Inhibition Of Viral DNA Synthesis Research Articles

Divide et impera: An In Silico Screening Targeting HCMV ppUL44 Processivity Factor Homodimerization Identifies Small Molecules Inhibiting Viral Replication.

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, 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 for 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, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-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 HCMV AD169 strain 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, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that 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 aryl hydrocarbon receptor and interferon gamma generate antiviral states via transcriptional repression.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activation induces the expression of numerous genes, with many effects on cells. However, AhR activation is not known to affect the replication of viruses. We show that AhR activation in macrophages causes a block to HIV-1 and HSV-1 replication. We find that AhR activation transcriptionally represses cyclin-dependent kinase (CDK)1/2 and their associated cyclins, thereby reducing SAMHD1 phosphorylation, cellular dNTP levels and both HIV-1 and HSV-1 replication. Remarkably, a different antiviral stimulus, interferon gamma (IFN-γ), that induces a largely non-overlapping set of genes, also transcriptionally represses CDK1, CDK2 and their associated cyclins, resulting in similar dNTP depletion and antiviral effects. Concordantly, the SIV Vpx protein provides complete and partial resistance to the antiviral effects of AhR and IFN-γ, respectively. Thus, distinct antiviral signaling pathways converge on CDK/cyclin repression, causing inhibition of viral DNA synthesis and replication.

Essential function of VCP/p97 in infection cycle of the nucleopolyhedrovirus AcMNPV in Spodoptera frugiperda Sf9 cells

The protein VCP/p97 (also named CDC48 and TER94) belongs to a type II subfamily of the AAA+ATPases and controls cellular proteostasis by acting upstream of proteasomes in the ubiquitin-proteasome protein degradation pathway. The function of VCP/p97 in the baculovirus infection cycle in insect cells remains unknown. Here, we identified VCP/p97 in the fall armyworm Spodoptera frugiperda (Sf9) cells and analyzed the replication of the Autographa californica multiple nucleopolyhedrovirus, AcMNPV, in Sf9 cells in which the VCP/p97 function was inhibited. The specific allosteric inhibitor of the VCP/p97 ATPase activity, NMS-873, did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA synthesis and efficiently suppressed expression of viral proteins and production of budded virions. NMS-873 caused accumulation of ubiquitinated proteins in a manner similar to the inhibitor of proteasome activity, Bortezomib. This suggests the essential function of VCP/p97 in the baculovirus infection cycle might be associated, at least in part, with the ubiquitin-proteasome system.

Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice

Chronic hepatitis B virus infection cannot be cured by current therapies, so new treatments are urgently needed. We recently identified novel inhibitors of the hepatitis B virus ribonuclease H that suppress viral replication in cell culture. Here, we employed immunodeficient FRG KO mice whose livers had been engrafted with primary human hepatocytes to ask whether ribonuclease H inhibitors can suppress hepatitis B virus replication in vivo. Humanized FRG KO mice infected with hepatitis B virus were treated for two weeks with the ribonuclease H inhibitors #110, an α-hydroxytropolone, and #208, an N-hydroxypyridinedione. Hepatitis B virus viral titers and S and e antigen plasma levels were measured. Treatment with #110 and #208 caused significant reductions in plasma viremia without affecting hepatitis B virus S or e antigen levels, and viral titers rebounded following treatment cessation. This is the expected pattern for inhibitors of viral DNA synthesis. Compound #208 suppressed viral titers of both hepatitis B virus genotype A and C isolates. These data indicate that Hepatitis B virus replication can be suppressed during infection in an animal by inhibiting the viral ribonuclease H, validating the ribonuclease H as a novel target for antiviral drug development.

Antiviral activity of macrophage‐activating Chinese mixed herb hot water extract

ABSTRACTAimMacrophage‐activating Chinese mixed herb (MACH) extract, a hot‐water decoction of Cucurbita moschata (pumpkin seed), Carthamus tinctorius (safflower flower), P. asiatica (psyllium seed), and L. japonica (Japanese honeysuckle flower), is able to induce the production of interferon and interleukin, and hence reduce viral load in patients with hepatitis C virus (HCV) infection. The aim of this study was therefore to analyze the antiviral activity in the available models and identify the mechanism of alleviation of HCV infection.MethodsThe antiviral and anti‐Herpes simplex virus (anti‐HSV) activity of MACH extracts was examined using the plaque reduction assay in a mouse model of cutaneous HSV infection.ResultsMACH extracts inhibited plaque formation of HSV, Varicella zoster virus, cytomegalovirus, and poliovirus at 50% effective concentration for plaque formation (EC50) of 3.09, 0.84, 1.54, and 3.3 mg/mL, respectively. MACH extract inhibited the late protein synthesis of HSV, indicating inhibition of viral DNA synthesis. MACH extract was given orally at 0, 40, and 400 mg in 200 μL water 7 days before infection in female BALB/c mice (6 weeks old) cutaneously infected with HSV. MACH extract was not toxic, as assessed by bodyweight changes, and significantly alleviated the development of skin lesions at days 6–8 (P < 0.05) but did not prolong the survival period.ConclusionOn plaque reduction assay, MACH extract had significant but mild therapeutic anti‐HSV activity. This anti‐viral activity might be associated with the therapeutic antiviral activity in animals and humans.

Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes.

Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. A prerequisite for CCC DNA formation is the uncoating (disassembly) of NCs to expose their RC DNA content for conversion to CCC DNA. We report here that in an immortalized mouse hepatocyte cell line, AML12HBV10, in which RC DNA exposure is enhanced, the exposed viral DNA could trigger an innate immune response that was able to modulate viral gene expression and replication. When viral gene expression and replication were low, the innate response initially stimulated these processes but subsequently acted to shut off viral gene expression and replication after they reached peak levels. Inhibition of viral DNA synthesis or cellular DNA sensing and innate immune signaling diminished the innate response. These results indicate that HBV DNA, when exposed in the host cell cytoplasm, can function to trigger an innate immune response that, in turn, modulates viral gene expression and replication. Chronic infection by hepatitis B virus (HBV) afflicts hundreds of millions worldwide and is sustained by the episomal covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. Release of viral genomic DNA from cytoplasmic nucleocapsids (NCs) (NC disassembly or uncoating) is a prerequisite for its conversion to CCC DNA, which can also potentially expose the viral DNA to host DNA sensors and trigger an innate immune response. We have found that in an immortalized mouse hepatocyte cell line in which efficient CCC DNA formation was associated with enhanced exposure of nucleocapsid-associated DNA, the exposed viral DNA indeed triggered host cytoplasmic DNA sensing and an innate immune response that was able to modulate HBV gene expression and replication. Thus, HBV can, under select conditions, be recognized by the host innate immune response through exposed viral DNA, which may be exploited therapeutically to clear viral persistence.

Transcriptional analysis of ORF amv133 of Amsacta moorei entomopoxvirus.

The open reading frame (ORF) amv133 of Amsacta moorei entomopoxvirus, encodes a putative lipase gene. Its temporal expression pattern was characterized by RT-PCR and found to start at 6 h postinfection (h p.i.) and reach a maximum level at 48 h p.i. While the ORF has a late promoter motif, the inhibition of viral DNA synthesis by Ara-C failed to inhibit transcription, but a general inhibitor of protein synthesis prevented its transcription, indicating that amv133 is an intermediate gene. 5'-RACE analysis showed that transcription was initiated at position -77 relative to the translational start site. To determine the size of the promoter, several truncations were generated and cloned upstream of the firefly luciferase reporter gene. The resulting constructs were tested in a dual assay. A fragment that contained 115 bp relative to the transcription start site exhibited optimum promoter length.

Site-Specific Association with Host and Viral Chromatin by Kaposi's Sarcoma-Associated Herpesvirus LANA and Its Reversal during Lytic Reactivation

Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and LBS2) on viral DNA and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here, we have examined LANA interactions with host chromatin on a genome-wide scale using chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LANA-binding site 1 (LBS1) motif in KSHV DNA. Comparison of the ChIP-seq profile with whole-transcriptome (high-throughput sequencing of RNA transcripts [RNA-seq]) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. Here, we employ complementary genome-wide analyses to evaluate the distribution of the highly abundant latency-associated nuclear antigen, LANA, on the host genome and its impact on host gene expression during KSHV latent infection. Combined, ChIP-seq and RNA-seq reveal that LANA accumulates at active gene promoters that harbor specific short DNA sequences that are highly reminiscent of its cognate binding sites in the virus genome. Unexpectedly, we found that such association does not lead to remodeling of global host transcription during latency. We also report for the first time that LANA's ability to bind host and viral chromatin is highly dynamic and is disrupted in cells undergoing an extensive lytic reactivation. This therefore suggests that the association of LANA to chromatin during a productive infection cycle is controlled by a new regulatory mechanism.

Acyclovir

Acyclovir is one of the oldest antiviral drugs, having been in use for three decades now. It is an acyclic guanosine derivative and has activity mainly against herpes virus-1 (HSV-1), herpes virus-2 (HSV-2), and varicella zoster virus (VZV). It is only weakly active against Epstein–Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus-6 (HHV-6), and not used for these infections. It acts by inhibition of viral DNA synthesis. Before it can act, it has to be phosphorylated by virus-specific thymidine kinase. This step gives it a high therapeutic index, but is also a major mechanism of viral resistance.

Resistance of Human Cytomegalovirus to Cyclopropavir Maps to a Base Pair Deletion in the Open Reading Frame of UL97

Human cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC50s] = 0.46 and 4.1 μM, respectively). We hypothesize that the mechanism of action of CPV is similar to that of GCV: phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation to a triphosphate by endogenous kinases, resulting in inhibition of viral DNA synthesis. To test this hypothesis, we isolated a CPV-resistant virus, sequenced its genome, and discovered that bp 498 of UL97 was deleted. This mutation caused a frameshift in UL97 resulting in a truncated protein that lacks a kinase domain. To determine if this base pair deletion was responsible for drug resistance, the mutation was engineered into the wild-type viral genome, which was then exposed to increasing concentrations of CPV. The results demonstrate that the engineered virus was approximately 72-fold more resistant to CPV (EC50 = 25.8 ± 3.1 μM) than the wild-type virus (EC50 = 0.36 ± 0.11 μM). We conclude, therefore, that this mutation is sufficient for drug resistance and that pUL97 is involved in the mechanism of action of CPV.

Murine Cytomegalovirus Protein pM79 Is a Key Regulator for Viral Late Transcription

Herpesvirus genes are temporally expressed during permissive infections, but how their expression is regulated at late times is poorly understood. Previous studies indicate that the human cytomegalovirus (CMV) gene, UL79, is required for late gene expression. However, the mechanism remains to be fully elucidated, and UL79 homologues in other CMVs have not been studied. Here, we characterized the role of the conserved murine CMV (MCMV) gene M79. We showed that M79 encoded a protein (pM79) which was expressed with early-late kinetics and localized to nuclear viral replication compartments. M79 transcription was significantly decreased in the absence of viral DNA synthesis but markedly stimulated by pM79. To investigate its role, we created the recombinant virus SMin79, in which pM79 expression was disrupted. While marker-rescued virus grew efficiently in fibroblasts, SMin79 failed to produce infectious progeny but was rescued by pM79 expression in trans. During SMin79 infection, representative viral immediate-early and early gene products as well as viral DNA accumulated sufficiently. Formation of viral replication compartments also appeared normal. Pulsed-field gel electrophoresis analysis indicated that the overall structure of replicating viral DNA was indistinguishable between wild-type and SMin79 infection. Viral tiled array and quantitative PCR analysis revealed that many late transcripts sensitive to a viral DNA synthesis inhibitor (phosphonoacetic acid) were markedly reduced by pM79 mutation. This study indicates that cytomegaloviruses use a conserved mechanism to promote transcription at late stages of infection and that pM79 is a critical regulator for at least a subset of viral DNA synthesis-dependent transcripts.

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

We report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb' region of the viral genome. A UL97-null (Δ97) mutant of strain TB40/E, which encodes a full-length ULb' region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a Δ97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb' region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(β-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb' regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ≈ 100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb' regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb' region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb' region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.

Amplification of JNK Signaling Is Necessary To Complete the Murine Gammaherpesvirus 68 Lytic Replication Cycle

Several studies have previously defined host-derived signaling events capable of driving lytic gammaherpesvirus replication or enhancing immediate-early viral gene expression. Yet signaling pathways that regulate later stages of the productive gammaherpesvirus replication cycle are still poorly defined. In this study, we utilized a mass spectrometric approach to identify c-Jun as an abundant cellular phosphoprotein present in late stages of lytic murine gammaherpesvirus 68 (MHV68) infection. Kinetically, c-Jun phosphorylation was enhanced as infection progressed, and this correlated with enhanced phosphorylation of the c-Jun amino-terminal kinases JNK1 and JNK2 and activation of AP-1 transcription. These events were dependent on progression beyond viral immediate-early gene expression, but not dependent on viral DNA replication. Both pharmacologic and dominant-negative blockade of JNK1/2 activity inhibited viral replication, and this correlated with inhibition of viral DNA synthesis and reduced viral gene expression. These data suggest a model in which MHV68 by necessity amplifies and usurps JNK/c-Jun signaling as infection progresses in order to facilitate late stages of the MHV68 lytic infection cycle.

Host Cell Nucleolin Is Required To Maintain the Architecture of Human Cytomegalovirus Replication Compartments

ABSTRACTDrastic 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. However, recent work with human cytomegalovirus (HCMV) showed that the viral DNA polymerase subunit UL44 concentrates and viral DNA synthesis occurs at the periphery of these compartments. Any cellular factors involved in replication compartment architecture are largely unknown. Previously, we found that nucleolin, a major protein component of nucleoli, associates with HCMV UL44 in infected cells and is required for efficient viral DNA synthesis. Here, we show that nucleolin binds to purified UL44. Confocal immunofluorescence analysis demonstrated colocalization of nucleolin with UL44 at the periphery of replication compartments. Pharmacological inhibition of viral DNA synthesis prevented the formation of replication compartments but did not abrogate association of UL44 and nucleolin. Thus, association of UL44 and nucleolin is unlikely to be a nonspecific effect related to development of replication compartments. No detectable colocalization of 5-ethynyl-2′-deoxyuridine (EdU)-labeled viral DNA with nucleolin was observed, suggesting that nucleolin is not directly involved in viral DNA synthesis. Small interfering RNA (siRNA)-mediated knockdown of nucleolin caused improper localization of UL44 and a defect in EdU incorporation into viral DNA. We propose a model in which nucleolin anchors UL44 at the periphery of replication compartments to maintain their architecture and promote viral DNA synthesis.

Leflunomide for cytomegalovirus: bench to bedside

Cytomegalovirus (CMV) remains a major cause of morbidity and mortality among transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide has been shown to have immunosuppressive activity in experimental allograft models together with antiviral activity inhibiting CMV both in vitro and in vivo. Data are emerging about its potential role in ganciclovir-sensitive and -resistant CMV, primarily by virtue of a unique mechanism inhibiting virion assembly, as opposed to inhibition of viral DNA synthesis by current agents. This review aims to put in perspective, the knowledge acquired in the last decade or so on leflunomide for CMV. Evidence suggests that it might have activity against human CMV with good oral bioavailability and, more importantly in the resource-poor setting, is economical. Although the data presented here are not from randomized trials, several relevant observations have been made that could influence future, more structured assessments of the drug. An immune suppressive compound with antiviral features and experimental activity in chronic rejection is an attractive combination for organ transplantation, and it appears that leflunomide may just fit that niche.

Safety and Efficacy of CMX001 as Salvage Therapy for Severe Adenovirus Infections in Immunocompromised Patients

No therapeutic agent has yet been established as the definitive therapy for adenovirus infections. We describe the clinical experience of 13 immunocompromised patients who received CMX001 (hexadecyloxypropyl cidofovir), an orally bioavailable lipid conjugate of cidofovir, for adenovirus disease. We retrospectively analyzed 13 patients with adenovirus disease and viremia treated with CMX001; data were available for ≥ 4 weeks after initiation of CMX001 therapy. Virologic response (VR) was defined as a 99% drop from baseline or undetectable adenovirus DNA in serum. The median age of the group was 6 years (range, 0.92-66 years). One patient had severe combined immunodeficiency, 1 patient was a small bowel transplant recipient, and 11 were allogeneic stem cell transplant recipients. Adenovirus disease was diagnosed at a median of 75 days (range, 15-720 days) after transplantation. All patients received i.v. cidofovir for a median of 21 days (range, 5-90 days) before CMX001 therapy. The median absolute lymphocyte count at CMX001 initiation was 300 cells/μL (range, 7-1500 cells/μL). Eight patients (61.5%) had a ≥ 1 log10 drop in viral load after the first week of therapy. By week 8, 9 patients (69.2%) demonstrated a VR, with a median time to achieve VR of 7 days (range, 3-35 days). The change in absolute lymphocyte count was inversely correlated with the change in log10 viral load only at week 6 (r = -0.74; P = .03). Patients with VR had longer survival than those without VR (median 196 days versus 54.5 days; P = .04). No serious adverse events were attributed to CMX001 during therapy. CMX001 may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients.

Cyclopropavir inhibits the normal function of the human cytomegalovirus UL97 kinase.

Cyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.

Alternative Splicing of the Human Cytomegalovirus Major Immediate-Early Genes Affects Infectious-Virus Replication and Control of Cellular Cyclin-Dependent Kinase

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.

Human Cytomegalovirus Early Protein pUL21a Promotes Efficient Viral DNA Synthesis and the Late Accumulation of Immediate-Early Transcripts

We have previously reported that a newly annotated gene of human cytomegalovirus (HCMV), UL21a, encodes an early viral protein termed pUL21a. Most notably, the virions of a UL21a deletion virus had markedly reduced infectivity, indicating that UL21a is required to establish an efficient productive infection. In this study, we infected fibroblasts with equal numbers of DNA-containing viral particles and identified where in the viral life cycle pUL21a acted. The UL21a deletion virus entered cells and initiated viral gene expression efficiently; however, it synthesized viral DNA poorly and accumulated several immediate-early (IE) transcripts at reduced levels at late times of infection. The defect in viral DNA synthesis preceded that in gene expression, and inhibition of viral DNA synthesis reduced the late accumulation of IE transcripts in both wild-type and mutant virus-infected cells to equivalent levels. This suggests that reduced viral DNA synthesis is the cause of reduced IE gene expression in the absence of UL21a. The growth of UL21a deletion virus was similar to that of recombinant HCMV in which pUL21a expression was abrogated by stop codon mutations, and the defect was rescued in pUL21a-expressing fibroblasts. pUL21a expression in trans was sufficient to restore viral DNA synthesis and gene expression of mutant virus produced from normal fibroblasts, whereas mutant virus produced from complementing cells still exhibited the defect in normal fibroblasts. Thus, pUL21a does not promote the functionality of HCMV virions; rather, its de novo synthesis facilitates viral DNA synthesis, which is necessary for the late accumulation of IE transcripts and establishment of a productive infection.

Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Kinetic studies with CPV as the substrate for pUL97 demonstrated a Km of 1,750+/-210 microM. Introduction of 1.0 or 10 nM maribavir, a known pUL97 inhibitor, and subsequent Lineweaver-Burk analysis demonstrated competitive inhibition of CPV phosphorylation, with a Ki of 3.0+/-0.3 nM. Incubation of CPV with pUL97 combined with GMP kinase [known to preferentially phosphorylate the (+)-enantiomer of CPV-MP] established that pUL97 stereoselectively phosphorylates CPV to its (+)-monophosphate. These results elucidate the mechanism of CPV phosphorylation and help explain its selective antiviral action.