Roseomics: a blank slate

Recent technological advances have led to an explosion in the system-wide profiling of biological processes in the study of herpesvirus biology, herein referred to as “-omics”. In many cases these approaches have revealed novel virus-induced changes to host cell biology that can be targeted with new antiviral therapeutics. Despite these successes, -omics approaches are not widely applied in the study of roseoloviruses. Here we describe examples of how -omics studies have shaped our understanding of herpesvirus biology, and discuss how these approaches might be used to identify host and viral factors that mediate roseolovirus pathogenesis

Corrigendum: The Expression of Human Cytomegalovirus MicroRNA MiR-UL148D during Latent Infection in Primary Myeloid Cells Inhibits Activin A-triggered Secretion of IL-6.

Scientific Reports 6: Article number: 31205; published online: 05 August 2016; updated: 26 September 2016. The original version of this Article contained an error in the spelling of the author Immaculada Montanuy which was incorrectly given as Immaculada Sellart. In addition, the Author Contributions Statement contained errors.</jats:p

The Human Cytomegalovirus Latency-Associated Gene Product Latency Unique Natural Antigen Regulates Latent Gene Expression

Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage

Revisiting promyelocytic leukemia protein targeting by human cytomegalovirus immediate-early protein 1

This work was supported by a grant (MR/P022146/1) from the Medical Research Council (https://mrc.ukri.org) to MMN, a grant (T16/28) from Tenovus Scotland (https://tenovus-scotland.org.uk) to CP, a European Union Erasmus+ grant (https://www.erasmusplus.org.uk) to BW and the Wellcome Trust Institutional Strategic Support Fund (https://wellcome.ac.uk) to CP and MMN.Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.Publisher PDFPeer reviewe

Human cytomegalovirus miR-UL112-1 promotes the down-regulation of viral immediate early-gene expression during latency to prevent T-cell recognition of latently infected cells.

Human cytomegalovirus, a member of the herpesvirus family, can cause significant morbidity and mortality in immune compromised patients resulting from either primary lytic infection or reactivation from latency. Latent infection is associated with a restricted viral transcription programme compared to lytic infection which consists of defined protein coding RNAs but also includes a number of virally encoded microRNAs (miRNAs). One of these, miR-UL112-1, is known to target the major lytic IE72 transcript but, to date, a functional role for miR-UL112-1 during latent infection has not been shown. To address this, we have analysed latent infection in myeloid cells using a virus in which the target site for miR-UL112-1 in the 3' UTR of IE72 was removed such that any IE72 RNA present during latent infection would no longer be subject to regulation by miR-UL112-1 through the RNAi pathway. Our data show that removal of the miR-UL112-1 target site in IE72 results in increased levels of IE72 RNA in experimentally latent primary monocytes. Furthermore, this resulted in induction of immediate early (IE) gene expression that is detectable by IE-specific cytotoxic T-cells (CTLs); no such CTL recognition of monocytes latently infected with wild-type virus was observed. We also recapitulated these findings in the more tractable THP-1 cell line model of latency. These observations argue that an important role for miR-UL112-1 during latency is to ensure tight control of lytic viral immediate early (IE) gene expression thereby preventing recognition of latently infected cells by the host's potent pre-existing anti-viral CTL response.Medical Research Council (Grant ID: G:0701279); National Institute for Health Research Biomedical Research CentreThis is the author accepted manuscript. The final version is available from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00054

An in vitro study of the effects of respiratory circuit setup and parameters on aerosol delivery during mechanical ventilation

IntroductionAerosol therapy is often prescribed concurrently during invasive mechanical ventilation (IMV). This study determines the effects of nebuliser position, circuit humidification source, and most importantly, lung health on the delivery of aerosol in simulated adult and paediatric IMV patients. Furthermore, the influence of closed suction catheters on aerosol delivery is also addressed.MethodsA vibrating mesh nebuliser was used to deliver Albuterol to simulated adult and paediatric IMV patients with differing states of lung health. Four different nebuliser positions and two types of humidification were analysed. Closed suction catheter mounts, a mainstay in IMV therapy, were incorporated into the circuits. The mean ± SD dose of aerosol (%) was assayed from a filter at the distal end of the endotracheal tube.ResultsNebuliser placement and circuit humidification source had no effect on the delivered dose (%) in adults, yet both significantly did in the simulated paediatric patients. The use of closed suction catheter mounts significantly reduced the delivered dose (%) in adults but not in paediatric patients. A simulated healthy lung state generated the largest delivered dose (%), irrespective of nebuliser position in the adult. However, different lung health and nebuliser positions yielded higher delivered doses (%) in paediatrics.ConclusionLung health and respiratory circuit composition significantly affect aerosol delivery in both adult and paediatric IMV patients. Nebuliser placement and respiratory circuit humidification source do not affect the delivered dose in adult but do in paediatric IMV patients

Virology under the microscope—a call for rational discourse

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology

The Natural Flavonoid Compound Deguelin Inhibits HCMV Lytic Replication within Fibroblasts

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus for which there is no vaccine or cure. This viral infection, once acquired, is life-long, residing latently in hematopoietic cells. However, latently infected individuals with weakened immune systems often undergo HCMV reactivation, which can cause serious complications in immunosuppressed and immunocompromised patients. Current anti-viral therapies target late stages of viral replication, and are often met with therapeutic resistance, necessitating the development of novel therapeutics. In this current study, we identified a naturally-occurring flavonoid compound, deguelin, which inhibits HCMV lytic replication. Our findings reveal that nanomolar concentrations of deguelin significantly suppress the production of the infectious virus. Further, we show that deguelin inhibits the lytic cycle during the phase of the replication cycle consistent with early (E) gene and protein expression. Importantly, our data reveal that deguelin inhibits replication of a ganciclovir-resistant strain of HCMV. Together, our findings identify a novel, naturally occurring compound that may prove useful in the treatment of HCMV replication

Host miRNA regulation of human cytomegalovirus immediate early protein translation promotes viral latency

Reactivation of human cytomegalovirus (HCMV) is a significant cause of disease and death in immunocompromised patients underscoring the need to understand how latency is controlled. Here we demonstrate that HCMV has evolved to utilize cellular miRNAs in cells that promote latency to regulate expression of a viral protein critical for viral reactivation. Our data reveal that hsa-miR-200 miRNA family members target the UL122 (IE2) 3′UTR, resulting in repression of this viral protein. Utilizing recombinant viruses that mutate the miRNA-binding site results in lytic rather than latent infections compared to wild type virus in ex vivo infections of primary CD34+ cells. Cells permissive for lytic replication demonstrate low levels of these miRNAs. We propose that cellular miRNA regulation of HCMV is critical for maintenance of viral latency. Importance Human cytomegalovirus (HCMV) is a herpesvirus that infects a majority of the population. Once acquired, individuals will harbor the virus for life, where the virus remains, for the most part, in a quiet or latent state. Under weakened immune conditions, the virus can reactivate, which can cause severe disease and often death. We have found that a family of small RNAs, termed microRNAs, encoded by human myeloid progenitor cells are capable of repressing a key viral protein, thus enabling the virus to ensure a quiet/latent state. As these progenitor cells mature further down the myeloid lineage towards cells that support active viral replication, the levels of these microRNAs decrease. Together, our data suggest that host cell microRNA regulation of HCMV is important for the quiet/latent state of this pathogen