Rubella Viruses Shift Cellular Bioenergetics to a More Oxidative and Glycolytic Phenotype with a Strain-Specific Requirement for Glutamine.

The flexible regulation of cellular metabolic pathways enables cellular adaptation to changes in energy demand under conditions of stress such as posed by a virus infection. To analyze such an impact on cellular metabolism, rubella virus (RV) was used in this study. RV replication under selected substrate supplementation with glucose, pyruvate, and glutamine as essential nutrients for mammalian cells revealed its requirement for glutamine. The assessment of the mitochondrial respiratory (based on the oxygen consumption rate) and glycolytic (based on the extracellular acidification rate) rate and capacity by respective stress tests through Seahorse technology enabled determination of the bioenergetic phenotype of RV-infected cells. Irrespective of the cellular metabolic background, RV infection induced a shift of the bioenergetic state of epithelial cells (Vero and A549) and human umbilical vein endothelial cells to a higher oxidative and glycolytic level. Interestingly there was a RV strain-specific, but genotype-independent demand for glutamine to induce a significant increase in metabolic activity. While glutaminolysis appeared to be rather negligible for RV replication, glutamine could serve as donor of its amide nitrogen in biosynthesis pathways for important metabolites. This study suggests that the capacity of RVs to induce metabolic alterations could evolve differently during natural infection. Thus, changes in cellular bioenergetics represent an important component of virus-host interactions and could complement our understanding of the viral preference for a distinct host cell population.IMPORTANCE RV pathologies, especially during embryonal development, could be connected with its impact on mitochondrial metabolism. With bioenergetic phenotyping we pursued a rather novel approach in virology. For the first time it was shown that a virus infection could shift the bioenergetics of its infected host cell to a higher energetic state. Notably, the capacity to induce such alterations varied among different RV isolates. Thus, our data add viral adaptation of cellular metabolic activity to its specific needs as a novel aspect to virus-host evolution. In addition, this study emphasizes the implementation of different viral strains in the study of virus-host interactions and the use of bioenergetic phenotyping of infected cells as a biomarker for virus-induced pathological alterations.

Mumps Virus SH Protein Inhibits NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor 1, Interleukin-1 Receptor 1, and Toll-Like Receptor 3 Complexes.

The mumps virus (MuV) small hydrophobic protein (SH) is a type I membrane protein expressed in infected cells. SH has been reported to interfere with innate immunity by inhibiting tumor necrosis factor alpha (TNF-α)-mediated apoptosis and NF-κB activation. To elucidate the underlying mechanism, we generated recombinant MuVs (rMuVs) expressing the SH protein with an N-terminal FLAG epitope or lacking SH expression due to the insertion of three stop codons into the SH gene. Using these viruses, we were able to show that SH reduces the phosphorylation of IKKß, IκBα, and p65 as well as the translocation of p65 into the nucleus of infected A549 cells. Reporter gene assays revealed that SH interferes not only with TNF-α-mediated NF-κB activation but also with IL-1ß- and poly(I·C)-mediated NF-κB activation, and that this inhibition occurs upstream of the NF-κB pathway components TRAF2, TRAF6, and TAK1. Since SH coimmunoprecipitated with tumor necrosis factor receptor 1 (TNFR1), RIP1, and IRAK1, we hypothesize that SH exerts its inhibitory function by interacting with TNFR1, interleukin-1 receptor type 1 (IL-1R1), and TLR3 complexes in the plasma membrane of infected cells.IMPORTANCE The MuV SH has been shown to impede TNF-α-mediated NF-κB activation and is therefore thought to contribute to viral immune evasion. However, the mechanisms by which SH mediates NF-κB inhibition remained largely unknown. In this study, we show that SH interacts with TNFR1, IL-1R1, and TLR3 complexes in infected cells. We thereby not only shed light on the mechanisms of SH-mediated NF-κB inhibition but also reveal that SH interferes with NF-κB activation induced by interleukin-1ß (IL-1ß) and double-stranded RNA.

Gene expression profiling of rubella virus infected primary endothelial cells of fetal and adult origin.

BACKGROUND: Rubella virus (RV) infection is usually a mild illness in children and adults. However, maternal infection during the first trimester of pregnancy can lead to congenital rubella syndrome (CRS) in the infant. Fetuses with CRS show damage to the endothelium of the heart and blood vessels; thus, it has been speculated that the clinical manifestations associated with CRS may be a result of endothelial cells persistently infected with RV. Here, we compared the effects of RV infection on gene expression in primary endothelial cells of fetal (HUVEC) and of adult (HSaVEC) origin by transcriptional profiling. RESULTS: More than 75 % of the genes differentially regulated following RV infection were identical in both cell types. Gene Ontology (GO) analysis of these commonly regulated genes showed an enrichment of terms involved in cytokine production and cytokine regulation. Increased accumulation of inflammatory cytokines following RV infection was verified by protein microarray. Interestingly, the chemokine CCL14, which is implicated in supporting embryo implantation at the fetal-maternal interface, was down-regulated following RV infection only in HUVEC. Most noticeably, when analyzing the uniquely regulated transcripts for each cell type, GO term-based cluster analysis of the down-regulated genes of HUVEC revealed an enrichment of the GO terms "sensory organ development", "ear development" and "eye development". CONCLUSION: Since impairment in vision and hearing are the most prominent clinical manifestations observed in CRS patients, the here detected down-regulated genes involved in the development of sensory organs sheds light on the molecular mechanisms that may contribute to the teratogenic effect of RV.

Rat cytomegalovirus (RCMV) English isolate and a newly identified Berlin isolate share similarities with but are separate as an anciently diverged clade from Mouse CMV and the Maastricht isolate of RCMV.

The genome of the rat cytomegalovirus (RCMV) English isolate (MuHV-8) differs significantly from the RCMV Maastricht isolate (MuHV-2) and other cytomegaloviruses (CMVs) in its size, base composition and genomic content. Analysis of the RCMV-Berlin isolate, MuHV-8, revealed that the two MuHV-8 isolates are highly similar in genome size and content, indicating that the smaller genome size (202 946 bp) compared to other known CMVs was not the result of an accidental deletion during passage in tissue culture. Surprisingly, the proteins encoded in MuHV-8 shared more overall similarity with their orthologues from mouse CMV (MuHV-1) compared to their orthologues in rat CMV (MuHV-2). Phylogenetic analyses of conserved viral genes showed that the two MuHV-8 isolates are from the same species and represent a unique clade that is distinct from other rodent CMVs.

Cytomegalovirus expresses the chemokine homologue vXCL1 capable of attracting XCR1+ CD4- dendritic cells.

Cytomegaloviruses (CMV) have developed various strategies to escape the immune system of the host. One strategy involves the expression of virus-encoded chemokines to modulate the host chemokine network. We have identified in the English isolate of rat CMV (murid herpesvirus 8 [MuHV8]) an open reading frame encoding a protein homologous to the chemokine XCL1, the only known C chemokine. Viral XCL1 (vXCL1), a glycosylated protein of 96 amino acids, can be detected 13 h postinfection in the supernatant of MuHV8-infected rat embryo fibroblasts. vXCL1 exclusively binds to CD4(-) rat dendritic cells (DC), a subset of DC that express the corresponding chemokine receptor XCR1. Like endogenous rat XCL1, vXCL1 selectively chemoattracts XCR1(+) CD4(-) DC. Since XCR1(+) DC in mice and humans have been shown to excel in antigen cross-presentation and thus in the induction of cytotoxic CD8(+) T lymphocytes, the virus has apparently hijacked this gene to subvert cytotoxic immune responses. The biology of vXCL1 offers an interesting opportunity to study the role of XCL1 and XCR1(+) DC in the cross-presentation of viral antigens.

Complete genome sequence of the english isolate of rat cytomegalovirus (Murid herpesvirus 8).

The complete genome of the English isolate of rat cytomegalovirus (RCMV-E) was determined. RCMV-E has a 202,946-bp genome with noninverting repeats but without terminal repeats. Thus, it differs significantly in size and genomic arrangement from closely related rodent cytomegaloviruses (CMVs). To account for the differences between the rat CMV isolates of Maastricht and England, RCMV-E was classified as Murid herpesvirus 8 by the International Committee on Taxonomy of Viruses.

Factor VIII Fc Fusion Protein but not FVIII Drives Human Monocyte-Derived Dendritic Cell Activation via FcγRIIa.

This study compares the effect of recombinant Factor VIII Fc fusion protein (rFVIII-Fc) with recombinant FVIII (rFVIII) on monocyte-derived dendritic cells (moDC's). Cells treated with rFVIII-Fc showed morphological changes typical for cell activation, had a significant up-regulation of cell activation markers and produced higher levels of pro-inflammatory cytokines. Even after stimulation with Lipopolysaccharides, the addition of rFVIII-Fc led to increased expression of activation markers, indicating that rFVIII-Fc is capable of amplifying the maturation signal. On the contrary, cultivation of moDC's with rFVIII did not alter cell morphology or increase surface activation marker expression and pro-inflammatory cytokine production. The binding of the Fc domain to the activating Fcγ receptor IIa (FcγRIIa) can cause cell activation. Therefore, the effect of rFVIII-Fc on FcγRIIa was analyzed in detail. Cultivation of moDC's with rFVIII-Fc led to increased phosphorylation of FcγRIIa, which was not detected for rFVIII. Blocking FcγRIIa prior to the cultivation with rFVIII-Fc significantly reduced the activating effect of rFVIII-Fc, indicating that rFVIII-Fc-induced moDC activation was caused by FcγRIIa. Moreover, rFVIII-Fc bound to FCGR2A-transfected human embryonic kidney 293 cells. Taken together, our data present a new mechanism of moDC activation by rFVIII-Fc via FcγRIIa.

Glycation of macrophages induces expression of pro-inflammatory cytokines and reduces phagocytic efficiency.

Glycation and the accumulation of advanced glycation end products (AGEs) are known to occur during normal aging but also in the progression of several diseases, such as diabetes. Diabetes type II and aging both lead to impaired wound healing. It has been demonstrated that macrophages play an important role in impaired wound healing, however, the underlying causes remain unknown. Elevated blood glucose levels as well as elevated methylglyoxal (MGO) levels in diabetic patients result in glycation and increase of AGEs. We used MGO to investigate the influence of glycation and AGEs on macrophages. We could show that glycation, but not treatment with AGE-modified serum proteins, increased expression of pro-inflammatory cytokines interleukin 1ß (IL-1ß) and IL-8 but also affected IL-10 and TNF-α expression, resulting in increased inflammation. At the same time, glycation reduced phagocytic efficiency and led to impaired clearance rates of invading microbes and cellular debris. Our data suggest that glycation contributes to changes of macrophage activity and cytokine expression and therefore could support the understanding of disturbed wound healing during aging and diabetes.

Novel DNA packaging recognition in the unusual bacteriophage N15.

Phage lambda's cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cos(N15)) is closely related to cos(λ), but whereas the cosB(N15) subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosB(λ). A bioinformatic study of N15-like phages indicates that cosB(N15) also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200 bp distal to R3. An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB-λ. The DNA binding domain of TerS-N15 is a dimer.

Mixed influenza A and B infections complicate the detection of influenza viruses with altered sensitivities to neuraminidase inhibitors.

Previously, three influenza A(H3N2) isolates with a reduced susceptibility to the neuraminidase inhibitors (NAIs) zanamivir and oseltamivir were identified during screening by the Neuraminidase Inhibitor Susceptibility Network (NISN). The isolates were from untreated patients from the first three years post licensure of the NAIs. We plaque-purified progeny from each of these isolates and determined the NAI sensitivity of each plaqued population. Sequencing and serology for each population revealed that the isolates contained a mix of wild type influenza A(H3N2) and influenza B. The NAI susceptibility reductions that had originally been reported were a consequence of influenza B neuraminidases that have lower relative NAI sensitivities, rather than being due to resistant influenza A(H3N2) viruses. Our study highlights the need to check for mixed influenza infections when isolates with potentially lower sensitivities to NAIs are identified.