The non-coding RNA interactome in joint health and disease.

Non-coding RNAs have distinct regulatory roles in the pathogenesis of joint diseases including osteoarthritis (OA) and rheumatoid arthritis (RA). As the amount of high-throughput profiling studies and mechanistic investigations of microRNAs, long non-coding RNAs and circular RNAs in joint tissues and biofluids has increased, data have emerged that suggest complex interactions among non-coding RNAs that are often overlooked as critical regulators of gene expression. Identifying these non-coding RNAs and their interactions is useful for understanding both joint health and disease. Non-coding RNAs regulate signalling pathways and biological processes that are important for normal joint development but, when dysregulated, can contribute to disease. The specific expression profiles of non-coding RNAs in various disease states support their roles as promising candidate biomarkers, mediators of pathogenic mechanisms and potential therapeutic targets. This Review synthesizes literature published in the past 2 years on the role of non-coding RNAs in OA and RA with a focus on inflammation, cell death, cell proliferation and extracellular matrix dysregulation. Research to date makes it apparent that 'non-coding' does not mean 'non-essential' and that non-coding RNAs are important parts of a complex interactome that underlies OA and RA.

Epigenetic Alterations in Pediatric Sleep Apnea.

Pediatric obstructive sleep apnea has significant negative effects on health and behavior in childhood including depression, failure to thrive, neurocognitive impairment, and behavioral issues. It is strongly associated with an increased risk for chronic adult disease such as obesity and diabetes, accelerated atherosclerosis, and endothelial dysfunction. Accumulating evidence suggests that adult-onset non-communicable diseases may originate from early life through a process by which an insult applied at a critical developmental window causes long-term effects on the structure or function of an organism. In recent years, there has been increased interest in the role of epigenetic mechanisms in the pathogenesis of adult disease susceptibility. Epigenetic mechanisms that influence adaptive variability include histone modifications, non-coding RNAs, and DNA methylation. This review will highlight what is currently known about the phenotypic associations of epigenetic modifications in pediatric obstructive sleep apnea and will emphasize the importance of epigenetic changes as both modulators of chronic disease and potential therapeutic targets.

CENP-B promotes the centromeric localization of ZFAT to control transcription of noncoding RNA.

The centromere is a chromosomal locus that is essential for the accurate segregation of chromosomes during cell division. Transcription of noncoding RNA (ncRNA) at the centromere plays a crucial role in centromere function. The zinc-finger transcriptional regulator ZFAT binds to a specific 8-bp DNA sequence at the centromere, named the ZFAT box, to control ncRNA transcription. However, the precise molecular mechanisms by which ZFAT localizes to the centromere remain elusive. Here we show that the centromeric protein CENP-B is required for the centromeric localization of ZFAT to regulate ncRNA transcription. The ectopic expression of CENP-B induces the accumulation of both endogenous and ectopically expressed ZFAT protein at the centromere in human cells, suggesting that the centromeric localization of ZFAT requires the presence of CENP-B. Coimmunoprecipitation analysis reveals that ZFAT interacts with the acidic domain of CENP-B, and depletion of endogenous CENP-B reduces the centromeric levels of ZFAT protein, further supporting that CENP-B is required for the centromeric localization of ZFAT. In addition, knockdown of CENP-B significantly decreased the expression levels of ncRNA at the centromere where ZFAT regulates the transcription, suggesting that CENP-B is involved in the ZFAT-regulated centromeric ncRNA transcription. Thus, we concluded that CENP-B contributes to the establishment of the centromeric localization of ZFAT to regulate ncRNA transcription.

Roles of noncoding RNAs in preeclampsia.

Preeclampsia (PE) is an idiopathic disease that occurs during pregnancy. It comprises multiple organ and system damage, and can seriously threaten the safety of the mother and infant throughout the perinatal period. As the pathogenesis of PE is unclear, there are few specific remedies. Currently, the only way to eliminate the clinical symptoms is to terminate the pregnancy. Although noncoding RNA (ncRNA) was once thought to be the "junk" of gene transcription, it is now known to be widely involved in pathological and physiological processes, including pregnancy-related disorders. Moreover, there is growing evidence that the unbalanced expression of specific ncRNA is involved in the pathogenesis of PE. In the present review, we summarize the expression patterns of ncRNAs, i.e., microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), and the functional mechanisms by which they affect the development of PE, and examine the clinical significance of ncRNAs as biomarkers for the diagnosis of PE. We also discuss the contributions made by genetic polymorphisms and epigenetic ncRNA regulation to PE. In the present review, we wish to explore and reinforce the clinical value of ncRNAs as noninvasive biomarkers of PE.

Methylation and Noncoding RNAs in Gastric Cancer: Everything Is Connected.

Despite recent progress, gastric cancer remains one of the most common cancers and has a high mortality rate worldwide. Aberrant DNA methylation pattern and deregulation of noncoding RNA expression appear in the early stages of gastric cancer. Numerous investigations have confirmed their significant role in gastric cancer tumorigenesis and their high potential as diagnostic and prognostic biomarkers. Currently, it is clear that these epigenetic regulators do not work alone but interact with each other, generating a complex network. The aim of our review was to summarize the current knowledge of this interaction in gastric cancer and estimate its clinical potential for the diagnosis, prognosis, and treatment of the disease.

Identification and validation of key non-coding RNAs and mRNAs using co-expression network analysis in pre-eclampsia.

ABSTRACT: Pre-eclampsia (PE) is a common complication of pregnancy, associated with maternal and fetal morbidity and mortality. In this study, we aimed to explore important long non-coding RNAs (lncRNAs) and their possible mechanisms in PE.GSE60438 expression profile including 25 PE samples and 23 normal samples were obtained from gene expression omnibus (GEO) database. After normalization with betaqn package in R, differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were identified using the limma package. Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway were analyzed using DAVID 6.7 and GSEA 3.0. LncRNAs-mRNAs coexpression was implemented using weighted gene co-expression network analysis (WGCNA). MicroRNAs linked with these DElncRNAs and DEmRNAs were predicted and a competitive endogenous RNA (ceRNA) network was built.A total of 53 DElncRNAs and 301 DEmRNAs were identified between control and PE samples. These DEmRNAs were enriched into pathways such as protein digestion and absorption, osteoclast differentiation. WGCNA constructed a lncRNA-mRNA coexpression network, among which SUMO1P3, NACAP1, NCF1C, ANXA2P1, GTF2IP1, NAPSB, OR7E37P were hub genes. ceRNA network was constructed together with microRNAs (miRNAs), and functional analysis indicated cellular membrane and sugar binding were involved in PE progression. Five lncRNAsANXA2P1, GTF2IP1, NACAP1, NCF1C and OR7E37P were successfully validated in our clinical specimens.The DElncRNAs, including ANXA2P1, GTF2IP1, NACAP1, NCF1C and OR7E37P might play important roles in PE. However, the exact mechanism of these lncRNAs in prediction and diagnosis of PE should be further explored.

CXCL8 chemokine in ulcerative colitis.

Ulcerative colitis (UC) is a major type of inflammatory bowel disease (IBD), which is characterized by diffuse inflammation of the mucosa of the colon and rectum. Abdominal pain, diarrhea, and hematochezia are UC's main clinical manifestations. Pathogenesis of UC has not yet been clearly elucidated, but it is considered to result from dysregulated expressions of molecules engaged in proinflammatory and anti-inflammatory processes. CXCL8 is one of the most important proinflammatory factors which play a vital role in many inflammatory diseases including UC. The CXCL8-CXCR1/2 axis participates in the pathogenesis of UC through multiple signaling pathways, including PI3k/Akt, MAPKs and NF-κB signaling pathways. Meanwhile, more and more studies in recent years have shown that UC patients have specific non-coding RNA (ncRNA) expression profiles, which may be involved in the occurrence and development of inflammation. In this article, we analyzed the CXCL8-CXCR1/2 axis related signaling pathways and ncRNAs in UC, as well as recent advances in our understanding of the CXCL8-CXCR1/2 axis inhibition as a therapeutic strategy against UC.

Computational methods for annotation of plant regulatory non-coding RNAs using RNA-seq.

Plant transcriptome encompasses numerous endogenous, regulatory non-coding RNAs (ncRNAs) that play a major biological role in regulating key physiological mechanisms. While studies have shown that ncRNAs are extremely diverse and ubiquitous, the functions of the vast majority of ncRNAs are still unknown. With ever-increasing ncRNAs under study, it is essential to identify, categorize and annotate these ncRNAs on a genome-wide scale. The use of high-throughput RNA sequencing (RNA-seq) technologies provides a broader picture of the non-coding component of transcriptome, enabling the comprehensive identification and annotation of all major ncRNAs across samples. However, the detection of known and emerging class of ncRNAs from RNA-seq data demands complex computational methods owing to their unique as well as similar characteristics. Here, we discuss major plant endogenous, regulatory ncRNAs in an RNA sample followed by computational strategies applied to discover each class of ncRNAs using RNA-seq. We also provide a collection of relevant software packages and databases to present a comprehensive bioinformatics toolbox for plant ncRNA researchers. We assume that the discussions in this review will provide a rationale for the discovery of all major categories of plant ncRNAs.

Involvement of GcvB small RNA in intrinsic resistance to multiple aminoglycoside antibiotics in Escherichia coli.

Deleting the gene for small RNA GcvB in Escherichia coli was found to increase the sensitivity to several aminoglycoside antibiotics, such as neomycin, streptomycin, kanamycin, kasugamycin and spectinomycin, at low concentrations. GcvB, conserved in gram-negative enteric bacteria, is known to negatively control the expression of many genes for amino acid incorporation systems, especially the periplasmic ABC-transporter proteins. Deletions of several amino acid transporter genes in ΔgcvB cells decreased the antibiotic sensitivity to the wild-type level, suggesting that those genes are involved in uptake of aminoglycosides into the cell. Since GcvB is constitutively synthesized in growing cells, repressing synthesis of amino acid transporters, it contributes to the intrinsic resistance to several aminoglycoside antibiotics.

Nucleolar RNA polymerase II drives ribosome biogenesis.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

Linc01555 promotes proliferation, migration and invasion of gastric carcinoma cells by interacting with Notch signaling pathway.

PURPOSE: To detect the expression level of long non-coding ribonucleic acid 01555 (linc01555) in gastric cancer (GC) tissues and cells, and its effects on the biological functions of GC cells. METHODS: The relative expression of linc01555 in 61 cases of GC and para-carcinoma tissues and GC cells was detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). GC cells were divided into experimental group (si-linc01555) and control group (si-NC), and the interference efficiency was detected through qRT-PCR. The effects of interference in linc01555 expression on GC cell proliferation, colony formation ability, migration and invasion were determined using cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and Transwell assay. Moreover, the expressions of molecular markers in the downstream Notch pathway were detected using western blotting. RESULTS: The results of qRT-PCR showed that the expression of linc01555 was upregulated in GC tissues and cells. The results of CCK-8 assay revealed that the proliferative activity of GC cells declined after interference in linc01555 expression. It was found in colony formation assay that the proliferation ability of GC cells declined after interference in linc01555 expression, and it was observed in wound healing assay that the cell migration ability in the experimental group was weakened compared with that in the control group. According to the results of transwell assay, both migration and invasion ability of GC cells declined after interference in linc01555 expression. Finally, the western blotting showed that there were changes in the expressions of molecular markers in the Notch signaling pathway after interference in linc01555 expression. CONCLUSIONS: The expression of linc01555 is upregulated in GC tissues and cells, and the highly-expressed linc01555 promotes the proliferation, invasion and metastasis of GC cells through the Notch signaling pathway.

PART1 and hsa-miR-429-Mediated SHCBP1 Expression Is an Independent Predictor of Poor Prognosis in Glioma Patients.

Gliomas are the most common primary brain tumors. Because of their high degree of malignancy, patient survival rates are unsatisfactory. Therefore, exploring glioma biomarkers will play a key role in early diagnosis, guiding treatment, and monitoring the prognosis of gliomas. We found two lncRNAs, six miRNAs, and nine mRNAs that were differentially expressed by analyzing genomic data of glioma patients. The diagnostic value of mRNA expression levels in gliomas was determined by receiver operating characteristic (ROC) curve analysis. Among the nine mRNAs, the area under the ROC curve values of only CEP55 and SHCBP1 were >0.7, specifically 0.834 and 0.816, respectively. Additionally, CEP55 and SHCBP1 were highly expressed in glioma specimens and showed increased expression according to the glioma grade, and outcomes of high expression patients were poor. CEP55 was enriched in the cell cycle, DNA replication, mismatch repair, and P53 signaling pathway. SHCBP1 was enriched in the cell cycle, DNA replication, ECM receptor interaction, and P53 signaling pathway. Age, grade, IDH status, chromosome 19/20 cogain, and SHCBP1 were independent factors for prognosis. Our findings suggest the PART1-hsa-miR-429-SHCBP1 regulatory network plays an important role in gliomas.

Novel approaches for efficient in vivo fermentation production of noncoding RNAs.

Genome-derived noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), play an essential role in the control of target gene expression underlying various cellular processes, and dysregulation of ncRNAs is involved in the pathogenesis and progression of various diseases in virtually all species including humans. Understanding ncRNA biology has opened new avenues to develop novel RNA-based therapeutics. Presently, ncRNA research and drug development is dominated by the use of ncRNA mimics that are synthesized chemically in vitro and supplemented with extensive and various types of artificial modifications and thus may not necessarily recapitulate the properties of natural RNAs generated and folded in living cells in vivo. Therefore, there are growing interests in developing novel technologies for in vivo production of RNA molecules. The two most recent major breakthroughs in achieving an efficient, large-scale, and cost-effective fermentation production of recombinant or bioengineered RNAs (e.g., tens of milligrams from 1 L of bacterial culture) are (1) using stable RNA carriers and (2) direct overexpression in RNase III-deficient bacteria, while other approaches offer a low yield (e.g., nano- to microgram scales per liter). In this article, we highlight these novel microbial fermentation-based technologies that have shifted the paradigm to the production of true biological ncRNA molecules for research and development.

Long intergenic noncoding RNA profiles of pheochromocytoma and paraganglioma: A novel prognostic biomarker.

Many long intergenic noncoding RNAs (lincRNAs) serve as cancer biomarkers for diagnosis or prognostication. To understand the role of lincRNAs in the rare neuroendocrine tumors pheochromocytoma and paraganglioma (PCPG), we performed first time in-depth characterization of lincRNA expression profiles and correlated findings to clinical outcomes of the disease. RNA-Seq data from patients with PCPGs and 17 other tumor types from The Cancer Genome Atlas and other published sources were obtained. Differential expression analysis and a machine-learning model were used to identify transcripts specific to PCPGs, as well as established PCPG molecular subtypes. Similarly, lincRNAs specific to aggressive PCPGs were identified, and univariate and multivariate analysis was performed for metastasis-free survival. The results were validated in independent samples using RT-PCR. From a pan-cancer context, PCPGs had a specific and unique lincRNA profile. Among PCPGs, five different molecular subtypes were identified corresponding to the established molecular classification. Upregulation of 13 lincRNAs was found to be associated with aggressive/metastatic PCPGs. RT-PCR validation confirmed the overexpression of four lincRNAs in metastatic compared to non-metastatic PCPGs. Kaplan-Meier analysis identified five lincRNAs as prognostic markers for metastasis-free survival of patients in three subtypes of PCPGs. Stratification of PCPG patients with a risk-score formulated using multivariate analysis of lincRNA expression profiles, presence of key driver mutations, tumor location, and hormone secretion profiles showed significant differences in metastasis-free survival. PCPGs thus exhibit a specific lincRNA expression profile that also corresponds to the established molecular subgroups and can be potential marker for the aggressive/metastatic PCPGs.

LncRNA PTCSC3 inhibits the proliferation, invasion and migration of cervical cancer cells via sponging miR-574-5p.

Dysregulation of long non-coding RNA papillary thyroid carcinoma susceptibility candidate 3 (lncRNA PTCSC3) has been found to correlate with various types of cancers. Quantitative RT-PCR showed a down-regulation of PTCSC3 in cervical cancer tissues compared with normal cervical tissues. The present study aimed to investigate the role of lncRNA PTCSC3 in cervical cancer and the underlying mechanisms. PTCSC3 was overexpressed in cervical cancer cell lines C-33A and Hela by transfection with pcDNA3.1-lncRNA PTCSC3 expressing plasmid. Overexpression of lncRNA PTCSC3 inhibited cell proliferation, induced cell cycle arrest, and suppressed cell invasion and migration using CCK8 assay, flow cytometry, Transwell assay and wound healing examination, respectively. Western blotting analysis showed that PTCSC3 overexpression decreased the expression of cyclinD1, matrix metalloproteinases 9 (MMP9), N-cadherin and ß-catenin and increased E-cadherin expression. Further, PTCSC3 negatively regulated miR-574-5p expression and dual-luciferase assay verified the binding activity between miR-574-5p and lncRNA PTCSC3. Enforced up-regulation of miR-574-5p abolished the inhibitory effect of lncRNA PTCSC3 on cervical cancer cell proliferation, invasiveness and mobility. Taken together, lncRNA PTCSC3 inhibited cell growth and metastasis via sponging miR-574-5p in cervical cancer. Therefore, we demonstrate the tumour-suppressive function of lncRNA PTCSC3 in cervical cancer and suggest that PTCSC3 is a potential therapeutic target for cervical cancer.

Regulatory RNA at the crossroads of carbon and nitrogen metabolism in photosynthetic cyanobacteria.

Cyanobacteria are photosynthetic bacteria that populate widely different habitats. Accordingly, cyanobacteria exhibit a wide spectrum of lifestyles, physiologies, and morphologies and possess genome sizes and gene numbers which may vary by up to a factor of ten within the phylum. Consequently, large differences exist between individual species in the size and complexity of their regulatory networks. Several non-coding RNAs have been identified that play crucial roles in the acclimation responses of cyanobacteria to changes in the environment. Some of these regulatory RNAs are conserved throughout the cyanobacterial phylum, while others exist only in a few taxa. Here we give an overview on characterized regulatory RNAs in cyanobacteria, with a focus on regulators of photosynthesis, carbon and nitrogen metabolism. However, chances are high that these regulators represent just the tip of the iceberg.

The Roles of Hypoxia-Inducible Factors and Non-Coding RNAs in Gastrointestinal Cancer.

Hypoxia-inducible factors (HIFs) are transcription factors that play central roles in cellular responses against hypoxia. In most cancers, HIFs are closely associated with tumorigenesis by regulating cell survival, angiogenesis, metastasis, and adaptation to the hypoxic tumor microenvironment. Recently, non-coding RNAs (ncRNAs) have been reported to play critical roles in the hypoxic response in various cancers. Here, we review the roles of hypoxia-response ncRNAs in gastrointestinal cancer, with a particular focus on microRNAs and long ncRNAs, and discuss the functional relationships and regulatory mechanisms between HIFs and ncRNAs.

Impact of Toxoplasma gondii Infection on Host Non-coding RNA Responses.

As an intracellular microbe, Toxoplasma gondii must establish a highly intimate relationship with its host to ensure success as a parasite. Many studies over the last decade-and-a-half have highlighted how the host reshapes its immunoproteome to survive infection, and conversely how the parasite regulates host responses to ensure persistence. The role of host non-protein-coding RNA during infection is a vast and largely unexplored area of emerging interest. The potential importance of this facet of the host-parasite interaction is underscored by current estimates that as much as 80% of the host genome is transcribed into non-translated RNA. Here, we review the current state of knowledge with respect to two major classes of non-coding RNA, microRNA (miRNA) and long non-coding RNA (lncRNA), in the host response to T. gondii infection. These two classes of regulatory RNA are known to have profound and widespread effects on cell function. However, their impact on infection and immunity is not well-understood, particularly for the response to T. gondii. Nevertheless, numerous miRNAs have been identified that are upregulated by Toxoplasma, and emerging evidence suggests a functional role during infection. While the field of lncRNA is in its infancy, it is already clear that Toxoplasma is also a strong trigger for this class of regulatory RNA. Non-coding RNA responses induced by T. gondii are likely to be major determinants of the host's ability to resist infection and the parasite's ability to establish long-term latency.

Network analysis of noncoding RNAs in pepper provides insights into fruit ripening control.

Pepper is an important vegetable worldwide and is a model plant for nonclimacteric fleshy fruit ripening. Drastic visual changes and internal biochemical alterations are involved in fruit coloration, flavor, texture, aroma, and palatability to animals during the pepper fruit ripening process. To explore the regulation of bell pepper fruit ripening by noncoding RNAs (ncRNAs), we examined their expression profiles; 43 microRNAs (miRNAs), 125 circular RNAs (circRNAs), 366 long noncoding RNAs (lncRNAs), and 3266 messenger RNAs (mRNAs) were differentially expressed (DE) in mature green and red ripe fruit. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the targets of the DE ncRNAs and DE mRNAs included several kinds of transcription factors (TFs) (ERF, bHLH, WRKY, MYB, NAC, bZIP, and ARF), enzymes involved in cell wall metabolism (beta-galactosidase, beta-glucosidase, beta-amylase, chitinase, pectate lyase (PL), pectinesterase (PE) and polygalacturonase (PG)), enzymes involved in fruit color accumulation (bifunctional 15-cis-phytoene synthase, 9-cis-epoxycarotenoid dioxygenase, beta-carotene hydroxylase and carotene epsilon-monooxygenase), enzymes associated with fruit flavor and aroma (glutamate-1-semialdehyde 2,1-aminomutase, anthocyanin 5-aromatic acyltransferase, and eugenol synthase 1) and enzymes involved in the production of ethylene (ET) (ACO1/ACO4) as well as other plant hormones such as abscisic acid (ABA), auxin (IAA), and gibberellic acid (GA). Based on accumulation profiles, a network of ncRNAs and mRNAs associated with bell pepper fruit ripening was developed that provides a foundation for further developing a more refined understanding of the molecular biology of fruit ripening.

Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression.

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level.