indicating their higher affinity for Kdw. These results are inconsistent with the hypothesis that immunodominance in the anti-influenza response reflects high affinity of the immunodominant peptide, but are consistent with skewing of the Tc-cell receptor repertoire.

Mice infected with an adenovirus mutant in which the E3 region is deleted, including TNF-resistance genes, develop fatal liver pathology within 3-4 days after infection. At least 10-fold more wild-type virus was needed to cause comparable pathology. These results indicate that the E3 region is critically involved in modulating the pathogenesis of adenovirus infection and that TNF may play a role in liver damage. To explore the latter possibility, the course of disease was examined in infected mice lacking TNFR-I and/or TNFRII, TNF only, or both TNF and lymphotoxin-alpha. Only mice lacking both TNFRI and TNFRII were protected from the lethal affects of the mutant adenovirus. Mice deficient in TNF or TNF and lymphotoxin-alpha displayed the fatal pathology. This outcome is consistent with the existence of another related ligand that binds TNFRI/II to mediate liver damage during infection with this mutant.

Vaccinia virus (VV)-encoded MHC class I Kd molecules which differ by a single amino acid change from glutamine (Kdw, wild type) to histidine (Kdm, mutant) at position 114 located in the floor of the peptide binding groove were compared in terms of peptide binding and cytotoxic T (Tc) cell recognition. Most anti-viral Tc cells were not affected or only marginally affected. However, the Kdm molecule did not detectably present the immunodominant peptide (NPP147-155) of influenza virus nucleoprotein (NP), encoded by the full-length NP gene either in influenza A virus or recombinant VV. This defect could be overcome by using exogenous synthetic NPP147-155 or translation from a minigene encoding NPP147-155 in VV. Kdw presented NPP147-155 encoded by the full-length NP gene, but Kdw-NPP147-155 complexes were at least 100-fold less abundant than after translation from a minigene.

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.

We have reviewed available data concerning the mechanism of somatic hypermutation in rearranged variable genes of Ig in B lymphocytes of mice and the gene conversion process which generates diversity in these genes in the B lymphocytes of chickens. In our view, these data are consistent with a unifying hypothesis of diversity generating mechanisms involving reverse transcription to produce cDNA from RNA transcripts followed by homologous recombination into chromosomal DNA. Thus, seemingly different processes in the mouse and chicken may have a common molecular basis.

We present here a unifying hypothesis for the molecular mechanism of somatic hypermutation and somatic gene conversion in IgV genes involving reverse transcription using RNA templates from the V-gene loci to produce cDNA which undergoes homologous recombination with chromosomal V(D)J DNA. Experimental evidence produced over the last 20 years is essentially consistent with this hypothesis. We also review evidence suggesting that somatically generated IgV sequences from B lymphocytes have been fed back to germline DNA over evolutionary time.

In human and mouse, the germline contains a tandem array of highly homologous variable (V) gene elements which encode part of the antigen-binding region of the antibody protein. During evolution this array apparently arose by gene duplication followed by diversification of duplicated genes via point mutation and recombination. Analysis of germline V gene sequences using a novel algorithm shows that major recombination sites coincide with the borders of the leader intron and the cap site, consistent with the hypothesis that over evolutionary time cDNA derived by reverse transcription of pre-mRNA in B lymphocytes has recombined with germline DNA.

Available data relevant to the mechanism of somatic hypermutation have been critically evaluated in the context of alternative models: (i) error-generating reverse transcription (RT) followed by homologous recombination; and (ii) error-prone DNA replication/repair. A set of basic principles concerning somatic hypermutation has also been formulated and a revised and expanded "RT-Mutatorsome" concept (analogous to telomerase) is presented which is consistent with these principles and all data on the distribution of somatic mutations in normal and Ig transgenic mice carrying particular V(D)J and flanking region constructs. It is predicted that in the mouse VH and Vk loci. the J-C intronic Enhancer-Nuclear Matrix Attachment Region (Ei/MAR) contains a unique sequence motif or secondary structure which ensures that only V(D)J sequences mutate whilst other regions of the genome are not mutated.

Infection of a wide variety of cells of human, mouse and other species' origin by flaviviruses such as WNV, YF, Den, MVE, KUN and JE, increases the cell-surface expression of MHC class I. This MHC class I up-regulation is not due to increased MHC class I synthesis per se, but the result of increased peptide availability in the ER for MHC class I assembly. This is most likely due to the interaction of the viral polyprotein with the ER membrane during viral replication. Flavivirus infection can overcome peptide deficiency in TAP-deficient or non-permissive cell lines such as RMA-S and Syrian hamster cells, BHK and NIL-2. The consequence of this increased MHC class I expression manifests itself in reduced susceptibility to NK cells and augmented lysis by Tc cells. In mice, long-term flavivirus-immune Tc cell memory formation is impaired, following the appearance of strong anti-self Tc cell reactivity observed in in vitro cultures from splenocytes of flavivirus-primed animals. We hypothesize that flavivirus-induced MHC class I up-regulation leads to transient T-cell autoimmunity, followed by down-regulation of both autoimmunity and virus-specific Tc cell memory. Furthermore, we speculate that flavivirus infections of humans in the tropics may be responsible for the observed lower incidence of overt autoimmunity in these geographic regions than in temperate climates where flaviviruses are not endemic.

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.

This article attempts to assemble theoretical-teleological argument to explore possible answers to the question of why class I MHC molecules bind smaller peptides than class II MHC molecules and the associated question of why the size of peptides binding to class I molecules is approaching the limit of the self-non-self discrimination. I propose that the small size of most class I-binding peptides precludes the production of 'MHC-restricted' antibodies. Such a strategy avoids the possibility of antibodies binding to the epitopes recognized by CD8+ T cells, thus blocking effector function required for clearance of potentially lethal infections.