-
Viruses Aug 2016The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was...
The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.
Topics: Blotting, Western; Cell Line; Computational Biology; Cyclin-Dependent Kinases; Cyclins; Cytomegalovirus; Host-Pathogen Interactions; Humans; Immunoprecipitation; Proteomics; Viral Proteins
PubMed: 27548200
DOI: 10.3390/v8080219 -
Journal of Virology Oct 2016Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by...
UNLABELLED
Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner.
IMPORTANCE
Transforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally mimics these viral oncoproteins through phosphorylation of Rb, fails to induce the production of nonlimiting amounts of dNTPs. This finding challenges the paradigm of the role of Rb inactivation during DNA virus infection and uncovers the existence of an alternative mechanism by which UL97 contributes to HCMV DNA synthesis. The ineffectiveness of the UL97 inhibitor maribavir in clinical trials might be better explained with a fuller understanding of the role of UL97 during infection. Furthermore, as the nucleoside analog ganciclovir is the current drug of choice for treating HCMV, knowing the provenance of the dNTPs incorporated into viral DNA may help inform antiviral therapeutic regimens.
Topics: Cells, Cultured; Cytomegalovirus; DNA, Viral; Deoxyribonucleotides; Humans; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Processing, Post-Translational; Retinoblastoma Protein; Viral Plaque Assay; Virus Replication
PubMed: 27440891
DOI: 10.1128/JVI.00731-16 -
Expert Review of Hematology Jun 2016Despite a remarkable reduction in the past decades, cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplant (HSCT) recipients remains a feared... (Review)
Review
Despite a remarkable reduction in the past decades, cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplant (HSCT) recipients remains a feared complication, still associated with significant morbidity and mortality. Today, first line treatment of CMV infection/reactivation is still based on dated antiviral compounds Ganciclovir (GCV), Foscarnet (FOS) and Cidofovir (CDF) with their burdensome weight of side effects. Maribavir (MBV), Letermovir (LMV) and Brincidofovir (BDF) are three new promising anti-CMV drugs without myelosuppressive properties or renal toxic effects that are under investigation in randomized phase II and III trials. Adoptive T-cell therapy (ATCT) in CMV infection possesses a strong rationale, demonstrated by several proof of concept studies; its feasibility is currently under investigation by clinical trials. ATCT from third-party and naïve donors could meet the needs of HSCT recipients of seronegative donors and cord blood grafts. In selected patients such as recipients of T-cell depleted grafts, ATCT, based on CMV-specific host T-cells reconstitution kinetics, would be of value in the prophylactic and/or preemptive CMV treatment. Vaccine-immunotherapy has the difficult task to reduce the incidence of CMV reactivation/infection in highly immunocompromised HSCT patients. Newer notions on CMV biology may represent the base to flush out the Troll of transplantation.
Topics: Animals; Antiviral Agents; Cytomegalovirus Infections; Cytomegalovirus Vaccines; Drug Resistance, Viral; Hematopoietic Stem Cell Transplantation; Humans; Immunotherapy, Adoptive; T-Lymphocytes; Transplantation, Homologous; Treatment Outcome
PubMed: 27043241
DOI: 10.1080/17474086.2016.1174571 -
Antiviral Research May 2016Human cytomegalovirus (HCMV) is the leading cause of congenital infections. Symptomatic newborns present with a range of sequelae including disorders of the CNS such as...
Human cytomegalovirus (HCMV) is the leading cause of congenital infections. Symptomatic newborns present with a range of sequelae including disorders of the CNS such as visual impairment, microcephaly, mental retardation and hearing loss. HCMV congenital infection causes gross changes in brain morphology and disturbances in glial and neuronal distribution, number and migration. In these studies, we have evaluated the effectiveness of the antiviral maribavir in inhibiting HCMV infections of ES cell-derived neuronal progenitor cells (NPC). We used EZ-spheres generated from H9 ES cells which are pre-rosette NPCs that retain long-term potential to differentiate into diverse central and peripheral neural lineages following directed differentiation. Our results demonstrate that the maribavir disrupts HCMV replication and viral yield in undifferentiated EZ-sphere-derived NPCs. In addition, we observed that maribavir limits HCMV replication and reduces the percentage of infected cells during differentiation of NPCs. Finally, early steps in differentiation are maintained during infection by treating with maribavir, likely an indirect effect resulting from decreased viral spread. Future studies of NPC proliferation and differentiation during infection treated with maribavir could provide the impetus for studying maribavir as an antiviral agent for congenital HCMV disease.
Topics: Antiviral Agents; Benzimidazoles; Cell Line; Embryonic Stem Cells; Humans; Neural Stem Cells; Neurogenesis; Neurons; Ribonucleosides; Virus Replication
PubMed: 26875788
DOI: 10.1016/j.antiviral.2016.02.007 -
Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection.Journal of Virology Oct 2015Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously...
UNLABELLED
Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21(Cip1). In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21(Cip1). Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21(Cip1) in wild-type virus but not a pUL27-deficient virus. We manipulated the p21(Cip1) levels to evaluate the functional consequence to MBV. Overexpression of p21(Cip1) restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21(Cip1) in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21(Cip1) and support the idea that CDKs can complement some activities of pUL97.
IMPORTANCE
HCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21(Cip1). In contrast, we provide evidence that p21(Cip1) functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21(Cip1) activators might be useful in combination with MBV to effectively inhibit HCMV infections.
Topics: Antiviral Agents; Astrocytes; Benzimidazoles; CDC2 Protein Kinase; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cytomegalovirus; Drug Resistance, Viral; Fibroblasts; Gene Expression Regulation; Host-Pathogen Interactions; Humans; Lamin Type A; Osteoblasts; Phosphorylation; Protein Kinase Inhibitors; Ribonucleosides; Signal Transduction; Viral Proteins
PubMed: 26223645
DOI: 10.1128/JVI.00986-15 -
Current Opinion in Infectious Diseases Aug 2015The purpose of this study is to provide updated information on diagnosis of cytomegalovirus (CMV) drug resistance, treatments for drug-resistant infection and potential... (Review)
Review
PURPOSE OF REVIEW
The purpose of this study is to provide updated information on diagnosis of cytomegalovirus (CMV) drug resistance, treatments for drug-resistant infection and potential uses of experimental antiviral compounds.
RECENT FINDINGS
For established CMV antivirals, uncommon viral UL97 kinase and UL54 DNA polymerase drug resistance mutations are sporadically described that expand an extensive existing database. Some novel mutations reported from treated patients have no drug-resistant phenotype and may be genotyping artefacts. Next-generation sequencing technology may enable earlier detection of emerging resistance mutations in treated patients. Management options for drug-resistant infection include optimization of host defenses, antiviral dose escalation, substitutions or combinations of standard or experimental antivirals. Maribavir and letermovir have antiviral targets distinct from the classic DNA polymerase. UL97 mutations elicited by ganciclovir and maribavir are different, although a single p-loop mutation can confer significant cross-resistance. High-grade resistance mutations in the UL56 terminase gene are readily selected in vitro under letermovir and await clinical correlation.
SUMMARY
Technical advancements can enhance the accurate and timely genotypic detection of drug resistance. Antivirals undergoing clinical trial offer the prospect of new viral targets and drug combinations, but unresolved issues exist with regard to their therapeutic potential for drug-resistant CMV and their genetic barriers to resistance.
Topics: Antiviral Agents; Cytomegalovirus; Cytomegalovirus Infections; Drug Resistance, Viral; Genotype; Genotyping Techniques; Humans; Immunocompromised Host; Molecular Diagnostic Techniques; Transplant Recipients; Transplantation
PubMed: 26098499
DOI: 10.1097/QCO.0000000000000170 -
Biology of Blood and Marrow... Jan 2015The 2015 Tandem American Society for Blood and Marrow Transplantation/Center for International Blood and Marrow Transplant Meetings provide an opportunity to review the... (Review)
Review
The 2015 Tandem American Society for Blood and Marrow Transplantation/Center for International Blood and Marrow Transplant Meetings provide an opportunity to review the current status and future perspectives on therapy for cytomegalovirus (CMV) infection in the setting of hematopoietic stem cell transplantation (HSCT). After many years during which we have seen few tangible advances in terms of new antiviral drugs, we are now experiencing an exciting period of late-stage drug development, characterized by a series of phase III trials incorporating a variety of novel agents. These trials have the potential to shift our current standard therapeutic strategies, which generally involve pre-emptive therapy based on sensitive molecular surveillance, towards the prophylactic approaches we see more generally with other herpes viruses such as herpes simplex and varicella zoster. This comes at a time when the promise of extensive preclinical research has been translated into encouraging clinical responses with several cellular immunotherapy strategies, which have also been moved towards definitive late-stage clinical trials. How these approaches will be integrated with the new wave of antiviral drugs remains open to conjecture. Although most of the focus of these cellular immunotherapy studies has been on adaptive immunity, and in particular T cells, an increasing awareness of the possible role of other cellular subsets in controlling CMV infection has developed. In particular, the role of natural killer (NK) cells is being revisited, along with that of γδ T cells. Depletion of NK cells in mice results in higher titers of murine CMV in tissues and increased mortality, whereas NK cell deficiency in humans has been linked to severe CMV disease. We will review recent progress in these areas.
Topics: Acetates; Adoptive Transfer; Animals; Antiviral Agents; Benzimidazoles; Cell- and Tissue-Based Therapy; Clinical Trials, Phase III as Topic; Cytomegalovirus; Cytomegalovirus Infections; Cytosine; Hematopoietic Stem Cell Transplantation; Humans; Isoxazoles; Killer Cells, Natural; Leflunomide; Mice; Organophosphonates; Quinazolines; Ribonucleosides; T-Lymphocytes
PubMed: 25452035
DOI: 10.1016/j.bbmt.2014.11.002 -
Journal of Virology Jan 2015Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for...
UNLABELLED
Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.
IMPORTANCE
Human cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.
Topics: Cell Nucleus; Cytomegalovirus; Host-Pathogen Interactions; Humans; Lamin Type A; Mass Spectrometry; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Processing, Post-Translational; Viral Proteins; Virus Release
PubMed: 25339763
DOI: 10.1128/JVI.02426-14 -
Journal of Virology Jun 2014We report that UL133-UL138 (UL133/8), a transcriptional unit within the ULb' region (ULb') of the human cytomegalovirus (HCMV) genome, and UL97, a viral protein kinase...
UNLABELLED
We report that UL133-UL138 (UL133/8), a transcriptional unit within the ULb' region (ULb') of the human cytomegalovirus (HCMV) genome, and UL97, a viral protein kinase encoded by HCMV, play epistatic roles in facilitating progression of the viral lytic cycle. In studies with HCMV strain TB40/E, pharmacological blockade or genetic ablation of UL97 significantly reduced the levels of mRNA and protein for IE2 and viral early and early-late genes during a second wave of viral gene expression that commenced at between 24 and 48 h postinfection. These effects were accompanied by significant defects in viral DNA synthesis and viral replication. Interestingly, deletion of UL133/8 likewise caused significant defects in viral DNA synthesis, viral gene expression, and viral replication, which were not exacerbated upon UL97 inhibition. When UL133/8 was restored to HCMV laboratory strain AD169, which otherwise lacks the locus, the resulting recombinant virus replicated similarly to the parental virus. However, during UL97 inhibitor treatment, the virus in which UL133/8 was restored showed significantly exacerbated defects in viral DNA synthesis, viral gene expression, and production of infectious progeny virus, thus recapitulating the differences between wild-type TB40/E and its UL133/8-null derivative. Phenotypic evaluation of mutants null for specific open reading frames within UL133/8 revealed a role for UL135 in promoting viral gene expression, viral DNA synthesis, and viral replication, which depended on UL97. Taken together, our findings suggest that UL97 and UL135 play interdependent roles in promoting the progression of a second phase of the viral lytic cycle and that these roles are crucial for efficient viral replication.
IMPORTANCE
A unique feature of the herpesviruses, such as human cytomegalovirus (HCMV), is that they can undergo latency, a state during which the virus silences its gene expression, which allows lifelong viral persistence in immunocompetent hosts. We have uncovered an unexpected link between a cluster of HCMV genes involved in latency, UL133-UL138, and a virally encoded protein kinase, UL97, which plays crucial roles in manipulating the cell cycle during HCMV lytic replication. Although viral immediate early (IE) gene expression is essential for HCMV lytic replication, the activation of IE gene expression in latently infected cells is not sufficient to result in production of infectious virus. Our findings here and in an accompanying study (M. Umashankar, M. Rak, F. Bughio, P. Zagallo, K. Caviness, and F. D. Goodrum, J. Virol. 88:5987-6002, 2014) show that proteins expressed from the UL133-UL138 latency locus and UL97 play interdependent roles in overcoming checkpoints that restrict the viral lytic replication cycle, findings which suggest intriguing implications for establishment of and reactivation from HCMV latency.
Topics: Benzimidazoles; Blotting, Western; Chromosomes, Artificial, Bacterial; Cytomegalovirus; DNA Primers; Epistasis, Genetic; Humans; Phosphotransferases (Alcohol Group Acceptor); Real-Time Polymerase Chain Reaction; Ribonucleosides; Viral Proteins; Virus Latency; Virus Replication
PubMed: 24623439
DOI: 10.1128/JVI.00447-14 -
Journal of Virology May 2014The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length...
UNLABELLED
The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir.
IMPORTANCE
The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo.
Topics: Antiviral Agents; Benzimidazoles; Cytomegalovirus; Humans; Microbial Sensitivity Tests; Phosphotransferases (Alcohol Group Acceptor); Protein Isoforms; Ribonucleosides; Virus Replication
PubMed: 24522923
DOI: 10.1128/JVI.00192-14