Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV.

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Herpes simplex virus 1 (HSV-1) infection triggers specific metabolic changes in its host cell. To explore the interactions between cellular metabolism and HSV-1 infection, we performed an siRNA screen of cellular metabolic genes, measuring their effect on viral replication. The screen identified multiple enzymes predicted to influence HSV-1 replication, including argininosuccinate synthetase 1 (AS1), which consumes aspartate as part of de novo arginine synthesis. Knockdown of AS1 robustly enhanced viral genome replication and the production of infectious virus. Using high-resolution liquid chromatography-mass spectrometry, we found that the metabolic phenotype induced by knockdown of AS1 in human fibroblasts mimicked multiple aspects of the metabolic program observed during HSV-1 infection, including an increase in multiple nucleotides and their precursors. Together with the observation that AS1 protein and mRNA levels decrease during wild-type infection, this work suggests that reduced AS1 activity is partially responsible for the metabolic program induced by infection.metabolomics | herpesviruses M any viruses, including herpes simplex virus 1 (HSV-1), human cytomegalovirus (HCMV), influenza, Dengue, hepatitis C, and hepatitis E (1-11), have been shown to significantly perturb metabolic homeostasis over the course of infection. HSV-1 is an intriguing case study, because the virus encodes several of its own metabolic enzymes, including a dUTPase, uracil-DNA glycosylase, ribonucleotide reductase, and thymidine kinase (12). Infection of host cells by multiple strains of HSV-1 induces alterations to central carbon metabolism, such as increased flux through upper glycolysis, and the anapleurotic feeding of carbons into the citric acid (TCA) cycle through the activity of pyruvate carboxylase (1). Infection also induces nucleotide synthesis, manifested by the increased flux of carbons through aspartate to nucleotides, as well as the increased pool levels of multiple nucleotides themselves (1).Although the metabolic reprogramming that occurs during HSV-1 infection has been described, the importance of specific host cell metabolic enzymes to infection is relatively unknown. Here we sought to further resolve the relationship between infection and metabolism by screening for cellular metabolic enzymes that modulate the efficiency of viral infection in primary human fibroblasts. The screen identified several dozen enzymes that modulate HSV-1 growth, including argininosuccinate synthetase (AS1), which antagonized viral replication.AS1 acts as a homotetramer to catalyze the synthesis of argininosuccinate from aspartate and citrulline (13,14). The enzyme plays a role in the production of urea in the kidney and liver, but functions primarily as the limiting step in the citrulline-nitric oxide (NO) cycle in other cell types (15). As a constituent of the citrulline-NO cycle, AS1 participates in the de novo production of the nonessential amino acid arginine as well as the soluble factor NO.We found that AS1 knockdown by siRNA in...

Background: The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for highthroughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. Results: To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5-50 Colony Forming Units (CFU)/ml).

BackgroundExamination of complex biological systems has long been achieved through methodical investigation of the system’s individual components. While informative, this strategy often leads to inappropriate conclusions about the system as a whole. With the advent of high-throughput “omic” technologies, however, researchers can now simultaneously analyze an entire system at the level of molecule (DNA, RNA, protein, metabolite) and process (transcription, translation, enzyme catalysis). This strategy reduces the likelihood of improper conclusions, provides a framework for elucidation of genotype-phenotype relationships, and brings finer resolution to comparative genomic experiments. Here, we apply a multi-omic approach to analyze the gene expression profiles of two closely related Pseudomonas aeruginosa strains grown in n-alkanes or glycerol.ResultsThe environmental P. aeruginosa isolate ATCC 33988 consumed medium-length (C10–C16) n-alkanes more rapidly than the laboratory strain PAO1, despite high genome sequence identity (average nucleotide identity >99%). Our data shows that ATCC 33988 induces a characteristic set of genes at the transcriptional, translational and post-translational levels during growth on alkanes, many of which differ from those expressed by PAO1. Of particular interest was the lack of expression from the rhl operon of the quorum sensing (QS) system, resulting in no measurable rhamnolipid production by ATCC 33988. Further examination showed that ATCC 33988 lacked the entire lasI/lasR arm of the QS response. Instead of promoting expression of QS genes, ATCC 33988 up-regulates a small subset of its genome, including operons responsible for specific alkaline proteases and sphingosine metabolism.ConclusionThis work represents the first time results from RNA-seq, microarray, ribosome footprinting, proteomics, and small molecule LC-MS experiments have been integrated to compare gene expression in bacteria. Together, these data provide insights as to why strain ATCC 33988 is better adapted for growth and survival on n-alkanes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3708-4) contains supplementary material, which is available to authorized users.