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 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.

Herpesviruses produce a plethora of pleomorphic and heterogeneous particle populations. The composition and biological role of these is not understood. Detailed analysis has been challenging due to the lack of multidimensional identification and purification methodologies. Fluorescence-activated cell sorting (FACS), originally developed to sort objects with at least ten thousand-fold larger volumes, has recently been applied to cellular exosomes as well as viral particles and has been dubbed nanoscale flow cytometry or “flow virometry”. In comparison to other nanoparticles, herpesvirus concentrations can be measured with high precision using simple culturing methods. Here, we used this unique capability to evaluate a standard FACS sorter. We demonstrate that detection and separation capabilities were insufficient to distinguish infectious fluorescent viral populations from populations lacking fluorescence and infectivity. Moreover, fluorescent populations did not contain single virus particles but mostly aggregates. On top, analysis of viral samples by flow cytometry was confounded by swarm detection, as multiple objects are measured simultaneously and interpreted as a single object. Despite these technical difficulties, comparison of crude supernatant to gradient purified HCMV revealed that infectious virus is a minor proportion of the particles released from infected cells. Our data stress the need for a set of standardized controls and protocols when applying FACS to biological nanoparticles and highlights technical challenges that need to be solved before flow virometry can achieve its full potential.

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets of therapeutic intervention. Among the latter, viral protein-protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle being required for oriLyt-dependent DNA replication, we performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB40-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited AD169 in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells with B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

The past few years have brought substantial progress toward understanding how human cytomegalovirus (HCMV) enters the remarkably wide spectrum of cell types and tissues that the virus infects. Neuropilin-2 and platelet-derived growth factor receptor alpha (PDGFRa) were identified as receptors, respectively, for the trimeric and pentameric glycoprotein H/glycoprotein L (gH/gL) complexes that in large part govern HCMV cell tropism, while CD90 and CD147 were also found to play roles during entry. X-ray crystal structures for the proximal viral fusogen, glycoprotein B (gB), and for the pentameric gH/gL complex (pentamer) have been solved. A novel virion gH complex consisting of gH bound to UL116 instead of gL was described, and findings supporting the existence of a stable complex between gH/gL and gB were reported. Additional work indicates that the pentamer promotes a mode of cell-associated spread that resists antibody neutralization, as opposed to the trimeric gH/gL complex (trimer), which appears to be broadly required for the infectivity of cell-free virions. Finally, viral factors such as UL148 and US16 were identified that can influence the incorporation of the alternative gH/gL complexes into virions. We will review these advances and their implications for understanding HCMV entry and cell tropism.