Characterization of the murine gammaherpesvirus 68 ORF74 product: a novel oncogenic G protein-coupled receptor.

Murine gammaherpesvirus (MHV-68) is well established as a small animal model for the study of gammaherpesviruses. The MHV-68 genome contains an open reading frame (ORF74) that has significant sequence homology with mammalian G-protein coupled receptors (GPCRs) and the GPCR from the related Kaposi's sarcoma-associated herpesvirus (KSHV). Here we show that the MHV-68 ORF74 is predicted to encode a GPCR since it has seven potential transmembrane helices and that it has other sequence motifs in common with GPCRS: Of interest is the observation that the sequence around a conserved arginine at the start of the second intracellular loop suggests that the ORF74 product may signal constitutively (agonist independent). Given that the ORF74 product is predicted to encode a GPCR we named it MHV-GPCR. In studies on the transcription of the MHV-GPCR, we determined that it was encoded on multiple early transcripts of 3.4, 4.4, 6.6 and 8.7 kb in size. At least one of these transcripts was bicistronic, containing the ORF encoding the Bcl-2 homologue also. In vivo, we found that MHV GPCR was expressed during acute infection but also during persistence, particularly in the lungs of infected mice. Immunofluorescence studies indicated that the MHV GPCR protein was expressed on the surface of cells in patches. Finally, like the KSHV GPCR, expression of the MHV GPCR resulted in transformation of NIH 3T3 cells. We surmise, therefore, that the MHV GPCR may act in concert with genes with which it is expressed such as vBcl-2 to enhance the growth and survival of MHV-68-infected cells.

Identification of a region of the virus genome involved in murine gammaherpesvirus 68-induced splenic pathology.

Infection with the murine gammaherpesvirus MHV-68 has profound effects on splenic and mediastinal lymph node pathology in mice which lack the interferon-gamma receptor (IFN-gamma R(-/-)). In these mice MHV-68 infection causes fibrosis and loss of lymphocytes in the spleen and the mediastinal lymph node as well as interstitial pulmonary fibrosis and fibrotic changes in the liver. The changes are associated with transient elevated latent virus loads in the spleen. Four independent virus mutants with insertions and/or deletions in the left end of the genome fail to induce the pathological changes and establish latency at normal levels in the spleen. The data indicate that the pathology does not correlate with any of the known genes encoded within this region of the genome, genes M1-M4 and the eight vtRNAs. Northern analysis of mRNAs transcribed by wild-type and mutant viruses shows that at least two uncharacterized transcripts are encoded within this region. These transcripts are absent in the mutant viruses and are candidates for the virus genes responsible for the aberrant pathology in IFN-gamma R(-/-) mice.

Transcriptome profile of murine gammaherpesvirus-68 lytic infection.

The murine gammaherpesvirus-68 genome encodes 73 protein-coding open reading frames with extensive similarities to human gamma(2) herpesviruses, as well as unique genes and cellular homologues. We performed transcriptome analysis of stage-specific viral RNA during permissive infection using an oligonucleotide-based microarray. Using this approach, M4, K3, ORF38, ORF50, ORF57 and ORF73 were designated as immediate-early genes based on cycloheximide treatment. The microarray analysis also identified 10 transcripts with early expression kinetics, 32 transcripts with early-late expression kinetics and 29 transcripts with late expression kinetics. The latter group consisted mainly of structural proteins, and showed high expression levels relative to other viral transcripts. Moreover, we detected all eight tRNA-like transcripts in the presence of cycloheximide and phosphonoacetic acid. Lytic infection with MHV-68 also resulted in a significant reduction in the expression of cellular transcripts included in the DNA chip. This global approach to viral transcript analysis offers a powerful system for examining molecular transitions between lytic and latent virus infections associated with disease pathogenesis.

Expression profiling reveals novel innate and inflammatory responses in the jejunal epithelial compartment during infection with Trichinella spiralis.

Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule beta (RELMbeta) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmbeta and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmbeta).

Chronic mild hypoxia protects heart-derived H9c2 cells against acute hypoxia/reoxygenation by regulating expression of the SUR2A subunit of the ATP-sensitive K+ channel.

Chronic exposure to lower oxygen tension may increase cellular resistance to different types of acute metabolic stress. Here, we show that 24-h-long exposure to slightly decreased oxygen tension (partial pressure of oxygen (PO2) of 100 mm Hg instead of normal 144 mm Hg) confers resistance against acute hypoxia/reoxygenation-induced Ca2+ loading in heart-derived H9c2 cells. The number of ATP-sensitive K+ (K(ATP)) channels were increased in cells exposed to PO2 = 100 mm Hg relative to cells exposed to PO2 = 144 mm Hg. This was due to an increase in transcription of SUR2A, a K(ATP) channel regulatory subunit, but not Kir6.2, a K(ATP) channel pore-forming subunit. PO2 = 100 mm Hg also increased the SUR2 gene promoter activity. Experiments with cells overexpressing wild type of hypoxia-inducible factor (HIF)-1alpha and dominant negative HIF-1beta suggested that the HIF-1-signaling pathway did not participate in observed PO2-mediated regulation of SUR2A expression. On the other hand, NADH inhibited the effect of PO2 = 100 mm Hg but not the effect of PO2 = 20 mm Hg. LY 294002 and PD 184 352 prevented PO2-mediated regulation of K(ATP) channels, whereas rapamycin was without any effect. HMR 1098 inhibited the cytoprotective effect of PO2 = 100 mm Hg, and a decrease of PO2 from 144 to 100 mm Hg did not change the expression of any other gene, including those involved in stress and hypoxic response, as revealed by Affymetrix high density oligonucleotide arrays. We conclude that slight hypoxia activates HIF-1alpha-independent signaling cascade leading to an increase in SUR2A protein, a higher density of K(ATP) channels, and a cellular phenotype more resistant to acute metabolic stress.