Human Cytomegalovirus Strains Research Articles

A Review of the Potential Role of Human Cytomegalovirus (HCMV) Infections in Breast Cancer Carcinogenesis and Abnormal Immunity.

Previously recognized classical human onco-viruses can regulate complex neoplastic events, and are estimated to play a role during carcinogenesis in 15–20% of cancer cases. Although the DNA and gene products of several viruses have been found in breast tumors, none of the classical onco-viruses have definitely been linked to the initiation of breast cancer. However, recent evidence shows that human cytomegalovirus (HCMV) gene products are found in >90% of tumors and metastases of breast cancers, and their increased expression can be correlated to a more aggressive breast cancer phenotype. Supporting the active role of HCMV in breast cancer, a specific HCMV strain, HCMV-DB, was recently shown to exert oncogenic transformational activity in breast epithelial cells in vitro, and to give rise to fast-growing, triple-negative breast tumors when injected into immune deficient mice. The same observation holds true for clinical studies implying increased HCMV protein expression in triple negative breast cancer biopsies. In addition to functionally being able to hijack tumor-promoting cellular events, HCMV is known to exhibit a wide range of immunosuppressive effects, which can have radical impact on the tumor microenvironment. HCMV infected cells can avoid recognition and elimination by the immune system by orchestrating polarization of immunosuppressive type II macrophages, preventing antigen presentation, by expressing T cell inhibitory molecules, and possibly, by the induction of regulatory T (Treg) cell responses. These actions would be especially deleterious for the antigenic activation and proliferation of tumor specific CD8+ cytotoxic T lymphocytes (CTLs), whose effector functions have recently been targeted by successful, experimental immunotherapy protocols. The recognition of alternative causes and drivers of breast cancer is a pivotal research topic for the development of diagnostics and novel, effective preventive and therapeutic strategies targeting both tumor cells and their microenvironments.

Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response

The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles.IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.

A42 Next-generation sequencing to analyze multiple-strain infections, genotype distribution, and antiviral resistance in hematopoietic stem cell transplantation recipients with human cytomegalovirus infection

Next-generation sequencing (NGS) produces comprehensive insights across the entire genome of the human cytomegalovirus (HCMV), which is an important opportunistic pathogen following hematopoietic stem cell transplantation (HSCT). To assess the clinical impact of HCMV diversity, genotype distribution, and resistance mutations, we performed NGS directly on plasma specimens from HSCT recipients with HCMV reactivation. Twenty-nine HCMV-positive plasma samples (median viral load 1.7 × 103 IU/ml) collected from a prospective allogenic HSCT recipient cohort (n = 16) between 21 and 80 days after transplantation were sequenced on an Illumina MiSeq after preparation of target-enriched sequencing libraries. Consensus HCMV genome sequences were assembled for 24 samples. The presence of multiple-strain infections and antiviral resistance mutations in genes UL54 and UL97 was determined by variant analysis. Genotype distribution was determined by specific marker analysis of several hypervariable genes (RL5A, RL6, RL12, RL13, UL1, UL9, UL11, UL73, UL74, UL120, UL146, and UL139). Associations between genomic and clinical features (e.g. graft-versus-host disease (GvHD), donor/recipient HCMV serostatus, dynamics of HCMV antigenemia, survival) were explored. Multiple infections involving up to 3 HCMV strains were detected in seven out of sixteen patients, with one patient analyzed at > 2 time points, showing a switch of the dominant HCMV population. No known antiviral resistance mutations were detected, which may be expected due to sample collection early after HSCT from patients without antiviral prophylaxis. Multiple-strain infection was associated with an earlier peak of HCMV-antigenemia (P = 0.054), but not with duration of viremia, antigenemia peak values, donor/recipient HCMV serostatus, T-cell depletion, acute or chronic GvHD, disease relapse, or reduced survival. Genotype distribution analysis revealed a potential link of one genotype of the immunomodulatory gene UL11 with GvHD incidence after HCMV reactivation. NGS of HCMV diversity directly from plasma samples, even with low viral loads, enables the acquisition of data of potential clinical interest. To identify reliable associations between clinical features and HCMV diversity, further patient cohorts with suitable sample sizes are required.

An overview of letermovir: a cytomegalovirus prophylactic option

ABSTRACTIntroduction: Human cytomegalovirus (HCMV) or human herpesvirus 5 (HHV-5) is a β-herpesvirus that causes widespread infection in nearly all members of the human population worldwide. Its persistence in humans after primary infection in a latent phase as well as a partial non–protective immune response is the basis for repeated re-activation/re-infection episodes occurring both in immunocompetent and immunocompromised subjects. In the latter patient populations, which include hematopoietic stem cell transplant (HSCT) recipients, HCMV reactivation episodes may be particularly severe, leading to both systemic and end-organ diseases. Since the 90s, at least four antiviral drugs targeting the DNA polymerase complex have been developed for the prevention and treatment of HCMV infections in transplant recipients, used as first-line (ganciclovir and valganciclovir) and second-line therapy (foscarnet and cidofovir). However, due to their toxicity and drug-resistance induction, new drugs with different targets were needed.Areas covered: In 2017, a new drug named letermovir (LTV), which targets the HCMV DNA terminase complex, was licensed for prophylaxis of HCMV infections in HSCT recipients. This is the focus of this review.Expert opinion: LTV safety and efficacy are promising. However, long-term adverse events and the emergence of drug-resistant HCMV strains must be investigated in extended clinical trials prior to drawing final conclusions.

Survey of cellular immune responses to human cytomegalovirus infection in the microenvironment of the uterine-placental interface.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, yet there are no established treatments for preventing maternal-fetal transmission. During first trimester, HCMV replicates in basal decidua that functions as a reservoir for virus and source of transmission to the attached placenta and fetal hemiallograft but also contains immune cells, including natural killer cells, macrophages, and T cell subsets, that respond to pathogens, protecting the placenta and fetus. However, the specific cellular and cytokine responses to infection are unknown, nor are the immune correlates of protection that guide development of therapeutic strategies. Here we survey immune cell phenotypes in intact explants of basal decidua infected with a clinical pathogenic HCMV strain ex vivo and identify specific changes occurring in response to infection in the tissue environment. Using 4-color immunofluorescence microscopy, we found that at 3days postinfection, virus replicates in decidual stromal cells and epithelial cells of endometrial glands. Infected cells and effector memory CD8+ T cells (TEM) in contact with them make IFN-γ. CD8+ TEM cells produce granulysin and cluster at sites of infection in decidua and the epithelium of endometrial glands. Quantification indicated expansion of two immune cell subtypes-CD8+ TEM cells and, to a lesser extent, iNKT cells. Approximately 20% of immune cells were found in pairs in both control and infected decidua, suggesting frequent cross-talk in the microenvironment of decidua. Our findings indicate a complex immune microenvironment in basal decidua and suggest CD8+ TEM cells play a role in early responses to decidual infection in seropositive women.

Multiple-Strain Infections of Human Cytomegalovirus With High Genomic Diversity Are Common in Breast Milk From Human Immunodeficiency Virus-Infected Women in Zambia.

BackgroundIn developed countries, human cytomegalovirus (HCMV) is a major pathogen in congenitally infected and immunocompromised individuals, where multiple-strain infection appears linked to disease severity. The situation is less documented in developing countries. In Zambia, breast milk is a key route for transmitting HCMV and carries higher viral loads in human immunodeficiency virus (HIV)–infected women. We investigated HCMV strain diversity.MethodsHigh-throughput sequence datasets were generated from 28 HCMV-positive breast milk samples donated by 22 mothers (15 HIV-infected and 7 HIV-negative) at 4–16 weeks postpartum, then analyzed by genome assembly and novel motif-based genotyping in 12 hypervariable HCMV genes.ResultsAmong the 20 samples from 14 donors (13 HIV-infected and one HIV-negative) who yielded data meeting quality thresholds, 89 of the possible 109 genotypes were detected, and multiple-strain infections involving up to 5 strains per person were apparent in 9 HIV-infected women. Strain diversity was extensive among individuals but conserved compartmentally and longitudinally within them. Genotypic linkage was maintained within hypervariable UL73/UL74 and RL12/RL13/UL1 loci for virus entry and immunomodulation, but not between genes more distant from each other.ConclusionsBreast milk from HIV-infected women contains multiple HCMV strains of high genotypic complexity and thus constitutes a major source for transmitting viral diversity.

Human Cytomegalovirus Genomes Sequenced Directly From Clinical Material: Variation, Multiple-Strain Infection, Recombination, and Gene Loss.

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.

Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination

Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) PLoS Pathog 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.

Copy-Paste Mutagenesis: A Method for Large-Scale Alteration of Viral Genomes.

The cloning of the large DNA genomes of herpesviruses, poxviruses, and baculoviruses as bacterial artificial chromosomes (BAC) in Escherichia coli has opened a new era in viral genetics. Several methods of lambda Red-mediated genome engineering (recombineering) in E. coli have been described, which are now commonly used to generate recombinant viral genomes. These methods are very efficient at introducing deletions, small insertions, and point mutations. Here we present Copy-Paste mutagenesis, an efficient and versatile strategy for scarless large-scale alteration of viral genomes. It combines gap repair and en passant mutagenesis procedures and relies on positive selection in all crucial steps. We demonstrate that this method can be used to generate chimeric strains of human cytomegalovirus (HCMV), the largest human DNA virus. Large (~15 kbp) genome fragments of HCMV strain TB40/E were tagged with an excisable marker and cloned (copied) in a low-copy plasmid vector by gap repair recombination. The cloned fragment was then excised and inserted (pasted) into the HCMV AD169 genome with subsequent scarless removal of the marker by en passant mutagenesis. We have done four consecutive rounds of this procedure, thereby generating an AD169-TB40/E chimera containing 60 kbp of the donor strain TB40/E. This procedure is highly useful for identifying gene variants responsible for phenotypic differences between viral strains. It can also be used for repair of incomplete viral genomes, and for modification of any BAC-cloned sequence. The method should also be applicable for large-scale alterations of bacterial genomes.

Exploration of (hetero)aryl Derived Thienylchalcones for Antiviral and Anticancer Activities.

Search for new antiviral and anticancer agents are essential because of the emergence of drug resistance in recent years. In continuation of our efforts in identifying the new small molecule antiviral and anticancer agents, we identified chalcones as potent antiviral and anticancer agents. With the aim of identifying the broad acting antiviral and anticancer agents, we discovered substituted aryl/heteroaryl derived thienyl chalcones as antiviral and anticancer agents. A focused set of thienyl chalcone derivaties II-VI was screened for selected viruses Hepatitis B virus (HBV), Herpes simplex virus 1 (HSV-1), Human cytomegalovirus (HCMV), Dengue virus 2 (DENV2), Influenza A (H1N1) virus, MERS coronavirus, Poliovirus 1 (PV 1), Rift Valley fever (RVF), Tacaribe virus (TCRV), Venezuelan equine encephalitis virus (VEE) and Zika virus (ZIKV) using the National Institute of Allergy and Infectious Diseases (NIAID)'s Division of Microbiology and Infectious Diseases (DMID) antiviral screening program. Additionally, a cyclopropylquinoline derivative IV has been screened for 60 human cancer cell lines using the Development Therapeutics Program (DTP) of NCI. All thienyl chalcone derivatives II-VI displayed moderate to excellent antiviral activity towards several viruses tested. Compounds V and VI were turned out be active compounds towards human cytomegalovirus for both normal strain (AD169) as well as resistant isolate (GDGr K17). Particularly, cyano derivative V showed very high potency (EC50: <0.05 µM) towards AD169 strain of HCMV compared to standard drug Ganciclovir (EC50: 0.12 µM). Additionally, it showed moderate activity in the secondary assay (AD169; EC50: 2.30 µM). The cyclopropylquinoline derivative IV displayed high potency towards Rift Valley fever virus (RVFV) and Tacaribe virus (TCRV) towards Rift Valley fever virus (RVFV). The cyclopropylquinoline derivative IV is nearly 28 times more potent in our initial in vitro visual assay (EC50: 0.39 µg/ml) and nearly 17 times more potent in neutral red assay (EC50: 0.71 μg/ml) compared to the standard drug Ribavirin (EC50: 11 µg/ml; visual assay and EC50: 12 µg/ml; neutral red assay). It is nearly 12 times more potent in our initial in vitro visual assay (EC50: >1 µg/ml) and nearly 8 times more potent in neutral red assay (EC50: >1.3 µg/ml) compared to the standard drug Ribavirin (EC50: 12 µg/ml; visual assay and EC50: 9.9 µg/ml; neutral red assay) towards Tacaribe virus (TCRV). Additionally, cyclopropylquinoline derivative IV has shown strong growth inhibitory activity towards three major cancers (colon, breast, and leukemia) cell lines and moderate growth inhibition shown towards other cancer cell lines screened. Compounds V and VI were demonstrated viral inhibition towards Human cytomegalovirus, whereas cyclopropylquinoline derivative IV towards Rift Valley fever virus and Tacaribe virus. Additionally, cyclopropylquinoline derivative IV has displayed very good cytotoxicity against colon, breast and leukemia cell lines in vitro.

The isoquinoline alkaloid berberine inhibits human cytomegalovirus replication by interfering with the viral Immediate Early-2 (IE2) protein transactivating activity.

The identification and validation of new small molecules able to inhibit the replication of human cytomegalovirus (HCMV) remains a priority to develop alternatives to the currently used DNA polymerase inhibitors, which are often burdened by long-term toxicity and emergence of cross-resistance. To contribute to this advancement, here we report on the characterization of the mechanism of action of a bioactive plant-derived alkaloid, berberine (BBR), selected in a previous drug repurposing screen expressly devised to identify early inhibitors of HCMV replication. Low micromolar concentrations of BBR were confirmed to suppress the replication of different HCMV strains, including clinical isolates and strains resistant to approved DNA polymerase inhibitors. Analysis of the HCMV replication cycle in infected cells treated with BBR then revealed that the bioactive compound compromised the progression of virus cycle at a stage prior to viral DNA replication and Early (E) genes expression, but after Immediate-Early (IE) proteins expression. Mechanistic studies in fact highlighted that BBR interferes with the transactivating functions of the viral IE2 protein, thus impairing efficient E gene expression and the progression of HCMV replication cycle. Finally, the mechanism of the antiviral activity of BBR appears to be conserved among different CMVs, since BBR suppressed murine CMV (MCMV) replication and inhibited the transactivation of the prototypic MCMV E1 gene by the IE3 protein, the murine homolog of IE2. Together, these observations warrant for further experimentation to obtain proof of concept that BBR could represent an attractive candidate for alternative anti-HCMV therapeutic strategies.

Expression of human miR-200b-3p and -200c-3p in cytomegalovirus-infected tissues.

Human cytomegalovirus (HCMV) infection can cause inflammatory tissue-invasive end-organ diseases upon lytic replication. In humans, mature miR-200b-3p and -200c-3p suppress the synthesis of HCMV immediate early 2 (IE2) protein by binding to the 3′-UTR of the mRNA encoded by the unique long (UL) 122-123 region in human foreskin fibroblasts and pre-transplant peripheral blood mononuclear cells stimulated with HCMV. The present study aimed to quantitate the expression of Homo sapiens (hsa)-miR-200b-3p and 200c-3p in HCMV-infected tissues. We collected 240 HCMV-infected and 154 HCMV-non-infected, formalin-fixed, paraffin-embedded tissue samples of the gastrointestinal (GI) tract and bronchi/lungs. MiRNAs, HCMV, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantitated by quantitative reverse transcription-PCR (qRT-PCR) and quantitative PCR (qPCR) on the basis of standard curves generated using miRNA mimics, the HCMV strain from National Institute for Biological Standards and Control (NIBSC) 09/162, and GAPDH control. To avoid the effect of cell counts on the qRT-PCR and qPCR results, the data were normalized to GAPDH levels. HCMV-infected tissues had significantly lower levels of 200b-3p/GAPDH (3.03 ± 1.50 compared with 3.98 ± 1.08 log10 copies/μl, P<0.001) and 200c-3p/GAPDH (4.67 ± 1.84 compared with 6.35 ± 1.47 log10 copies/μl, P<0.001) than normal tissues. The values for 200b-3p/GAPDH (r = −0.51, P<0.001) and 200c-3p/GAPDH (r = −0.54, P<0.001) were significantly inversely correlated with HCMV load. Low tissue levels of 200b-3p and 200c-3p in humans are associated with cytopathic inflammation due to HCMV infection.

A gB/CD3 bispecific BiTE antibody construct for targeting Human Cytomegalovirus-infected cells

Bispecific T cell engager (BiTE) antibody constructs are successfully used as cancer therapeutics. We hypothesized that this treatment strategy could also be applicable for therapy of human cytomegalovirus (HCMV) infection, since HCMV-encoded proteins are abundantly expressed on the surface of infected cells. Here we show that a BiTE antibody construct directed against HCMV glycoprotein B (gB) and CD3 efficiently triggers T cells to secrete IFN-γ and TNF upon co-culture with fibroblasts infected with HCMV strain AD169, Towne or Toledo. Titration of gB expression levels in non-infected cells confirmed that already low levels of gB are sufficient for efficient triggering of T cells in presence of the BiTE antibody construct. Comparison of redirecting T cells with the bispecific antibody versus a chimeric antigen receptor (CAR) based on the same scFv showed a similar sensitivity for gB expression. Although lysis of infected target cells was absent, the BiTE antibody construct inhibited HCMV replication by triggering cytokine production. Notably, even strongly diluted supernatants of the activated T cells efficiently blocked the replication of HCMV in infected primary fibroblasts. In summary, our data prove the functionality of the first BiTE antibody construct targeting an HCMV-encoded glycoprotein for inhibiting HCMV replication in infected cells.

The Human Cytomegalovirus US27 Gene Product Constitutively Activates Antioxidant Response Element-Mediated Transcription through Gβγ, Phosphoinositide 3-Kinase, and Nuclear Respiratory Factor 1.

Human cytomegalovirus (HCMV) is a widespread pathogen that modulates host chemokine signaling during persistent infection in the host. HCMV encodes four proteins with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. Each of the four receptors modulates host CXCR4 signaling. US28, UL33, and UL78 impair CXCR4 signaling outcomes, while US27 enhances signaling, as evidenced by increased calcium mobilization and cell migration to CXCL12. To investigate the effects of US27 on CXCR4 during virus infection, fibroblasts were infected with bacterial artificial chromosome-derived clinical strain HCMV TB40/E-mCherry (wild type [WT]), mutants lacking US27 (TB40/E-mCherry-US27Δ [US27Δ]) or all four GPCRs (TB40 E-mCherry-allΔ), or mutants expressing only US27 but not US28, UL33, or UL78 (TB40/E-mCherry-US27wt [US27wt]). CXCR4 gene expression was significantly higher in WT- and US27wt-infected fibroblasts. This effect was evident at 3 h postinfection, suggesting that US27 derived from the parental virion enhanced CXCR4 expression. Reporter gene assays demonstrated that US27 increased transcriptional activity regulated by the antioxidant response element (ARE), and small interfering RNA treatment indicated that this effect was mediated by NRF-1, the primary transcription factor for CXCR4. Increased translocation of NRF-1 into the nucleus of WT-infected cells compared to mock- or US27Δ-infected cells was confirmed by immunofluorescence microscopy. Chemical inhibitors targeting Gβγ and phosphoinositide 3-kinase (PI3K) ablated the increase in ARE-driven transcription, implicating these proteins as mediators of US27-stimulated gene transcription. This work identifies the first signaling pathway activated by HCMV US27 and may reveal a novel regulatory function for this orphan viral receptor in stimulating stress response genes during infection.IMPORTANCE Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, causing deafness, blindness, and other serious birth defects. CXCR4 is a human chemokine receptor that is crucial for both fetal development and immune responses. We found that the HCMV protein US27 stimulates increased expression of CXCR4 through activation of the transcription factor nuclear respiratory factor 1 (NRF-1). NRF-1 regulates stress response genes that contain the antioxidant response element (ARE), and HCMV infection is associated with increased expression of many stress response genes when US27 is present. Our results show that the US27 protein activates the NRF-1/ARE pathway, stimulating higher expression of CXCR4 and other stress response genes, which is likely to be beneficial for virus replication and/or immune evasion.

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.

Large-Scale Screening of HCMV-Seropositive Blood Donors Indicates that HCMV Effectively Escapes from Antibodies by Cell-Associated Spread.

Immunoglobulins are only moderately effective for the treatment of human cytomegalovirus (HCMV) infections, possibly due to ineffectiveness against cell-associated virus spread. To overcome this limitation, we aimed to identify individuals with exceptional antibodies in their plasma that can efficiently block the cell-associated spread of HCMV. A Gaussia luciferase-secreting mutant of the cell-associated HCMV strain Merlin was generated, and luciferase activity evaluated as a readout for the extent of cell-associated focal spread. This reporter virus-based assay was then applied to screen plasma samples from 8400 HCMV-seropositive individuals for their inhibitory effect, including direct-acting antiviral drugs as positive controls. None of the plasmas reduced virus spread to the level of these controls. Even the top-scoring samples that partially reduced luciferase activity in the screening assay failed to inhibit focal growth when reevaluated with a more accurate, immunofluorescence-based assay. Selected sera with high neutralizing capacity against free viruses were analyzed separately, and none of them prevented the focal spread of three recent clinical HCMV isolates nor reduced the number of particles transmitted, as demonstrated with a fluorescent Merlin mutant. We concluded that donors with cell-to-cell-spread-inhibiting plasma are nonexistent or extremely rare, emphasizing cell-associated spread as a highly efficient immune escape mechanism of HCMV.

Multiantigenic Modified Vaccinia Virus Ankara Vaccine Vectors To Elicit Potent Humoral and Cellular Immune Reponses against Human Cytomegalovirus in Mice.

As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in protection against HCMV infection and disease. Here we describe a vaccine approach based on the well-characterized modified vaccinia virus Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show that in mice, multiantigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the individual antigens. In addition, we demonstrate that the PC/gB antigen combination of these multiantigenic MVA vectors can enhance the stimulation of humoral immune responses that mediate in vitro neutralization of different HCMV strains and antibody-dependent cellular cytotoxicity. These results support the use of MVA to develop a multiantigenic vaccine candidate for controlling HCMV infection and disease in different target populations, such as pregnant women and transplant recipients.IMPORTANCE The development of a human cytomegalovirus (HCMV) vaccine to prevent congenital disease and transplantation-related complications is an unmet medical need. While many HCMV vaccine candidates have been developed, partial success in preventing or controlling HCMV infection in women of childbearing age and transplant recipients has been observed with an approach based on envelope glycoprotein B (gB). We introduce a novel vaccine strategy based on the clinically deployable modified vaccinia virus Ankara (MVA) vaccine vector to elicit potent humoral and cellular immune responses by multiple immunodominant HCMV antigens, including gB, phosphoprotein 65, and all five subunits of the pentamer complex. These findings could contribute to development of a multiantigenic vaccine strategy that may afford more protection against HCMV infection and disease than a vaccine approach employing solely gB.

Infection of endotheliotropic human cytomegalovirus of trabecular meshwork cells.

Human cytomegalovirus (HCMV) infections can cause endotheliitis which is associated with an elevation in the intraocular pressure (IOP). However, the mechanism of the IOP elevation has not been established. The purpose of this study was to determine whether HCMV strains which are capable of infecting corneal endothelial cells can also replicate and induce anti-viral responses, and can reorganize the actin cytoskeleton in trabecular meshwork cells. Experimental study design. Cultured primary human trabecular meshwork cells (HTMCs) were infected with the Towne or TB40/E strains of HCMV. TB40/E is trophic for vascular endothelial and corneal endothelial cells. Real-time PCR, western blot, and fluorescent immunostaining have been used to determine whether HCMV-infected HTMCs will support the expression of viral mRNA and protein, allow viral replication, and elicit anti-viral host responses. We also determined whether lytic replication was present after HCMV infection. HCMV infection led to the expression of viral mRNA and proteins of IE1, glycoprotein B(gB), and pp65. TB40/E infection induced interferon-β, a sign of host anti-viral immune response and monocyte chemotactic protein-1 (MCP-1) as IOP-related chemokine. Together with the induction of the regulators of actin cytoskeleton, myosin phosphatase Rho interacting protein (MPRIP) and MCP-1, TB40/E induced a high level of expression of viral proteins, including IE1, gB, and pp65 as well as actin stress fiber formation, and achieved pathogenically high viral titers. Human trabecular meshwork cells support the replication of endotheliotropic TB40/E strain of HCMV which indicates that this strain may have high virulence for trabecular meshwork.

The transcriptome of human mammary epithelial cells infected with the HCMV-DB strain displays oncogenic traits

Increasing evidence indicates that human cytomegalovirus (HCMV) populations under the influence of host environment, can either be stable or rapidly differentiating, leading to tissue compartment colonization. We isolated previously from a 30-years old pregnant woman, a clinical isolate of HCMV, that we refered to as the HCMV-DB strain (accession number KT959235). The HCMV-DB clinical isolate demonstrated its ability to infect primary macrophages and to upregulate the proto-oncogene Bcl-3. We observed in this study that the genome of HCMV-DB strain is close to the genomes of other primary clinical isolates including the Toledo and the JP strains with the later having been isolated from a glandular tissue, the prostate. Using a phylogenetic analysis to compare the genes involved in virus entry, we observed that the HCMV-DB strain is close to the HCMV strain Merlin, the prototype HCMV strain. HCMV-DB infects human mammary epithelial cells (HMECs) which in turn display a ER−/PR−/HER2− phenotype, commonly refered to as triple negative. The transcriptome of HCMV-DB-infected HMECs presents the characteristics of a pro-oncogenic cellular environment with upregulated expression of numerous oncogenes, enhanced activation of pro-survival genes, and upregulated markers of cell proliferation, stemcellness and epithelial mesenchymal transition (EMT) that was confirmed by enhanced cellular proliferation and tumorsphere formation in vitro. Taken together our data indicate that some clinical isolates could be well adapted to the mammary tissue environment, as it is the case for the HCMV-DB strain. This could influence the viral fitness, ultimately leading to breast cancer development.

The Human Cytomegalovirus Protein UL148A Downregulates the NK Cell-Activating Ligand MICA To Avoid NK Cell Attack.

Natural killer (NK) cells are lymphocytes of the innate immune system capable of killing hazardous cells, including virally infected cells. NK cell-mediated killing is triggered by activating receptors. Prominent among these is the activating receptor NKG2D, which binds several stress-induced ligands, among them major histocompatibility complex (MHC) class I-related chain A (MICA). Most of the human population is persistently infected with human cytomegalovirus (HCMV), a virus which employs multiple immune evasion mechanisms, many of which target NK cell responses. HCMV infection is mostly asymptomatic, but in congenitally infected neonates and in immunosuppressed patients it can lead to serious complications and mortality. Here we discovered that an HCMV protein named UL148A whose role was hitherto unknown is required for evasion of NK cells. We demonstrate that UL148A-deficient HCMV strains are impaired in their ability to downregulate MICA expression. We further show that when expressed by itself, UL148A is not sufficient for MICA targeting, but rather acts in concert with an unknown viral factor. Using inhibitors of different cellular degradation pathways, we show that UL148A targets MICA for lysosomal degradation. Finally, we show that UL148A-mediated MICA downregulation hampers NK cell-mediated killing of HCMV-infected cells. Discovering the full repertoire of HCMV immune evasion mechanisms will lead to a better understanding of the ability of HCMV to persist in the host and may also promote the development of new vaccines and drugs against HCMV.IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen which is usually asymptomatic but that can cause serious complications and mortality in congenital infections and in immunosuppressed patients. One of the difficulties in developing novel vaccines and treatments for HCMV is its remarkable ability to evade our immune system. In particular, HCMV directs significant efforts to thwarting cells of the innate immune system known as natural killer (NK) cells. These cells are crucial for successful control of HCMV infection, and yet our understanding of the mechanisms which HCMV utilizes to elude NK cells is partial at best. In the present study, we discovered that a protein encoded by HCMV which had no known function is important for preventing NK cells from killing HCMV-infected cells. This knowledge can be used in the future for designing more-efficient HCMV vaccines and for formulating novel therapies targeting this virus.