Use of a Murine Cytomegalovirus K181-Derived bacterial artificial chromosome as a vaccine vector for immunocontraception

Cytomegaloviruses (CMVs) are members of the Betaherpesvirinae subfamily of the Herpesviridae, and their properties of latency, large DNA size, gene redundancy, and ability to be cloned as bacterial artificial chromosomes (BACs) suggest their utility as vaccine vectors. While the K181 strain of murine CMV (MCMV) is widely used to study MCMV biology, a BAC clone of this virus had not previously been produced. We report here the construction of a BAC clone of the K181Perth strain of MCMV. The in vivo and in vitro growth characteristics of virus derived from the K181 BAC were similar to those of wild-type K181. The utility of the K181 BAC as a method for the rapid production of vaccine vectors was assessed. A vaccine strain of BAC virus, expressing the self-fertility antigen, murine zona pellucida 3, was produced rapidly using standard bacterial genetics techniques and rendered female BALB/c mice infertile with a single intraperitoneal inoculation. In addition, attenuated vaccine strains lacking the open reading frames m07 to m12 exhibited no reduction in efficacy compared to the full-length vaccine strain. In conclusion, we describe the production of a K181-based BAC virus which behaved essentially as wild-type K181 and allowed the rapid production of effective viral vaccine vectors

Redetermination of the crystal structure of trans-dichloro-syn-bis[meso- ethane-1,2-diylbis(methylphenyl-phosphane)]ruthenium(II), Ru((C6H5)(CH3)P(C2H4)P(CH3)(C6H5))2Cl2

Tbe distribution ofthe functional subsets ofporcine T cells, the cytolytic/suppressor (Tc/s) and the helper/inducer (Tb/i) cells was studied in cryostat sections ofthymus, Iymph nodes, tonsils, Peyer's patches, spleen and liver using the indirect immunoperoxidase technique. Tbree murine monoclonal antibodies (mAb) were used. Tbe mAb 8/1 reacts with an antigen present on all T cells and on cells of the myeloid Iineage; the antigen has not yet been characterized biochemically. Tbe mAb 295/33 (antiT8) binds to the porcine T8 antigen and defines the Tc/s subset, while mAb PT -4 (anti-T4) detects the porcine T4 antigen and defines the Tb/i subset. Practically all thymocytes were stained by mAb 8/1. Tbe majority of corticaI thymocytes apparently co-expressed T8 and T 4, whereas distinct fractions of medullary cells were labelIed by either anti-T8 or anti-T4. In peripherallymphoid organs all three mAb reacted with cells in the thymus-dependent areas and with cells scattered in the lymphoid follic1es. In Iymph nodes, tonsils and Peyer's patches, anti-T8 and anti-T4 each labelIed approximately half of the cells stained by mAb 8/ I. In the periarteriolar lymphoid sheath of the spleen, anti-T 4 labelled more cells than did anti-T8. The reactivity of mAb 8/1 with the Kupffer cells of the Iiver demonstrated the expression of the 8/1 antigen on cells of the monocyte lineage. Tbe T8 and T4 antigens could not be detected in acetone-fixed and paraffin-embedded sections, while the antigen recognized by mAb 8/1 remained preserved. Altogether, despite an inverted microanatomical structure ofporcine Iymph nodes, the frequency and distribution ofT8+ and T4+ cells in thymusdependent areas proved to be similar to those found in other species

Systemic and local infection routes govern different cellular dissemination pathways during gammaherpesvirus infection <em>in vivo</em>.

Human gammaherpesviruses cause morbidity and mortality associated with infection and transformation of lymphoid and endothelial cells. Knowledge of cell types involved in virus dissemination from primary virus entry to virus latency is fundamental for the understanding of gammaherpesvirus pathogenesis. However, the inability to directly trace cell types with respect to virus dissemination pathways has prevented definitive conclusions regarding the relative contribution of individual cell types. Here, we describe that the route of infection affects gammaherpesvirus dissemination pathways. We constructed a recombinant murine gammaherpesvirus 68 (MHV-68) variant harboring a cassette which switches fluorescent markers in a Cre-dependent manner. Since the recombinant virus which was constructed on the wild-type background was attenuated, in this study we used an M1-deleted version, which infected mice with normal kinetics. Infection of Cre-transgenic mice with this convertible virus was used to estimate the quantitative contribution of defined cell types to virus productivity and dissemination during the acute phase of MHV-68 infection. In systemic infection, we found splenic vascular endothelial cells (EC) among the first and main cells to produce virus. After local infection, the contribution of EC to splenic virus production did not represent such early kinetics. However, at later time points, B cell-derived viruses dominated splenic productivity independently of systemic or local infection. Systemic versus local infection also governed the cell types involved in loading peritoneal exudate cells, leading to latency in F4/80- and CD11b-positive target cells. Systemic infection supported EC-driven dissemination, whereas local infection supported B cell-driven dissemination

Dendritic Cells under Influence of Mouse Cytomegalovirus Have a Physiologic Dual Role : To Initiative and to Restrict T Cell Activation.

The aim of this study is to analyze the dynamics of the mouse cytomegalovirus (MCMV)-dendritic cell (DC) interaction. Immature and mature DCs derived from the mouse stem cell line factor-dependent cell Paterson mixed potential were infected with a recombinant MCMV expressing green fluorescent protein. Infection of immature DCs resulted in DC activation and virus production, both of which may contribute to viral dissemination. The infection of mature DCs was nonproductive and was restricted to immediate-early and early viral protein expression. During early stages of MCMV infection, mature DCs up-regulated major histocompatibility complex (MHC) and costimulatory molecules and activated autologous, but not allogeneic, naive T cells. At later times of MCMV infection, DCs prevented T cell activation by down-regulation of MHC and costimulatory molecules. Thus, DCs under the influence of MCMV have a physiologic dual role: to initiate and to restrict T cell activation. The lack of immunostimulation in allogeneic settings may explain the increased risk of MCMV morbidity after allogeneic transplantation

Proteins of the secretory pathway govern virus productivity during lytic gammaherpesvirus infection.

Diseases caused by gammaherpesviruses continue to be a challenge for human health and antiviral treatment. Most of the commonly used antiviral drugs are directed against viral gene products. However, the emergence of drug-resistant mutations ma limit the effectiveness of these drugs. Since viruses require a host cell to propagate, the search for host cell targets is an interestin alternative. Methods: In this study, we infected three different cell types (fibroblasts, endothelial precursor cells and macrophages with a murine gammaherpesvirus and analysed the host cell response for changes either common to all or unique to a particular cell type using oligonucleotide microarrays. Results: The analysis revealed a number of genes whose transcription was significantly up- or down-regulated in either one or two of the cell types tested. After infection, only two genes, Lman1 (also known as ERGIC53) an synaptobrevin-like 1 (sybl1) were significantly up-regulated in all three cell types, suggestive for a general role for the virus life cycl independent of the cell type. Both proteins have been implicated in cellular exocytosis and transport of glycoproteins through the secre tory pathway. To test the significance of the observed up-regulation, the functionality of these proteins was modulated, and the effect on virus replication was monitored. Inhibition of either Lman1 or sybl1 resulted in a significant reduction in virus production. Conclusions: This suggests that proteins of the secretory pathway which appear to be rate limiting for virus production may represent new targets for intervention

A viral ER resident glycoprotein inactivates the MHC encoded peptide transporter.

AbstractHuman cytomegalovirus inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in human cytomegalovirus–infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is ER restricted and is identical with TAP. US6 protein is found in complexes with TAP1/2, MHC class I heavy chain, β2-microglobulin, calnexin, calreticulin, and tapasin. TAP inhibition, however, is independent of the presence of class I heavy chain and tapasin. The results establish a new mechanism for viral immune escape and a novel role for ER-resident proteins to regulate TAP via its luminal face