Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV) and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq). We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases. We have conducted a comprehensive analysis of the murine cytomegalovirus and host cell transcriptomes during lytic infection. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. Infection alters expression of many unexpected host genes, including many noncoding RNA genes. Correspondingly, many cluster in unexpected biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases.

Sphingolipids are present in membranes of all eukaryotic cells. Bioactive sphingolipids also function as signaling molecules that regulate cellular processes such as proliferation, migration, and apoptosis. Human cytomegalovirus (HCMV) exploits a variety of cellular signaling pathways to promote its own replication. However, whether HCMV modulates lipid signaling pathways is an essentially unexplored area of research in virus-host cell interactions. In this study, we examined the accumulation of the bioactive sphingolipids and the enzymes responsible for the biosynthesis and degradation of these lipids. HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK), the enzyme that generates sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). We also utilized a mass spectrometry approach to generate a sphingolipidomic profile of HCMV-infected cells. We show that HCMV infection results in increased levels of dhS1P and ceramide at 24 h, suggesting an enhancement of de novo sphingolipid synthesis. Subsequently dihydrosphingosine and dhS1P decrease at 48 h consistent with attenuation of de novo sphingolipid synthesis. Finally, we present evidence that de novo sphingolipid synthesis and sphingosine kinase activity directly impact virus gene expression and virus growth. Together, these findings demonstrate that host cell sphingolipids are dynamically regulated upon infection with a herpes virus in a manner that impacts virus replication.

Human cytomegalovirus (HCMV) infections are prevalent in human populations and can cause serious diseases, especially in those with compromised or immature immune systems. The HCMV genome of 230 kb is among the largest of the herpesvirus genomes. Although the entire sequence of the laboratory-adapted AD169 strain of HCMV has been available for 18 years, the precise number of viral genes is still in question. We undertook an analysis of the HCMV transcriptome as an approach to enumerate and analyze the gene products of HCMV. Transcripts of HCMV-infected fibroblasts were isolated at different times after infection and used to generate cDNA libraries representing different temporal classes of viral genes. cDNA clones harboring viral sequences were selected and subjected to sequence analysis. Of the 604 clones analyzed, 45% were derived from genomic regions predicted to be noncoding. Additionally, at least 55% of the cDNA clones in this study were completely or partially antisense to known or predicted HCMV genes. The remarkable accumulation of antisense transcripts during infection suggests that currently available genomic maps based on open-reading-frame and other in silico analyses may drastically underestimate the true complexity of viral gene products. These findings also raise the possibility that aspects of both the HCMV life cycle and genome organization are influenced by antisense transcription. Correspondingly, virus-derived noncoding and antisense transcripts may shed light on HCMV pathogenesis and may represent a new class of targets for antiviral therapies.

Interferons (IFNs) activate the first lines of defense against viruses, and promote innate and adaptive immune responses to viruses. We report that the immediate early 1 (IE1) protein of human cytomegalovirus (HCMV) disrupts signaling by IFNγ. The carboxyl-terminal region of IE1 is required for this function. We found no defect in the initial events in IFNγ signaling or in nuclear accumulation of signal transducer and activator of transcription 1 (STAT1) in IE1-expressing cells. Moreover, we did not observe an association between disruption of IFNγ signaling and nuclear domain 10 (ND10) disruption. However, there is reduced binding of STAT1 homodimers to target gamma activated sequence (GAS) elements in the presence of IE1. Co-immunoprecipitation studies failed to support a direct interaction between IE1 and STAT1, although these studies revealed that the C-terminal region of IE1 was required for interaction with STAT2. Together, these results indicate that IE1 disrupts IFNγ signaling by interfering with signaling events in the nucleus through a novel mechanism.