Cardiac glycosides inhibit early and late vaccinia virus protein expression.

Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses.

Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses.

We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors.

Inhibition of human cytomegalovirus replication by interferon alpha can involve multiple anti-viral factors.

The shortcomings of current direct-acting anti-viral therapy against human cytomegalovirus (HCMV) has led to interest in host-directed therapy. Here we re-examine the use of interferon proteins to inhibit HCMV replication utilizing both high and low passage strains of HCMV. Pre-treatment of cells with interferon alpha (IFNα) was required for robust and prolonged inhibition of both low and high passage HCMV strains, with no obvious toxicity, and was associated with an increased anti-viral state in HCMV-infected cells.

Learning from the experiences of pregnant women participating in a research study investigating human cytomegalovirus shedding: A qualitative study.

Transmission of human cytomegalovirus (CMV), from a pregnant woman to her fetus can cause congenital CMV infection, with life-long problems in some infected children. The presence of CMV in an infected individual's bodily fluid is known as shedding. An individual can become infected with CMV through contact with another individual who is shedding CMV in their bodily fluid, and the avoidance of contact with infected fluids may reduce the risk of infection.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV).

Toward inhibition of human cytomegalovirus replication with compounds targeting cellular proteins.

Antiviral therapy for human cytomegalovirus (HCMV) currently relies upon direct-acting antiviral drugs. However, it is now well known that these drugs have shortcomings, which limit their use. Here I review the identification and investigation of compounds targeting cellular proteins that have anti-HCMV activity and could supersede those anti-HCMV drugs currently in use.

A systematic review and meta-analysis of the prevalence of human cytomegalovirus shedding in seropositive pregnant women.

The detection of human cytomegalovirus (HCMV) in an individual's bodily fluid by culture techniques or through HCMV DNA detection by polymerase chain reaction, is known as HCMV shedding. Human cytomegalovirus shedding has the potential to transmit HCMV infection, where an individual can become infected with HCMV through contact with the bodily fluid of another individual containing HCMV. Human cytomegalovirus shedding can occur in primary infection and in non-primary infection for individuals with prior infection (HCMV seropositive).

Identification and characterization of bisbenzimide compounds that inhibit human cytomegalovirus replication.

The shortcomings of current anti-human cytomegalovirus (HCMV) drugs has stimulated a search for anti-HCMV compounds with novel targets. We screened collections of bioactive compounds and identified a range of compounds with the potential to inhibit HCMV replication. Of these compounds, we selected bisbenzimide compound RO-90-7501 for further study.

Limited replication of human cytomegalovirus in a trophoblast cell line.

Several viruses, including human cytomegalovirus (HCMV), are thought to replicate in the placenta. However, there is little understanding of the molecular mechanisms involved in HCMV replication in this tissue. We investigated replication of HCMV in the extravillous trophoblast cell line SGHPL-4, a commonly used model of HCMV replication in the placenta.

Metagenomic profiling of placental tissue suggests DNA virus infection of the placenta is rare.

It is widely recognized that pathogens can be transmitted across the placenta from mother to foetus. Recent re-evaluation of metagenomic studies indicates that the placenta has no unique microbiome of commensal bacteria. However, viral transmission across the placenta, including transmission of DNA viruses such as the human herpesviruses, is possible.

Identification of lead anti-human cytomegalovirus compounds targeting MAP4K4 via machine learning analysis of kinase inhibitor screening data.

Chemogenomic approaches involving highly annotated compound sets and cell based high throughput screening are emerging as a means to identify novel drug targets. We have previously screened a collection of highly characterized kinase inhibitors (Khan et al., Journal of General Virology, 2016) to identify compounds that increase or decrease expression of a human cytomegalovirus (HCMV) protein in infected cells.

RO0504985 is an inhibitor of CMGC kinase proteins and has anti-human cytomegalovirus activity.

Public-private partnerships allow many previously unavailable compounds to be screened for antiviral activity. Here a screening method was used to identify an oxindole compound, RO0504985, from a Roche kinase inhibitor library that inhibited human cytomegalovirus (HCMV) protein production. RO0504985 was previously described as an inhibitor of cyclin-dependent kinase 2 (CDK2).

High-throughput screening of a GlaxoSmithKline protein kinase inhibitor set identifies an inhibitor of human cytomegalovirus replication that prevents CREB and histone H3 post-translational modification.

To identify new compounds with anti-human cytomegalovirus (HCMV) activity and new anti-HCMV targets, we developed a high-throughput strategy to screen a GlaxoSmithKline Published Kinase Inhibitor Set. This collection contains a range of extensively characterized compounds grouped into chemical families (chemotypes). From our screen, we identified compounds within chemotypes that impede HCMV protein production and identified kinase proteins associated with inhibition of HCMV protein production that are potential novel anti-HCMV targets.

Identification of compounds with anti-human cytomegalovirus activity that inhibit production of IE2 proteins.

Using a high throughput screening methodology we surveyed a collection of largely uncharacterized validated or suspected kinase inhibitors for anti-human cytomegalovirus (HCMV) activity. From this screen we identified three structurally related 5-aminopyrazine compounds (XMD7-1, -2 and -27) that inhibited HCMV replication in virus yield reduction assays at low micromolar concentrations. Kinase selectivity assays indicated that each compound was a kinase inhibitor capable of inhibiting a range of cellular protein kinases.

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions.

Unlabelled: High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls.

Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells.

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα.

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells.

In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipulation of subnuclear structures. This review outlines the involvement of several viral and cellular subnuclear structures in areas of HCMV replication and virus-host interaction that include viral transcription, viral DNA synthesis and the production of DNA-filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and promyelocytic leukaemia nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles).

Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli.

Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44.

A mutation deleting sequences encoding the amino terminus of human cytomegalovirus UL84 impairs interaction with UL44 and capsid localization.

Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro.

Host cell nucleolin is required to maintain the architecture of human cytomegalovirus replication compartments.

Unlabelled: Drastic reorganization of the nucleus is a hallmark of herpesvirus replication. This reorganization includes the formation of viral replication compartments, the subnuclear structures in which the viral DNA genome is replicated. The architecture of replication compartments is poorly understood.

Human cytomegalovirus UL44 concentrates at the periphery of replication compartments, the site of viral DNA synthesis.

The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells.

Sites and roles of phosphorylation of the human cytomegalovirus DNA polymerase subunit UL44.

The human cytomegalovirus DNA polymerase subunit UL44 is a phosphoprotein, but its sites and roles of phosphorylation have not been investigated. We compared sites of phosphorylation of UL44 in vitro by the viral protein kinase UL97 and cyclin-dependent kinase 1 with those in infected cells. Transient treatment of infected cells with a UL97 inhibitor greatly reduced labeling of two minor UL44 phosphopeptides.

The carboxy-terminal segment of the human cytomegalovirus DNA polymerase accessory subunit UL44 is crucial for viral replication.

The amino-terminal 290 residues of UL44, the presumed processivity factor of human cytomegalovirus DNA polymerase, possess all of the established biochemical activities of the full-length protein, while the carboxy-terminal 143 residues contain a nuclear localization signal (NLS). We found that although the amino-terminal domain was sufficient for origin-dependent synthesis in a transient-transfection assay, the carboxy-terminal segment was crucial for virus replication and for the formation of DNA replication compartments in infected cells, even when this segment was replaced with a simian virus 40 NLS that ensured nuclear localization. Our results suggest a role for this segment in viral DNA synthesis.

Association of human cytomegalovirus proteins IRS1 and TRS1 with the viral DNA polymerase accessory subunit UL44.

Multiple proteins interacting with DNA polymerases orchestrate DNA replication. Human cytomegalovirus (HCMV) encodes a DNA polymerase that includes the presumptive processivity factor UL44. UL44 is structurally homologous to the eukaryotic DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous proteins.