Semliki Forest virus as an expression vector in insect cell lines

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.

From sabotage to camouflage: viral evasion of cytotoxic T lymphocyte and natural killer cell-mediated immunity

The outcome of a virus infection is strongly influenced by interactions between host immune defences and virus 'anti-defence' mechanisms. For many viruses, their continued survival depends on, the speed of their attach: their capacity to replicate and transmit to uninfected hosts prior to their elimination by an effective immune response. In contrast, the success of persistent viruses lies in their capacity for immunological subterfuge: the evasion of host defence mechanisms by either mutation (covered elsewhere in this issue, by Gould and Bangham, pp. 321-328) or interference with the action of host cellular proteins that are important components of the immune response. This review will focus on the strategies employed by persistent viruses against two formidable host defences against virus infection: the CD8+ cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4(+) T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2(q)) or F1(C57BV6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further,E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7(+) tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in split tolerance to that autoantigen.

Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest

DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Tev sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. Ln support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the Rm-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork. (C) 1999 Academic Press.

Murine cytomegalovirus homologues of cellular immunomodulatory genes

The study of 'molecular mimicry' or 'genetic piracy', with respect to the utilisation of cellular genes captured and modified during the course of virus evolution, has been an area of increasing research with the expansion in virus genome sequencing. Examples of cellular immunomodulatory genes which have been captured from hosts have been identified in a number of viruses. This review concentrates upon studies of murine cytomegalovirus (MCMV), investigating the functions of viral genes homologous to G protein-coupled receptors, MHC class I and chemokines, The study of recombinant MCMV engineered with specific disruptions of these genes has revealed their significance during virus replication and dissemination within the host, In the case of the latter two classes of genes, evidence suggests they interfere with cellular immune responses, although the detailed mechanisms underlying this interference have yet to be delineated. Copyright (C) 2000 S. Karger AG, Basel.

Function of CMV-encoded MHC class I homologues

Homologues of MHC class I proteins have been identified in the genomes of human, murine and rat cytomegaloviruses (CMVs). Given the pivotal role of the MHC class I protein in cellular immunity, it has been postulated that the viral homologues subvert the normal antiviral immune response of the host, thus promoting virus replication and dissemination in an otherwise hostile environment. This review focuses on recent studies of the CMV MHC class I homologues at the molecular, cellular and whole animal level and presents current hypotheses for their roles in the CMV life cycle.

Transgenic expression of a viral oncoprotein exclusively in K14(+) epithelial cells results in peripheral but not central CD8 T cell tolerance

The role of thymic versus peripheral epithelial cells in the negative selection of the peptide-specific CD8 T cell repertoire is still largely unresolved. We have generated TCRb chain transgenic mice in which 20–35% of peripheral CD8 T cells recognize an epitope from a viral, nuclear oncoprotein (human papillomavirus type 16 E7) in the context ofMHC class I, H-2Db. When T cells from these transgenic mice develop through the thymus of a second transgenic mouse expressing E7 from a keratin 14 promoter, no major perturbation to thymic T cell development is observed over a 7 month period. In contrast, peripheral CD8 T cell responses in these same mice (E7TCRxK14E7 double transgenic) become reduced over time. This data suggests that peripheral tolerance mechanisms predominate over thymic negative selection in controlling CD8 T cell responses to this epithelial, nuclear oncoprotein.

Influence of Lactic Acid on Endogenous and Viral RNA-Induced Immune Mediator Production by Vaginal Epithelial Cells

OBJECTIVE: To evaluate the influence of lactic acid on immune mediator release from vaginal epithelial cells. METHODS: The human vaginal epithelial cell line, VK2/E6E7, was cultured in the presence or absence of physiological concentrations of lactic acid, and in the presence or absence of the viral Toll-like receptor 3 agonist, poly (inosinic acid: cytidylic acid). Supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-1 beta, IL-6, IL-8, IL-23, transforming growth factor (TGF)-beta and secretory leukocyte protease inhibitor. RESULTS: Vaginal epithelial cells spontaneously released IL-1 beta (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-beta (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-beta production was marginally enhanced (49%) by addition of lactic acid alone. Poly (inosinic acid: cytidylic acid) by itself stimulated the release of IL-6 (305 pg/mL) and enhanced IL-8 production (2.8 ng/mL). The combination of poly (inosinic acid: cytidylic acid) and lactic acid markedly increased IL-8 production (5.0 ng/mL) and induced the release of IL-1 beta (96.2 pg/mL). The poly (inosinic acid: cytidylic acid)-mediated lactic acid effect on IL-1 beta and IL-8 release was abrogated when the lactic acid was neutralized or if acetic acid was substituted for lactic acid. CONCLUSION: Lactic acid enhances the release of selective mediators from vaginal epithelial cells and stimulates antiviral immune responses. (Obstet Gynecol 2011;118:840-6) DOI: 10.1097/AOG.0b013e31822da9e9

Viral Load Predicts New World Health Organization Stage 3 and 4 Events in HIV-Infected Children Receiving Highly Active Antiretroviral Therapy, Independent of CD4 T Lymphocyte Value

Background. Many resource-limited countries rely on clinical and immunological monitoring without routine virological monitoring for human immunodeficiency virus (HIV)-infected children receiving highly active antiretroviral therapy (HAART). We assessed whether HIV load had independent predictive value in the presence of immunological and clinical data for the occurrence of new World Health Organization (WHO) stage 3 or 4 events (hereafter, WHO events) among HIV-infected children receiving HAART in Latin America. Methods. The NISDI (Eunice Kennedy Shriver National Institute of Child Health and Human Development International Site Development Initiative) Pediatric Protocol is an observational cohort study designed to describe HIV-related outcomes among infected children. Eligibility criteria for this analysis included perinatal infection, age ! 15 years, and continuous HAART for >= 6 months. Cox proportional hazards modeling was used to assess time to new WHO events as a function of immunological status, viral load, hemoglobin level, and potential confounding variables; laboratory tests repeated during the study were treated as time-varying predictors. Results. The mean duration of follow-up was 2.5 years; new WHO events occurred in 92 (15.8%) of 584 children. In proportional hazards modeling, most recent viral load 15000 copies/mL was associated with a nearly doubled risk of developing a WHO event (adjusted hazard ratio, 1.81; 95% confidence interval, 1.05-3.11; P = 033), even after adjustment for immunological status defined on the basis of CD4 T lymphocyte value, hemoglobin level, age, and body mass index. Conclusions. Routine virological monitoring using the WHO virological failure threshold of 5000 copies/mL adds independent predictive value to immunological and clinical assessments for identification of children receiving HAART who are at risk for significant HIV-related illness. To provide optimal care, periodic virological monitoring should be considered for all settings that provide HAART to children.

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

A tale of two terminators: Crystal structures sharpen the debate on DNA replication fork arrest mechanisms

The structure of the Tus-Ter DNA replication fork arrest complex of Escherichia coli reveals a novel architecture for the bound Tus protein and a new type of DNA-binding motif, The structure of the complex may explain how Tus can block movement of a replication fork approaching from one direction and not the other.

Reorganization of terminator DNA upon binding replication terminator protein: Implications for the functional replication fork arrest complex

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B. subtilis DNA terminator, Terl, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of Terl causes the DNA to become slightly unwound and bent by similar to 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to similar to 60 degrees. We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner, in the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry at the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo

Herpesviruses, such as murine and human cytomegalovirus (MCMV and HCMV), can establish a persistent infection within the host and have diverse mechanisms as protection from host immune defences'. Several herpesvirus genes that are homologous to host immune modulators have been identified, and are implicated in viral evasion of the host immune response(2,3). The discovery of a viral major histocompatibility complex (MHC) class I homologue, encoded by HCMV(4), led to speculation that it might function as an immune modulator and disrupt presentation of peptides by MHC class I to cytotoxic T cells(5). However, there is no evidence concerning the biological significance of this gene during viral infection. Recent analysis of the MCMV genome has also demonstrated the presence of a MHC class I homologue(6). Here we show that a recombinant MCMV,in which. the gene encoding the class I homologue has been disrupted, has severely restricted replication during the acute stage of infection compared with wild-type MCMV, We demonstrate by in vivo depletion studies that natural killer (NK) cells are responsible for the attenuated phenotype of the mutant. Thus the viral MHC dass I homologue contributes to immune evasion through interference with NK cell-mediated clearance.