Maintenance of long term gamma-herpesvirus B cell latency is dependent on CD40-mediated development of memory B cells

It has been proposed that the gamma-herpesviruses maintain lifelong latency in B cells by gaining entry into the memory B cell pool and taking advantage of host mechanisms for maintaining these cells. We directly tested this hypothesis by kinetically monitoring viral latency in CD40(+) and CD40(-) B cells from CD40(+)CD40(-) mixed bone marrow chimera mice after infection with a murine gamma-herpesvirus, MHV-68. CD40(+) B cells selectively entered germinal centers and differentiated into memory B cells. Importantly, latency was progressively lost in the CD40(-) B cells and preferentially maintained in the long-lived, isotype-switched CD40(+) B cells. These data directly demonstrate viral exploitation of the normal B cell differentiation pathway to maintain latency.     

Persistence and turnover of antigen-specific CD4 T cells during chronic tuberculosis infection in the mouse

CD4 T cells are critical for resistance to Mycobacterium tuberculosis infection, but how effective T cell responses are maintained during chronic infection is not well understood. To address this question we examined the CD4 T cell response to a peptide from ESAT-6 during tuberculosis infection in the mouse. The ESAT-6(1-20)/IA(b)-specific CD4 T cell response in the lungs, mediastinal lymph nodes, and spleen reached maxima 3-4 wk postinfection, when the bacteria came under the control of the immune response. Once chronic infection was established, the relative frequencies of Ag-specific CD4 T cells were maintained at nearly constant levels for at least 160 days. ESAT-6(1-20)/IA(b)-specific CD4 T cells that responded in vitro expressed activation markers characteristic of chronically activated effector cells and used a limited Vbeta repertoire that was clonally stable in vivo for at least 12 wk. 5-Bromo-2-deoxyuridine incorporation studies indicated a relatively high rate of cell division among both total CD4 and ESAT-6(1-20)/IA(b)-specific CD4 T cells during acute infection, but the degree of 5-bromo-2-deoxyuridine incorporation by both the CD4 T cells and the Ag-specific cells declined at least 3-fold during chronic infection. The data indicate that the peripheral ESAT-6(1-20)/IA(b)-specific CD4 T cell response to M. tuberculosis is characterized during the acute phase of infection by a period of extensive proliferation, but once bacterial control is achieved, this is followed during chronic infection by an extended containment phase that is associated with a persistent response of activated, yet more slowly proliferating, T cells.     

Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis

This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.     

A single malaria merozoite serine protease mediates shedding of multiple surface proteins by juxtamembrane cleavage

Erythrocyte invasion by the malaria merozoite is accompanied by the regulated discharge of apically located secretory organelles called micronemes. Plasmodium falciparum apical membrane antigen-1 (PfAMA-1), which plays an indispensable role in invasion, translocates from micronemes onto the parasite surface and is proteolytically shed in a soluble form during invasion. We have previously proposed, on the basis of incomplete mass spectrometric mapping data, that PfAMA-1 shedding results from cleavage at two alternative positions. We now show conclusively that the PfAMA-1 ectodomain is shed from the merozoite solely as a result of cleavage at a single site, just 29 residues away from the predicted transmembrane-spanning sequence. Remarkably, this cleavage is mediated by the same membrane-bound parasite serine protease as that responsible for shedding of the merozoite surface protein-1 (MSP-1) complex, an abundant, glycosylphosphatidylinositol-anchored multiprotein complex. Processing of MSP-1 is essential for invasion. Our results indicate the presence on the merozoite surface of a multifunctional serine sheddase with a broad substrate specificity. We further demonstrate that translocation and shedding of PfAMA-1 is an actin-independent process.     

Microsatellite variation in cyclically parthenogenetic populations of Myzus persicae in south-eastern Australia

We examined the population structure of the introduced aphid, Myzus persicae collected mainly from its primary host, Prunus persica, in south-east Australia. Myzus persicae has been present in Australia since at least 1893. Samples were collected in the spring of 1998 from two mainland and three Tasmanian localities and isofemale lines were established in the laboratory. The reproductive mode (life cycle), karyotype and 17-locus microsatellite genotype of each clone were determined. All populations showed significant population differentiation (F(ST) 0.058-0.202) even over small geographic distances (<50 km). All clones were karyotypically normal except for a subset of clones from one site that was exposed to the carbamate insecticide, Pirimor, the week prior to sampling. Those clones were heterozygous for an autosomal 1,3 translocation frequently associated in M. persicae with insecticide resistance. In contrast to other loci and despite being on different chromosomes, loci myz2(A) and M55(A) showed general and significant linkage disequilibrium. These loci may be affected by epistatic selection. We discuss the observed high clonal diversity, moderate but significant population differentiation, general conformance to Hardy-Weinberg equilibria and low linkage disequilibria with particular focus on the global population biology of M. persicae.     

Host cell invasion by malaria parasites

The complex life cycle of the malaria parasite includes three specialized invasive stages, distinct both in terms of their cellular architecture and in their choice of target host cell. Despite the dissimilarities between these forms, there are clear parallels in the manner by which they enter their respective host cells. Advances in the area of erythrocyte invasion by the malaria merozoite, outlined here by Chetan Chitnis and Mike Blackman and discussed at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000, will undoubtedly impact on our understanding of mechanisms of cell entry by the other invasive forms. Similarly, recent progress in dissecting the functional role of surface proteins expressed by sporozoite and ookinete stages has provided fascinating insights into general aspects of invasion by all invasive stages of apicomplexan parasites.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Modular Skeletal Evolution in Sticklebacks Is Controlled by Additive and Clustered Quantitative Trait Loci

Understanding the genetic architecture of evolutionary change remains a long-standing goal in biology. In vertebrates, skeletal evolution has contributed greatly to adaptation in body form and function in response to changing ecological variables like diet and predation. Here we use genome-wide linkage mapping in threespine stickleback fish to investigate the genetic architecture of evolved changes in many armor and trophic traits. We identify >100 quantitative trait loci (QTL) controlling the pattern of serially repeating skeletal elements, including gill rakers, teeth, branchial bones, jaws, median fin spines, and vertebrae. We use this large collection of QTL to address long-standing questions about the anatomical specificity, genetic dominance, and genomic clustering of loci controlling skeletal differences in evolving populations. We find that most QTL (76%) that influence serially repeating skeletal elements have anatomically regional effects. In addition, most QTL (71%) have at least partially additive effects, regardless of whether the QTL controls evolved loss or gain of skeletal elements. Finally, many QTL with high LOD scores cluster on chromosomes 4, 20, and 21. These results identify a modular system that can control highly specific aspects of skeletal form. Because of the general additivity and genomic clustering of major QTL, concerted changes in both protective armor and trophic traits may occur when sticklebacks inherit either marine or freshwater alleles at linked or possible “supergene” regions of the stickleback genome. Further study of these regions will help identify the molecular basis of both modular and coordinated changes in the vertebrate skeleton.     

The function of communities in protein interaction networks at multiple scales

Background  

If biology is modular then clusters, or communities, of proteins derived using only protein interaction network structure should define protein modules with similar biological roles. We investigate the link between biological modules and network communities in yeast and its relationship to the scale at which we probe the network.     

A replication-deficient murine gamma-herpesvirus blocked in late viral gene expression can establish latency and elicit protective cellular immunity

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, are widely disseminated and are associated with the onset of a variety of malignancies. Thus, the development of prophylactic and therapeutic vaccination strategies is an important goal. The experimental mouse gamma-herpesvirus, gammaHV68 (or MHV-68), has provided an in vivo model for studying immune control of these persistent viruses. In the current studies, we have examined infectivity, immunogenicity, and protective efficacy following infection with a replication-deficient gammaHV68 blocked in late viral gene expression, ORF31STOP. The data show that ORF31STOP was able to latently infect B cells. However, the anatomical site and persistence of the infection depended on the route of inoculation, implicating a role for viral replication in viral spread but not the infectivity per se. Furthermore, i.p. infection with ORF31STOP elicited strong cellular immunity but a non-neutralizing Ab response. In contrast, intranasal infection was poorly immunogenic. Consistent with this, mice infected i.p. had enhanced control of both the lytic and latent viral loads following challenge with wild-type gammaHV68, whereas intranasal infected mice were not protected. These data provide important insight into mechanisms of infection and protective immunity for the gamma-herpesviruses and demonstrate the utility of replication-deficient mutant viruses in direct testing of "proof of principal" vaccination strategies.