Chemokine Binding Protein M3 of Murine Gammaherpesvirus 68 Modulates the Host Response to Infection in a Natural Host

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology

The complement regulatory proteins CD55 (decay accelerating factor) and CD59 are expressed on the inner acrosomal membrane of human spermatozoa as well as CD46 (membrane cofactor protein)

The complement regulatory proteins CD55 and CD59 are expressed on the plasma membrane of human spermatozoa, whereas CD46 is only on the inner acrosomal membrane (IAM) which becomes surfaced exposed after the acrosome reaction when sperm assume fertilisation-competence. CD55 & CD59, two glycosylphosphatidylinositol (GPI)-anchored proteins, have been detected previously in some studies also in the acrosomal region of chemically fixed spermatozoa but never demonstrated at this site on unfixed spermatozoa. Dual labelling immunofluorescence and confocal microscopy on fresh unfixed spermatozoa, with minimal subsequent time to fixation, has shown CD55 to be markedly expressed on the IAM, more than on the plasma membrane. However, unlike for CD46, CD55 displayed patchy staining over the acrosome, with some variation between individual spermatozoa. All IAM-associated CD55 was localised within GM1-containing lipid rafts. CD59 was expressed also on the IAM, but in a pronounced granular pattern with more variation observed from one spermatozoa to another. Both CD55 & CD59 were released from the IAM by PI-PLC, demonstrating them to be GPI-anchored. Analysis of acrosome-reacted spermatozoal CD55 by Western blotting revealed a novel single 55 kDa protein lacking significant oligosaccharides susceptible to glycosidases. Antibody-induced membrane rafting and release of CD55 & CD59 in vitro may have influenced previous results. Significant coexpression of CD55 & CD46 on the IAM suggests some functional cooperation at this site

Transgenic plants are an alternative to oily fish for providing omega-3 polyunsaturated fatty acids in the human diet: A summary of the findings of a BBSRC funded project

The n-3 polyunsaturated fatty acids (PUFA) present primarily in oily fish, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are important components of cell membranes and are needed for normal development and cell function. Humans have very limited capacity for EPA and DHA synthesis from α-linolenic acid and so they must be obtained preformed from the diet. However, perceived unpalatability of oily fish and fish oil, concerns about contamination with environmental pollutants, dietary choices that exclude fish and animal products, and price limit the effectiveness of recommendations for EPA and DHA intakes. Moreover, marine sources of EPA and DHA are diminishing in the face of increasing demands. Therefore, an alternative source of EPA and DHA is needed that is broadly acceptable, can be upscaled and is sustainable. This review discusses these challenges and, using findings from recent nutritional trials, explains how they may be overcome by seed oils from transgenic plants engineered to produce EPA and DHA. Trials in healthy men and women assessed the acute uptake and appearance in blood over 8 hours of EPA and DHA from transgenic Camelina sativa compared to fish oil, and the incorporation of these PUFA into blood lipids after dietary supplementation. The findings showed that postprandial EPA and DHA incorporation into blood lipids and accumulation in plasma lipids after dietary supplementation was as good as that achieved with fish oil. The oil derived from this transgenic plant was well tolerated. This review also discusses the implications for human nutrition, marine ecology and agriculture

Influence of M3 on T and B cell numbers in the lungs of acutely infected wood mice.

<p>All data were from mice infected with either M3.MR or M3.stop virus at either 7 or 14 days p.i. as indicated. Quantification of B and T cell infiltrates was performed by counting the number of cells in corresponding sequential sections from the same histopathological sections as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001321#ppat-1001321-g003" target="_blank">Fig. 3</a>. The number of cells per unit area (138138 µm²) was then calculated for the interstitial infiltrate (Panels A and B) and the proportion of B and T cells for each area of perivascular/peribronchiolar (PV/PB) infiltration (Panels C, D) and iBALT (Panel E). Data are shown as the mean values ± SEM and compared between groups using a two sample <i>t</i>-test.</p

Influence of M3 on the changes in chemokine levels in the lungs of infected wood mice.

<p>Protein from lung lysates of wood mice infected with M3.stop or M3.MR MHV-68 were analysed for the concentration of specific chemokines by ELISA. Data were normalized relative to total protein concentration as determined by a modified Bradford assay. Error bars depict the standard error of the means for 4 individual wood mice. Statistical analysis was performed using Student's <i>t</i>-test and where significance was found this is indicated above the bars.</p

Comparative analysis of <i>M3</i> RNA expression in lungs of infected wood mice.

<p>Quantification by qRT-PCR of RNA expressed from the MHV-68 genome within lungs; <i>ORF50</i> RNA levels were assessed as a general reference for lytic-cycle gene expression. The copy numbers of individual viral-gene RNAs (as cDNAs) were normalized to those for cellular <i>RPL8</i>. Error bars represent the standard error of the mean from three wood mice per time point. (A) analysis of expression from the <i>M1-M4</i> locus of infected wood mice lungs. (B) analysis of M3 expression from the lungs of BALB/c and wood mice. Note that, although <i>M3</i> expression is similar for both species at 7 days p.i., after 14 days p.i., <i>M3</i> expression in BALB/c mouse lungs is drastically reduced compared to wood mice.</p