Impact of new cyclooxygenase 2 inhibitors on human cytomegalovirus replication in vitro

Background Human cytomegalovirus (HCMV) is involved in complications on immunocompromised patients. Current therapeutics are associated with several drawbacks, such as nephrotoxicity. Purpose: As HCMV infection affects inflammation pathways, especially prostaglandin E2 (PGE2) production via cyclooxygenase 2 enzyme (COX-2), we designed 2'-hydroxychalcone compounds to inhibit human cytomegalovirus. Study design We first selected the most efficient new synthetic chalcones for their effect against COX-2-catalyzed PGE2. Study sample Among the selected compounds, we assessed the antiviral efficacy against different HCMV strains, such as the laboratory strain AD169 and clinical strains (naïve or multi-resistant to conventional drugs) and toxicity on human cells. Results The most efficient and less toxic compound (chalcone 7) was tested against HCMV in combination with other antiviral molecules: artesunate (ART), baicalein (BAI), maribavir (MBV), ganciclovir (GCV), and quercetin (QUER) using Compusyn software. Association of chalcone 7 with MBV and BAI is synergistic, antagonistic with QUER, and additive with GCV and ART. Conclusion These results provide a promising search path for potential bitherapies against HCMV.

Selection of Human Cytomegalovirus Mutants with Resistance against PDGFRα-Derived Entry Inhibitors

The human cytomegalovirus (HCMV) infects fibroblasts via an interaction of its envelope glycoprotein gO with the cellular platelet-derived growth factor receptor alpha (PDGFRα), and soluble derivatives of this receptor can inhibit viral entry. We aimed to select mutants with resistance against PDGFRα-Fc and the PDGFRα-derived peptides GT40 and IK40 to gain insight into the underlying mechanisms and determine the genetic barrier to resistance. An error-prone variant of strain AD169 was propagated in the presence of inhibitors, cell cultures were monitored weekly for signs of increased viral growth, and selected viruses were tested regarding their sensitivity to the inhibitor. Resistant virus was analyzed by DNA sequencing, candidate mutations were transferred into AD169 clone pHB5 by seamless mutagenesis, and reconstituted virus was again tested for loss of sensitivity by dose-response analyses. An S48Y mutation in gO was identified that conferred a three-fold loss of sensitivity against PDGFRα-Fc, a combination of mutations in gO, gH, gB and gN reduced sensitivity to GT40 by factor 4, and no loss of sensitivity occurred with IK40. The resistance-conferring mutations support the notion that PDGFRα-Fc and GT40 perturb the interaction of gO with its receptor, but the relatively weak effect indicates a high genetic barrier to resistance.

Human cytomegalovirus protein pUL36: A dual cell death pathway inhibitor

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.

Complete Genome Sequence of a Human Cytomegalovirus Strain AD169 Bacterial Artificial Chromosome Clone

The complete sequence of the human cytomegalovirus strain AD169 (variant ATCC) cloned as a bacterial artificial chromosome (AD169-BAC, also known as HB15 or pHB15) was determined. The viral genome has a length of 230,290 bp and shows 52 nucleotide differences compared to a previously sequenced AD169varATCC clone.

Recombinant Phenotyping of Cytomegalovirus UL97 Kinase Sequence Variants for Ganciclovir Resistance

ABSTRACT A strain of human cytomegalovirus, T2211, modified from standard laboratory strain AD169 to contain a secreted alkaline phosphatase reporter gene for rapid viral quantitation, was cloned as a bacterial artificial chromosome, BA1, and then mutagenized to create recombinant viruses containing viral UL97 kinase sequence variants found in clinical specimens after ganciclovir treatment, but with no phenotypic data to determine their role in drug resistance. Seven control strains and 14 other recombinant strains were phenotyped for ganciclovir resistance and compared with similar strains created using prior technology to show a good concordance of findings. Sequence changes V466M, H469Y, A478V, N510S, A588V, K599R, L600I, G623S, T659I, and V665I were found to confer no significant ganciclovir resistance, while mutations L405P, M460T, A594E, and C603R conferred 3- to 9-fold increases in ganciclovir 50% inhibitory concentrations. Different mutations at codons 594 (A594V, A594E) and 603 (C603W, C603S) conferred varied amounts of ganciclovir resistance. Advances in recombinant phenotyping make it easier to show that many uncharacterized UL97 sequence variants do not confer ganciclovir resistance, but some are newly confirmed as resistance associated, including one (L405P) which is outside the codon range where such mutations are usually found. This information should improve the interpretation of genotypic data generated by diagnostic laboratories.

Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

ABSTRACT Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.

Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993–2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

Cytomegalovirus Destruction of Focal Adhesions Revealed in a High-Throughput Western Blot Analysis of Cellular Protein Expression

ABSTRACT Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and posttranslation regulation of cellular gene expression. High-throughput Western blotting (BD Biosciences Powerblot technology) with 1,009 characterized antibodies was therefore used to analyze and compare the effects of infection with attenuated high-passage strain AD169 and virulent low-passage strain Toledo at 72 hpi across gels run in triplicate for each sample. Six hundred ninety-four proteins gave a positive signal in the screen, of which 68 from strain AD169 and 71 from strain Toledo were defined as being either positively or negatively regulated by infection with the highest level of confidence (BD parameters). In follow-up analyses, a subset of proteins was selected on the basis of the magnitude of the observed effect or their potential to contribute to defense against immune recognition. In analyses performed at 24, 72, and 144 hpi, connexin 43 was efficiently downregulated during HCMV infection, implying a breakdown of intercellular communication. Mitosis-associated protein Eg-5 was found to be differentially upregulated in the AD169 and Toledo strains of HCMV. Focal adhesions link the actin cytoskeleton to the extracellular matrix and have key roles in initiating signaling pathways and substrate adhesion and regulating cell migration. HCMV suppressed expression of the focal-adhesion-associated proteins Hic-5, paxillin, and α-actinin. Focal adhesions were clearly disrupted in HCMV-infected fibroblasts, with their associated intracellular and extracellular proteins being dispersed. Powerblot shows potential for rapid screening of the cellular proteome during HCMV infection.

Deletion of gpUL132, a Structural Component of Human Cytomegalovirus, Results in Impaired Virus Replication in Fibroblasts

ABSTRACT The coding capacity of human cytomegalovirus (HCMV) for glycoproteins by far exceeds that of other herpesviruses. Few of these proteins have been characterized so far. We have investigated the gene product of reading frame UL132. The putative protein product of UL132 is a glycoprotein with a theoretical mass of 29.8 kDa. Transcription analysis revealed that the gene is transcribed with a true late kinetics from the laboratory-adapted strain AD169 and the low-passage isolate TB40E. Two proteins of 22 to 28 kDa and 45 to 60 kDa were detected in virus-infected cells as well as in extracellular virions. The larger protein carried N-linked carbohydrates. Both protein forms were present in laboratory-adapted strains as well as in low-passage isolates of HCMV. Recombinant viruses with the UL132 gene deleted were constructed in the low-passage HCMV isolate PAN as well as the high-passage isolate AD169. Deletion of UL132 from either genome resulted in a pronounced replication deficit with a reduction of approximately 100-fold for HCMV strain AD169. Thus, the protein product of the UL132 reading frame represents a structural viral glycoprotein of HCMV that has an important function for viral replication in tissue culture.