RESUMO
The terminase complex of human cytomegalovirus (HCMV) is required for viral genome packaging and cleavage. Critical to the terminase functions is a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We have previously reported metal-chelating N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human immunodeficiency virus 1 (HIV-1) RNase H. In the current work, we have synthesized new analogs and resynthesized known analogs of two isomeric HtPD subtypes, anti-HtPD (13), and syn-HtPD (14), and characterized them as inhibitors of pUL89-C. Remarkably, the vast majority of analogs strongly inhibited pUL89-C in the biochemical endonuclease assay, with IC50 values in the nM range. In the cell-based antiviral assay, a few analogs inhibited HCMV in low µM concentrations. Selected analogs were further characterized in a biophysical thermal shift assay (TSA) and in silico molecular docking, and the results support pUL89-C as the protein target of these inhibitors. Collectively, the biochemical, antiviral, biophysical, and in silico data reported herein indicate that the isomeric HtPD chemotypes 13-14 can serve as valuable chemical platforms for designing improved inhibitors of HCMV pUL89-C.
Assuntos
Antivirais , Citomegalovirus , Endonucleases , Proteínas Virais , Humanos , Antivirais/farmacologia , Antivirais/química , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Endonucleases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Desenho de FármacosRESUMO
Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.
Assuntos
Caseína Quinase II/genética , Proteínas Imediatamente Precoces/genética , Fosforilação/genética , Proteína SUMO-1/genética , Sumoilação/genética , Transativadores/genética , Sítios de Ligação , Caseína Quinase II/química , Citomegalovirus/enzimologia , Citomegalovirus/genética , Regulação Viral da Expressão Gênica/genética , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Cinética , Domínios e Motivos de Interação entre Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Transativadores/química , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Replicação Viral/genéticaRESUMO
Many cellular processes pertinent for viral infection are regulated by the addition of small ubiquitin-like modifiers (SUMO) to key regulatory proteins, making SUMOylation an important mechanism by which viruses can commandeer cellular pathways. Epstein-Barr virus (EBV) is a master at manipulating of cellular processes, which enables life-long infection but can also lead to the induction of a variety of EBV-associated cancers. To identify new mechanisms by which EBV proteins alter cells, we screened a library of 51 EBV proteins for global effects on cellular SUMO1 and SUMO2 modifications (SUMOylation), identifying several proteins not previously known to manipulate this pathway. One EBV protein (BRLF1) globally induced the loss of SUMOylated proteins, in a proteasome-dependent manner, as well as the loss of promeylocytic leukemia nuclear bodies. However, unlike its homologue (Rta) in Kaposi's sarcoma associated herpesvirus, it did not appear to have ubiquitin ligase activity. In addition we identified the EBV SM protein as globally upregulating SUMOylation and showed that this activity was conserved in its homologues in herpes simplex virus 1 (HSV1 UL54/ICP27) and cytomegalovirus (CMV UL69). All three viral homologues were shown to bind SUMO and Ubc9 and to have E3 SUMO ligase activity in a purified system. These are the first SUMO E3 ligases discovered for EBV, HSV1 and CMV. Interestingly the homologues had different specificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1 modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide new insights into the function of this family of conserved herpesvirus proteins, and the conservation of this SUMO E3 ligase activity across diverse herpesviruses suggests the importance of this activity for herpesvirus infections.
Assuntos
Citomegalovirus/enzimologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 4/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Estudo de Associação Genômica Ampla , Humanos , SumoilaçãoRESUMO
Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro.
Assuntos
Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Ganciclovir/farmacologia , Proteínas Quinases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citomegalovirus/genética , Farmacorresistência Viral/genética , Humanos , Mutação/genética , Fases de Leitura Aberta/genética , Fosforilação , Proteínas Quinases/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Infections with human cytomegalovirus (HCMV) are highly prevalent in the general population as the virus has evolved the capacity to undergo distinct replication strategies resulting in lytic, persistent, and latent infections. During the latent life cycle, HCMV resides in subsets of cells within the hematopoietic cell compartment, including hematopoietic progenitor cells (HPCs) and peripheral blood monocytes. Since only a small fraction of these cell types harbor viral genomes during natural latency, identification and analysis of distinct changes mediated by viral infection are difficult to assess. In order to characterize latent infections of HPCs, we used an approach that involves complementation of deficiencies within the human pyrimidine salvage pathway, thus allowing for conversion of labeled uracil into rUTP. Here, we report the development of a recombinant HCMV that complements the defective human pyrimidine salvage pathway, allowing incorporation of thiol containing UTP into all RNA species that are synthesized within an infected cell. This virus grows to wild-type kinetics and can establish a latent infection within two distinct culture models of HCMV latency. Using this recombinant HCMV, we report the specific labeling of transcripts only within infected cells. These transcripts reveal a transcriptional landscape during HCMV latency that is distinct from uninfected cells. The utility of this labeling system allows for the identification of distinct changes within host transcripts and will shed light on characterizing how HCMV establishes and maintains latency.IMPORTANCE HCMV is a significant pathogen that accounts for a substantial amount of complications within the immunosuppressed and immunocompromised. Of particular significance is the capacity of HCMV to reactivate within solid tissue and bone marrow transplant recipients. While it is known that HCMV latency resides within a fraction of HPCs and monocytes, the exact subset of cells that harbor latent viral genomes during natural infections remain uncharacterized. The capacity to identify changes within the host transcriptome during latent infections is critical for developing approaches that therapeutically or physically eliminate latent viral genome containing cells and will represent a major breakthrough for reducing complications due to HCMV reactivation posttransplant. In this report, we describe the generation and use of a recombinant HCMV that allows specific and distinct labeling of RNA species that are produced within virally infected cells. This is a critical first step in identifying how HCMV affects the host cell during latency and more importantly, allows one to characterize cells that harbor latent HCMV.
Assuntos
Citomegalovirus/genética , Pentosiltransferases/genética , RNA Viral/genética , Coloração e Rotulagem/métodos , Tiouracila/análogos & derivados , Uracila/química , Células Cultivadas , Citomegalovirus/enzimologia , Infecções por Citomegalovirus , Humanos , Tiouracila/química , Latência Viral/genéticaRESUMO
Over the past few decades, a large number of studies have identified herpesvirus sequences from many mammalian species around the world. Among the different nonhuman primate species tested so far for cytomegaloviruses (CMVs), only a few were from the New World. Seeking to identify CMV homologues in New World monkeys (NWMs), we carried out molecular screening of 244 blood DNA samples from 20 NWM species from Central and South America. Our aim was to reach a better understanding of their evolutionary processes within the Platyrrhini parvorder. Using PCR amplification with degenerate consensus primers targeting highly conserved amino acid motifs encoded by the herpesvirus DNA polymerase gene, we characterized novel viral sequences from 12 species belonging to seven genera representative of the three NWM families. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, and phylogenetic analyses confirmed that they all belonged to the Cytomegalovirus genus. Previously determined host taxa allowed us to demonstrate a good correlation between the distinct monophyletic clades of viruses and those of the infected primates at the genus level. In addition, the evolutionary branching points that separate NWM CMVs were congruent with the divergence dates of their hosts at the genus level. These results significantly expand our knowledge of the host range of this viral genus and strongly support the occurrence of cospeciation between these viruses and their hosts. In this respect, we propose that NWM CMV DNA polymerase gene sequences may serve as reliable molecular markers with which to infer Platyrrhini phylogenetics.IMPORTANCE Investigating evolutionary processes between viruses and nonhuman primates has led to the discovery of a large number of herpesviruses. No study published so far on primate cytomegaloviruses has extensively studied New World monkeys (NWMs) at the subspecies, species, genus, and family levels. The present study sought to identify cytomegalovirus homologues in NWMs and to decipher their evolutionary relationships. This led us to characterize novel viruses from 12 of the 20 primate species tested, which are representative of the three NWM families. The identification of distinct viruses in these primates not only significantly expands our knowledge of the host range of this viral genus but also sheds light on its evolutionary history. Phylogenetic analyses and molecular dating of the sequences obtained support a virus-host coevolution.
Assuntos
Citomegalovirus/classificação , Citomegalovirus/genética , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Doenças dos Macacos/virologia , Filogenia , Platirrinos/virologia , Proteínas Virais/genética , Animais , América Central/epidemiologia , Citomegalovirus/enzimologia , DNA Viral/sangue , DNA Viral/genética , DNA Viral/isolamento & purificação , Evolução Molecular , Doenças dos Macacos/sangue , Doenças dos Macacos/epidemiologia , Reação em Cadeia da Polimerase/métodos , América do Sul/epidemiologiaRESUMO
Several viruses have been found to encode a deubiquitinating protease (DUB). These viral DUBs are proposed to play a role in regulating innate immune or inflammatory signaling. In human cytomegalovirus (HCMV), the largest tegument protein encoded by UL48 contains DUB activity, but its cellular targets are not known. Here, we show that UL48 and UL45, an HCMV-encoded inactive homolog of cellular ribonucleotide reductase (RNR) large subunit (R1), target receptor-interacting protein kinase 1 (RIP1) to inhibit NF-κB signaling. Transfection assays showed that UL48 and UL45, which binds to UL48, interact with RIP1 and that UL48 DUB activity and UL45 cooperatively suppress RIP1-mediated NF-κB activation. The growth of UL45-null mutant virus was slightly impaired with showing reduced accumulation of viral late proteins. Analysis of a recombinant virus expressing HA-UL45 showed that UL45 interacts with both UL48 and RIP1 during virus infection. Infection with the mutant viruses also revealed that UL48 DUB activity and UL45 inhibit TNFα-induced NF-κB activation at late times of infection. UL48 cleaved both K48- and K63-linked polyubiquitin chains of RIP1. Although UL45 alone did not affect RIP1 ubiquitination, it could enhance the UL48 activity to cleave RIP1 polyubiquitin chains. Consistently, UL45-null virus infection showed higher ubiquitination level of endogenous RIP1 than HA-UL45 virus infection at late times. Moreover, UL45 promoted the UL48-RIP1 interaction and re-localization of RIP1 to the UL48-containing virion assembly complex. The mouse cytomegalovirus (MCMV)-encoded DUB, M48, interacted with mouse RIP1 and M45, an MCMV homolog of UL45. Collectively, our data demonstrate that cytomegalovirus-encoded DUB and inactive R1 homolog target RIP1 and cooperatively inhibit RIP1-mediated NF-κB signaling at the late stages of HCMV infection.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/enzimologia , Enzimas Desubiquitinantes/metabolismo , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleotídeo Redutases/metabolismo , Proteínas Virais/metabolismo , Citomegalovirus/genética , Enzimas Desubiquitinantes/genética , Humanos , NF-kappa B/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Ligação a RNA/genética , Ribonucleotídeo Redutases/genética , Transdução de Sinais , Proteínas Virais/genéticaRESUMO
Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme.IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.
Assuntos
Citomegalovirus/enzimologia , Endodesoxirribonucleases/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular/genética , Linhagem Celular , Cromossomos Artificiais Bacterianos/genética , Citomegalovirus/genética , Citomegalovirus/metabolismo , DNA Viral , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Fibroblastos/virologia , Genoma Viral , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
Previous studies identified the nuclear domain 10 (ND10) components promyelocytic leukemia protein (PML), hDaxx, and Sp100 as factors of an intrinsic immune response against human cytomegalovirus (HCMV). This antiviral function of ND10, however, is antagonized by viral effector proteins like IE1p72, which induces dispersal of ND10. Furthermore, we have shown that both major immediate early proteins of HCMV, IE1p72 and IE2p86, transiently colocalize with ND10 subnuclear structures and undergo modification by the covalent attachment of SUMO. Since recent reports indicate that PML acts as a SUMO E3 ligase, we asked whether the SUMOylation of IE1p72 and IE2p86 is regulated by PML. To address this, PML-depleted fibroblasts, as well as cells overexpressing individual PML isoforms, were infected with HCMV. Western blot experiments revealed a clear correlation between the degree of IE1p72 SUMO conjugation and the abundance of PML. On the other hand, the SUMOylation of IE2p86 was not affected by PML. By performing in vitro SUMOylation assays, we were able to provide direct evidence that IE1p72 is a substrate for PML-mediated SUMOylation. Interestingly, disruption of the RING finger domain of PML, which is proposed to confer SUMO E3 ligase activity, abolished PML-induced SUMOylation of IE1p72. In contrast, IE1p72 was still efficiently SUMO modified by a SUMOylation-defective PML mutant, indicating that intact ND10 bodies are not necessary for this effect. Thus, this is the first report that the E3 ligase PML is capable of stimulating the SUMOylation of a viral protein which is supposed to serve as a cellular mechanism to compromise specific functions of IE1p72.IMPORTANCE The major immediate early proteins of human cytomegalovirus, termed IE1p72 and IE2p86, have previously been shown to undergo posttranslational modification by covalent coupling to SUMO moieties at specific lysine residues. However, the enzymatic activities that are responsible for this modification have not been identified. Here, we demonstrate that the PML protein, which mediates an intrinsic immune response against HCMV, specifically serves as an E3 ligase for SUMO modification of IE1p72. Since SUMO modification of IE1p72 has previously been shown to interfere with STAT factor binding, thus compromising the interferon-antagonistic function of this viral effector protein, our finding highlights an additional mechanism through which PML is able to restrict viral infections.
Assuntos
Citomegalovirus/genética , Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/química , Proteína da Leucemia Promielocítica/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Citomegalovirus/enzimologia , Fibroblastos/virologia , Humanos , Proteínas Imediatamente Precoces/genética , Mutação , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica/química , Proteína SUMO-1/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Collapsing focal segmental glomerulosclerosis (FSGS) is a variant of FSGS and is associated with severe nephrotic syndrome and acute kidney injury and can occur after kidney transplantation. The exact mechanism of collapsing FSGS after kidney transplantation is unknown, but potential causes include autoimmune diseases, certain malignancies, bisphosphonates, m-TOR inhibitors, interferon-alpha, HIV infection, and other viruses. We describe a case of de novo Cytomegalovirus (CMV)-associated collapsing FSGS in a kidney transplant recipient with a UL97 phosphotransferase mutation that was successfully treated with intravenous ganciclovir, intravenous immunoglobulin, and steroids.
Assuntos
Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/virologia , Glomerulosclerose Segmentar e Focal/virologia , Transplante de Rim/efeitos adversos , Adulto , Citomegalovirus/enzimologia , Citomegalovirus/genética , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/etiologia , Progressão da Doença , Ganciclovir/administração & dosagem , Ganciclovir/uso terapêutico , Glomerulosclerose Segmentar e Focal/etiologia , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Imunoglobulinas Intravenosas/uso terapêutico , Masculino , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esteroides/administração & dosagem , Esteroides/uso terapêutico , Resultado do TratamentoRESUMO
The tripartite terminase complex of herpesviruses assembles in the cytoplasm of infected cells and exploits the host nuclear import machinery to gain access to the nucleus, where capsid assembly and genome-packaging occur. Here we analyzed the structure and conservation of nuclear localization signal (NLS) sequences previously identified in herpes simplex virus 1 (HSV-1) large terminase and human cytomegalovirus (HCMV) small terminase. We found a monopartite NLS at the N terminus of large terminase, flanking the ATPase domain, that is conserved only in α-herpesviruses. In contrast, small terminase exposes a classical NLS at the far C terminus of its helical structure that is conserved only in two genera of the ß-subfamily and absent in α- and γ-herpesviruses. In addition, we predicted a classical NLS in the third terminase subunit that is partially conserved among herpesviruses. Bioinformatic analysis revealed that both location and potency of NLSs in terminase subunits evolved more rapidly than the rest of the amino acid sequence despite the selective pressure to keep terminase gene products active and localized in the nucleus. We propose that swapping NLSs among terminase subunits is a regulatory mechanism that allows different herpesviruses to regulate the kinetics of terminase nuclear import, reflecting a mechanism of virus:host adaptation.
Assuntos
Citomegalovirus , Endodesoxirribonucleases , Evolução Molecular , Herpesvirus Humano 1 , Sinais de Localização Nuclear , Animais , Citomegalovirus/enzimologia , Citomegalovirus/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Humanos , Camundongos , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Estrutura Terciária de ProteínaRESUMO
Many antiviral and anticancer drugs are nucleoside analogs that target polymerases and cause DNA chain termination. Interestingly, ganciclovir (GCV), the first line of therapy for human cytomegalovirus (HCMV) infections, induces chain termination despite containing the equivalent of a 3'-hydroxyl group. Certain HCMV GCV resistance (GCV(r)) mutations, including ones associated with treatment failures, result in substitutions in the 3'-5' exonuclease (Exo) domain of the catalytic subunit of the viral DNA polymerase (Pol). To investigate how these mutations confer resistance, we overexpressed and purified wild-type (WT) HCMV Pol and three GCV(r) Exo mutants. Kinetic studies provided little support for resistance being due to effects on Pol binding or incorporation of GCV-triphosphate. The mutants were defective for Exo activity on all primer templates tested, including those with primers terminating with GCV, arguing against the mutations increasing excision of the incorporated drug. However, although the WT enzyme terminated DNA synthesis after incorporation of GCV-triphosphate and an additional nucleotide (N+1), the Exo mutants could efficiently synthesize DNA to the end of such primer templates. Notably, the Exo activity of WT Pol rapidly and efficiently degraded N+2 primer templates to N+1 products that were not further degraded. On N+1 primer templates, WT Pol, much more than the Exo mutants, converted the incoming deoxynucleoside triphosphate to its monophosphate, indicative of rapid addition and removal of incorporated nucleotides ("idling"). These results explain how GCV induces chain termination and elucidate a previously unidentified mechanism of antiviral drug resistance.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Farmacorresistência Viral , Exonucleases/metabolismo , Ganciclovir/química , Mutação , Antivirais/química , Antivirais/farmacocinética , Sequência de Bases , Domínio Catalítico , Citomegalovirus/enzimologia , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/química , Exonucleases/genética , Ganciclovir/farmacocinética , Proteínas Virais/químicaRESUMO
Cyclopropavir (CPV) is a promising antiviral drug against human cytomegalovirus (HCMV). As with ganciclovir (GCV), the current standard for HCMV treatment, activation of CPV requires multiple steps of phosphorylation and is enantioselective. We hypothesized that the resulting CPV triphosphate (CPV-TP) would stereoselectively target HCMV DNA polymerase and terminate DNA synthesis. To test this hypothesis, we synthesized both enantiomers of CPV-TP [(+) and (-)] and investigated their action on HCMV polymerase. Both enantiomers inhibited HCMV polymerase competitively with dGTP, with (+)-CPV-TP exhibiting a more than 20-fold lower apparent Ki than (-)-CPV-TP. Moreover, (+)-CPV-TP was a more potent inhibitor than GCV-TP. (+)-CPV-TP also exhibited substantially lower apparent Km and somewhat higher apparent kcat values than (-)-CPV-TP and GCV-TP for incorporation into DNA by the viral polymerase. As is the case for GCV-TP, both CPV-TP enantiomers behaved as nonobligate chain terminators, with the polymerase terminating DNA synthesis after incorporation of one additional nucleotide. These results elucidate how CPV-TP acts on HCMV DNA polymerase and help explain why CPV is more potent against HCMV replication than GCV.
Assuntos
Antivirais/farmacologia , Ciclopropanos/farmacologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Replicação do DNA/efeitos dos fármacos , DNA Viral/genética , Farmacorresistência Viral/genética , Guanina/farmacologia , Cinética , Estrutura Molecular , Fosforilação/efeitos dos fármacosRESUMO
UNLABELLED: DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet, requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir, and ganciclovir (3-, 14-, and 3-fold increases in the 50% effective concentrations [EC50s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC50) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant values of foscarnet against mutant UL30 and UL54 DNA polymerases were 45- and 4.9-fold higher, respectively, than those against their wild-type counterparts. Structural evaluation of the tryptophan position in the UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a tendency toward the closed conformation greater than that for UL30 and explains the modest effect of the W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N in conferring HCMV and especially HSV-1 susceptibility to foscarnet and the possible contribution of other residues localized at the interface between the fingers and N-terminal domains. IMPORTANCE: DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The W781V mutation in HSV-1 induced resistance to foscarnet, whereas the W780V mutation in HCMV slightly decreased drug susceptibility. This study suggests that the different profiles of susceptibility to foscarnet of the HSV-1 and HCMV mutants could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.
Assuntos
Citomegalovirus/enzimologia , DNA Polimerase Dirigida por DNA/genética , Farmacorresistência Viral/genética , Herpesvirus Humano 1/enzimologia , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Estrutura Secundária de Proteína/genética , Aciclovir , Análise de Variância , Animais , Antivirais/farmacologia , Chlorocebus aethiops , DNA Polimerase Dirigida por DNA/química , Foscarnet , Ganciclovir , Técnicas de Transferência de Genes , Humanos , Cinética , Células VeroRESUMO
UNLABELLED: We report that the human cytomegalovirus (HCMV) high-molecular-weight tegument protein (HMWP, pUL48; 253 kDa) and the HMWP-binding protein (hmwBP, pUL47; 110 kDa) can be recovered as a complex from virions disrupted by treatment with 50 mM Tris (pH 7.5), 0.5 M NaCl, 0.5% NP-40, and 10 mM dithiothreitol [DTT]. The subunit ratio of the complex approximates 1:1, with a shape and structure consistent with an elongated heterodimer. The HMWP/hmwBP complex was corroborated by reciprocal coimmunoprecipitation experiments using antipeptide antibodies and lysates from both infected cells and disrupted virus particles. An interaction of the amino end of pUL48 (amino acids [aa] 322 to 754) with the carboxyl end of pUL47 (aa 693 to 982) was identified by fragment coimmunoprecipitation experiments, and a head-to-tail self-interaction of hmwBP was also observed. The deubiquitylating activity of pUL48 is retained in the isolated complex, which cleaves K11, K48, and K63 ubiquitin isopeptide linkages. IMPORTANCE: Human cytomegalovirus (HCMV, or human herpesvirus 5 [HHV-5]) is a large DNA-containing virus that belongs to the betaherpesvirus subfamily and is a clinically important pathogen. Defining the constituent elements of its mature form, their organization within the particle, and the assembly process by which it is produced are fundamental to understanding the mechanisms of herpesvirus infection and developing drugs and vaccines against them. In this study, we report isolating a complex of two large proteins encoded by HCMV open reading frames (ORFs) UL47 and UL48 and identifying the binding domains responsible for their interaction with each other and of pUL47 with itself. Our calculations indicate that the complex is a rod-shaped heterodimer. Additionally, we determined that the ubiquitin-specific protease activity of the ORF UL48 protein was functional in the complex, cleaving K11-, K48-, and K63-linked ubiquitin dimers. This information builds on and extends our understanding of the HCMV tegument protein network that is required to interface the HCMV envelope and capsid.
Assuntos
Citomegalovirus/enzimologia , Multimerização Proteica , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Vírion/enzimologia , Linhagem Celular , Citomegalovirus/química , Humanos , Imunoprecipitação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteases Específicas de Ubiquitina/isolamento & purificação , Proteínas Virais/isolamento & purificação , Vírion/químicaRESUMO
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.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Citomegalovirus/enzimologia , Citomegalovirus/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ribonucleosídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Human cytomegalovirus (HCMV) kinase UL97 is required for efficient nuclear lamina disruption during nuclear egress. However, cellular protein kinase C (PKC) has been implicated in this process in other systems. Comparing the effects of UL97 and cellular kinase inhibitors on HCMV nuclear egress confirms a role for UL97 in lamina disruption and nuclear egress. A pan-PKC inhibitor did not affect lamina disruption but did reduce the number of cytoplasmic capsids more than the number of nuclear capsids.
Assuntos
Núcleo Celular/virologia , Infecções por Citomegalovirus/enzimologia , Citomegalovirus/enzimologia , Lâmina Nuclear/virologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Liberação de Vírus/efeitos dos fármacos , Capsídeo/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Humanos , Lâmina Nuclear/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína Quinase C/metabolismo , Montagem de Vírus/efeitos dos fármacosRESUMO
Methylenecyclopropane nucleoside (MCPN) analogs are being investigated for treatment of human cytomegalovirus (HCMV) infection because of favorable preclinical data and limited ganciclovir cross-resistance. Monohydroxymethyl MCPNs bearing ether and thioether functionalities at the purine 6 position have antiviral activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) in addition to HCMV. The role of the HCMV UL97 kinase in the mechanism of action of these derivatives was examined. When tested against a kinase-inactive UL97 K355M virus, a moderate 5- to 7-fold increase in 50% effective concentration (EC50) was observed, in comparison to a 13- to 25-fold increase for either cyclopropavir or ganciclovir. Serial propagation of HCMV under two of these compounds selected for three novel UL97 mutations encoding amino acid substitutions D456N, C480R,and Y617del. When transferred to baseline laboratory HCMV strains, these mutations individually conferred resistance to all of the tested MCPNs, ganciclovir, and maribavir. However, the engineered strains also demonstrated severe growth defects and abnormal cytopathic effects similar to the kinase-inactive mutant. Expressed and purified UL97 kinase showed in vitro phosphorylation of the newly tested MCPNs. Thus, HCMV UL97 kinase is involved in the antiviral action of these MCPNs, but the in vitro selection of UL97-defective viruses suggests that their activity against more typical ganciclovir-resistant growth-competent UL97 mutants may be relatively preserved.
Assuntos
Antivirais/química , Antivirais/farmacologia , Ciclopropanos/química , Ciclopropanos/farmacologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Éter/química , Sulfetos/química , Linhagem Celular , HumanosRESUMO
Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 µM, with suppression at 50 µM reaching â¼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 µM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Nucleotidiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/antagonistas & inibidores , Aciclovir/farmacologia , Animais , Chlorocebus aethiops , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Citomegalovirus/crescimento & desenvolvimento , Replicação do DNA/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , DNA Viral/genética , DNA Viral/metabolismo , Relação Dose-Resposta a Droga , Farmacorresistência Viral/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 2/enzimologia , Herpesvirus Humano 2/crescimento & desenvolvimento , Humanos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Cultura Primária de Células , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
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-(ß-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.