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1.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31896597

RESUMO

TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis.IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.


Assuntos
Adenosina Trifosfatases/metabolismo , Nucleocapsídeo/metabolismo , Nucleopoliedrovírus/fisiologia , Replicação Viral , Animais , Núcleo Celular/virologia , Citoplasma/virologia , DNA Helicases/metabolismo , DNA Viral/biossíntese , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Células Sf9/virologia , Vacúolos/virologia , Proteínas Virais/metabolismo , Vírion
2.
PLoS One ; 15(1): e0228028, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945138

RESUMO

Poxviruses replicate in cytoplasmic structures called factories and each factory begins as a single infecting particle. Sixty-years ago Cairns predicted that this might have effects on vaccinia virus (VACV) recombination because the factories would have to collide and mix their contents to permit recombination. We've since shown that factories collide irregularly and that even then the viroplasm mixes poorly. We've also observed that while intragenic recombination occurs frequently early in infection, intergenic recombination is less efficient and happens late in infection. Something inhibits factory fusion and viroplasm mixing but what is unclear. To study this, we've used optical and electron microscopy to track factory movement in co-infected cells and correlate these observations with virus development and recombinant formation. While the technical complexity of the experiments limited the number of cells that are amenable to extensive statistical analysis, these studies do show that intergenic recombination coincides with virion assembly and when VACV replication has declined to ≤10% of earlier levels. Along the boundaries between colliding factories, one sees ER membrane remnants and other cell constituents like mitochondria. These collisions don't always cause factory fusion, but when factories do fuse, they still entrain cell constituents like mitochondria and ER-wrapped microtubules. However, these materials wouldn't seem to pose much of a further barrier to DNA mixing and so it's likely that the viroplasm also presents an omnipresent impediment to DNA mixing. Late packaging reactions might help to disrupt the viroplasm, but packaging would sequester the DNA just as the replication and recombination machinery goes into decline and further reduce recombinant yields. Many factors thus appear to conspire to limit recombination between co-infecting poxviruses.


Assuntos
Replicação do DNA , DNA Viral/biossíntese , Recombinação Genética , Vírus Vaccinia , Vírion/fisiologia , Montagem de Vírus , Animais , Linhagem Celular , Citosol/imunologia , Retículo Endoplasmático/imunologia , Vírus Vaccinia/genética , Vírus Vaccinia/fisiologia
3.
BMC Infect Dis ; 19(1): 802, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31510934

RESUMO

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is a serious global health problem. Persistence of the virus occurs as a result of stability of the replication intermediate comprising covalently closed circular DNA (cccDNA). Development of drugs that are capable of disabling this cccDNA is vital. METHODS: To investigate an epigenetic approach to inactivating viral DNA, we engineered transcriptional repressors that comprise an HBV DNA-binding domain of transcription activator like effectors (TALEs) and a fused Krüppel Associated Box (KRAB). These repressor TALEs (rTALEs) targeted the viral surface open reading frame and were placed under transcription control of constitutively active or liver-specific promoters. RESULTS: Evaluation in cultured cells and following hydrodynamic injection of mice revealed that the rTALEs significantly inhibited production of markers of HBV replication without evidence of hepatotoxicity. Increased methylation of HBV DNA at CpG island II showed that the rTALEs caused intended epigenetic modification. CONCLUSIONS: Epigenetic modification of HBV DNA is a new and effective means of inactivating the virus in vivo. The approach has therapeutic potential and avoids potentially problematic unintended mutagenesis of gene editing.


Assuntos
DNA Viral/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/genética , Hepatite B/terapia , Hepatite B/virologia , Proteínas Repressoras/metabolismo , Replicação Viral/genética , Animais , Linhagem Celular , Ilhas de CpG , Metilação de DNA , DNA Circular/genética , DNA Viral/biossíntese , Epigênese Genética , Feminino , Fígado/metabolismo , Fígado/virologia , Camundongos , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética
4.
Biochem Soc Trans ; 47(4): 1013-1027, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31296733

RESUMO

Vertebrate protein SAMHD1 (sterile-α-motif and HD domain containing protein 1) regulates the cellular dNTP (2'-deoxynucleoside-5'-triphosphate) pool by catalysing the hydrolysis of dNTP into 2'-deoxynucleoside and triphosphate products. As an important regulator of cell proliferation and a key player in dNTP homeostasis, mutations to SAMHD1 are implicated in hypermutated cancers, and germline mutations are associated with Chronic Lymphocytic Leukaemia and the inflammatory disorder Aicardi-Goutières Syndrome. By limiting the supply of dNTPs for viral DNA synthesis, SAMHD1 also restricts the replication of several retroviruses, such as HIV-1, and some DNA viruses in dendritic and myeloid lineage cells and resting T-cells. SAMHD1 activity is regulated throughout the cell cycle, both at the level of protein expression and post-translationally, through phosphorylation. In addition, allosteric regulation further fine-tunes the catalytic activity of SAMHD1, with a nucleotide-activated homotetramer as the catalytically active form of the protein. In cells, GTP and dATP are the likely physiological activators of two adjacent allosteric sites, AL1 (GTP) and AL2 (dATP), that bridge monomer-monomer interfaces to stabilise the protein homotetramer. This review summarises the extensive X-ray crystallographic, biophysical and molecular dynamics experiments that have elucidated important features of allosteric regulation in SAMHD1. We present a comprehensive mechanism detailing the structural and protein dynamics components of the allosteric coupling between nucleotide-induced tetramerization and the catalysis of dNTP hydrolysis by SAMHD1.


Assuntos
Antivirais , Proteína 1 com Domínio SAM e Domínio HD/fisiologia , Replicação Viral/fisiologia , Regulação Alostérica , Catálise , Proliferação de Células/fisiologia , DNA Viral/biossíntese , Homeostase , Humanos , Mutação , Nucleotídeos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
5.
Science ; 364(6438): 399-402, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31023926

RESUMO

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.


Assuntos
Vírus da Hepatite B/crescimento & desenvolvimento , Hepatócitos/fisiologia , Hepatócitos/virologia , Cultura Primária de Células/métodos , Cultura de Vírus/métodos , Antivirais/isolamento & purificação , Antivirais/farmacologia , DNA Circular/biossíntese , DNA Circular/isolamento & purificação , DNA Viral/biossíntese , DNA Viral/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Transcriptoma , Vírion/efeitos dos fármacos , Vírion/crescimento & desenvolvimento
6.
J Virol ; 93(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30814291

RESUMO

Chromatin-based modifications of herpesviral genomes play a crucial role in dictating the outcome of infection. Consistent with this, host cell multiprotein complexes, such as polycomb repressive complexes (PRCs), were proposed to act as epigenetic regulators of herpesviral latency. In particular, PRC2 has recently been shown to contribute to the silencing of human cytomegalovirus (HCMV) genomes. Here, we identify a novel proviral role of PRC1 and PRC2, the two main polycomb repressive complexes, during productive HCMV infection. Western blot analyses revealed strong HCMV-mediated upregulation of RING finger protein 1B (RING1B) and B lymphoma Moloney murine leukemia virus insertion region 1 homolog (BMI1) as well as of enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED), which constitute the core components of PRC1 and PRC2, respectively. Furthermore, we observed a relocalization of PRC components to viral replication compartments, whereas histone modifications conferred by the respective PRCs were specifically excluded from these sites. Depletion of individual PRC1/PRC2 proteins by RNA interference resulted in a significant reduction of newly synthesized viral genomes and, in consequence, a decreased release of viral particles. Furthermore, accelerated native isolation of protein on nascent DNA (aniPOND) revealed a physical association of EZH2 and BMI1 with nascent HCMV DNA, suggesting a direct contribution of PRC proteins to viral DNA replication. Strikingly, substances solely inhibiting the enzymatic activity of PRC1/2 did not exert antiviral effects, while drugs affecting the abundance of PRC core components strongly compromised HCMV genome synthesis and particle release. Taken together, our data reveal an enzymatically independent, noncanonical function of both PRC1 and PRC2 during HCMV DNA replication, which may serve as a novel cellular target for antiviral therapy.IMPORTANCE Polycomb group (PcG) proteins are primarily known as transcriptional repressors that modify chromatin and contribute to the establishment and maintenance of cell fates. Furthermore, emerging evidence indicates that overexpression of PcG proteins in various types of cancers contributes to the dysregulation of cellular proliferation. Consequently, several inhibitors targeting PcG proteins are presently undergoing preclinical and clinical evaluation. Here, we show that infection with human cytomegalovirus also induces a strong upregulation of several PcG proteins. Our data suggest that viral DNA replication depends on a noncanonical function of polycomb repressor complexes which is independent of the so-far-described enzymatic activities of individual PcG factors. Importantly, we observe that a subclass of inhibitory drugs that affect the abundance of PcG proteins strongly interferes with viral replication. This principle may serve as a novel promising target for antiviral treatment.


Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Replicação do DNA , DNA Viral/biossíntese , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Replicação Viral , Células Cultivadas , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/terapia , DNA Viral/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética
7.
Virus Genes ; 55(3): 274-279, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30767118

RESUMO

The temporal coordination of viral gene expression is imperative for the regulation of the herpesvirus replication cycle. While the main factors of this transcriptional coordination are known, the subtler control mechanisms of gene expression remain elusive. Recent long read sequencing-based approached have revealed an intricate meshwork of overlaps between the herpesvirus transcripts and the overlap of the replication origins with noncoding RNAs. It has been shown that the transcriptional apparatuses can physically interfere with one another while transcribing overlapping regions. We hypothesize that transcriptional interference regulates the global gene expression across the herpesvirus genome. Additionally, an overall decrease in transcriptional activity in individual viral genes has been observed following the onset of DNA replication. An overlap of the replication origins with specific transcripts has also been described in several herpesviruses. The genome-wide interactions between the transcriptional apparatuses and between the replication and transcriptional machineries suggest the existence of novel layers of genetic regulation.


Assuntos
DNA Viral/biossíntese , Herpesviridae/genética , RNA Viral/biossíntese , Origem de Replicação/genética , Replicação do DNA/genética , DNA Viral/genética , Regulação Viral da Expressão Gênica , Redes Reguladoras de Genes/genética , Genoma Viral/genética , RNA Viral/genética
8.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30700606

RESUMO

The Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication in cis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.


Assuntos
Epigênese Genética/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Herpesvirus Humano 4/fisiologia , Histonas/metabolismo , Transcrição Genética/fisiologia , Replicação Viral/fisiologia , Replicação do DNA/fisiologia , DNA Viral/biossíntese , Humanos
9.
Arch Dis Child Fetal Neonatal Ed ; 104(6): F604-F608, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30728181

RESUMO

OBJECTIVES: Breast milk (BM) is the primary source of cytomegalovirus (CMV) transmission to premature infants with potentially harmful consequences. We therefore wanted to evaluate temperature and duration of short-term BM pasteurisation with respect to CMV inactivation, effect on CMV-IgG antibodies and BM enzyme activities. METHODS: 116 artificially CMV-spiked BM and 15 wild-type virus-infected samples were subjected for 5 s to different temperatures (55°C-72°C). CMV-IE-1 expression in fibroblast nuclei was assessed using the milk whey fraction in short-term microculture. BM lipase and alkaline phosphatase (AP) activities and CMV binding using CMV-recomLine immunoblotting and neutralising antibodies using epithelial target cells were analysed before and after heating. RESULTS: A minimum of 5 s above 60°C was necessary for CMV inactivation in both CMV-AD-169 spiked and wild-type infected BM. Lipase was very heat sensitive (activities of 54% at 55°C, 5% at 60°C and 2% at 65°C). AP showed activities of 77%, 88% and 10%, respectively. CMV-p150 IgG antibodies were mostly preserved at 62°C for 5 s. CONCLUSION: Our results show that short-term pasteurisation of BM at 62°C for 5 s might be efficient for CMV inactivation and reduces loss of enzyme activities, as well as CMV binding, and functional CMV antibodies.


Assuntos
Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/transmissão , Temperatura Alta , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Leite Humano/virologia , Fosfatase Alcalina/metabolismo , Anticorpos Antivirais/imunologia , Aleitamento Materno , Citomegalovirus , Infecções por Citomegalovirus/imunologia , DNA Viral/biossíntese , Humanos , Lipase/metabolismo , Leite Humano/enzimologia
10.
Proc Natl Acad Sci U S A ; 116(3): 1033-1042, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598436

RESUMO

Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase-primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.


Assuntos
Replicação do DNA/fisiologia , DNA Viral/biossíntese , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 1/fisiologia , Recombinação Genética/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Mutação , Mutação de Sentido Incorreto , Células Vero , Proteínas Virais/genética
11.
Mol Biol Cell ; 30(1): 146-159, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30403545

RESUMO

Using a mixture of 10 purified DNA replication and DNA recombination proteins encoded by the bacteriophage T4 genome, plus two homologous DNA molecules, we have reconstituted the genetic recombination-initiated pathway that initiates DNA replication forks at late times of T4 bacteriophage infection. Inside the cell, this recombination-dependent replication (RDR) is needed to produce the long concatemeric T4 DNA molecules that serve as substrates for packaging the shorter, genome-sized viral DNA into phage heads. The five T4 proteins that catalyze DNA synthesis on the leading strand, plus the proteins required for lagging-strand DNA synthesis, are essential for the reaction, as are a special mediator protein (gp59) and a Rad51/RecA analogue (the T4 UvsX strand-exchange protein). Related forms of RDR are widespread in living organisms-for example, they play critical roles in the homologous recombination events that can restore broken ends of the DNA double helix, restart broken DNA replication forks, and cross over chromatids during meiosis in eukaryotes. Those processes are considerably more complex, and the results presented here should be informative for dissecting their detailed mechanisms.


Assuntos
Bacteriófago T4/genética , Replicação do DNA , DNA Viral/biossíntese , Modelos Biológicos , Recombinação Genética , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/ultraestrutura , DNA Viral/ultraestrutura , Moldes Genéticos , Proteínas Virais/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-30397065

RESUMO

Human adenovirus (AdV) can cause fatal disease in immune-suppressed individuals, but treatment options are limited, in part because the antiviral cytidine analog cidofovir (CDV) is nephrotoxic. The investigational agent brincidofovir (BCV) is orally bioavailable, nonnephrotoxic, and generates the same active metabolite, cidofovir diphosphate (CDVpp). However, its mechanism of action against AdV is poorly understood. Therefore, we have examined the effect of CDVpp on DNA synthesis by a purified adenovirus 5 (AdV5) DNA polymerase (Pol). CDVpp was incorporated into nascent DNA strands and promoted a nonobligate form of chain termination (i.e., AdV5 Pol can extend, albeit inefficiently, a DNA chain even after the incorporation of a first CDVpp molecule). Moreover, unlike a conventional mismatched base pair, misincorporated CDVpp was not readily excised by the AdV5 Pol. At elevated concentrations, CDVpp inhibited AdV5 Pol in a manner consistent with both chain termination and direct inhibition of Pol activity. Finally, a recombinant AdV5 was constructed, containing Pol mutations (V303I and T87I) that were selected following an extended passage of wild-type AdV5 in the presence of BCV. This virus had a 2.1-fold elevated 50% effective concentration (EC50) for BCV and a 1.9-fold increased EC50 for CDV; thus, these results confirmed that viral resistance to BCV and CDV can be attributed to mutations in the viral Pol. These findings show that the anti-AdV5 activity of CDV and BCV is mediated through the viral DNA Pol and that their antiviral activity may occur via both (nonobligate) chain termination and (at high concentration) direct inhibition of AdV5 Pol activity.


Assuntos
Adenovírus Humanos/efeitos dos fármacos , Antivirais/farmacologia , Cidofovir/farmacologia , Citosina/análogos & derivados , DNA Viral/antagonistas & inibidores , DNA Polimerase Dirigida por DNA/genética , Organofosfonatos/farmacologia , Proteínas Virais/genética , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/enzimologia , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Citosina/metabolismo , Citosina/farmacologia , Primers do DNA/síntese química , Primers do DNA/genética , DNA Viral/biossíntese , DNA Viral/genética , DNA Polimerase Dirigida por DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Cinética , Mutação , Organofosfonatos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
13.
Antiviral Res ; 162: 30-38, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30543830

RESUMO

Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and reduces the risk of cytomegalovirus (CMV) infection in transplant recipients. Everolimus inhibits mTOR complex 1, which regulates factors involved in several crucial cellular functions and is required for CMV replication. However, it is not clear how everolimus regulates CMV replication and prevents and alleviates CMV infection. Effects of everolimus on CMV infection, spread, and DNA synthesis and release from infected cells were assessed by plaque formation, infectious centre assay, real-time PCR of infected cells, and culture supernatant in CMV-infected cultures with and without everolimus. Everolimus enhanced plaque formation by 3.6 times, but the size of the plaques was reduced to 36.4% of untreated cultures in the absence of a pretreatment period. Everolimus reduced viral adsorption but enhanced the replication efficiency of inoculated virus, resulting in an increase in plaque number in the early phase of infection. Preinfection treatment of cells with everolimus efficiently exhibited its antiviral efficacy, and everolimus delayed and suppressed viral DNA synthesis and release from infected cells. Everolimus had suppressed the spread of infection and reduced the number of total infected cells to 40% of untreated cells on day 9, indicating reduction of the size of CMV lesions to one-sixth in 2-3 replication cycles. Preinfection treatment of the cells with everolimus augmented its suppressive effect on CMV infection and replication. Everolimus reduced the total number of infected cells and limited the CMV lesions, and this reduction in the spread of CMV infection would alleviate CMV infection in transplant recipients.


Assuntos
Antivirais/farmacologia , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Everolimo/farmacologia , Replicação Viral/efeitos dos fármacos , Células Cultivadas , Citomegalovirus/fisiologia , DNA Viral/biossíntese , Humanos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaio de Placa Viral
14.
Nature ; 564(7735): 283-286, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30518855

RESUMO

The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.


Assuntos
Bacteriófagos/efeitos dos fármacos , Bacteriófagos/genética , Metabolismo Secundário , Streptomyces/química , Streptomyces/virologia , Replicação Viral/efeitos dos fármacos , Bacteriófago lambda/efeitos dos fármacos , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/fisiologia , Bacteriófagos/crescimento & desenvolvimento , Evolução Biológica , DNA Viral/biossíntese , DNA Viral/genética , Daunorrubicina/farmacologia , Escherichia coli/virologia , Pseudomonas aeruginosa/virologia , Streptomyces/metabolismo
15.
Antiviral Res ; 160: 126-136, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30393013

RESUMO

A small molecule chemical inhibitor CGP57380 that blocks activation of MAPK interacting kinase 1 (MNK1) was found to significantly suppress buffalopox virus (BPXV) replication. BPXV infection was shown to induce MNK1 activation. Depletion of MNK1 by small interfering RNA (siRNA), blocking activation of extracellular regulated kinase (ERK, an upstream activator of MNK1) and disruption of eIF4E/eIF4G interaction (downstream substrate of MNK1 which plays a central role in cap-dependent translation initiation), resulted in reduced BPXV replication, suggesting that ERK/MNK1/eIF4E signaling is a prerequisite for BPXV replication. With the help of time-of-addition and virus step-specific assays, CGP57380 treatment was shown to decrease synthesis of viral genome (DNA). Disruption of ERK/MNK1/eIF4E signaling resulted in reduced synthesis of viral proteins, suggesting that BPXV utilizes cap-dependent mechanism of translation initiation. Therefore, we concluded that decreased synthesis of viral genome in presence of MNK1 inhibitor is the result of reduced synthesis of viral proteins. Furthermore, BPXV was sequentially passaged (P = 40) in presence of CGP57380 or vehicle control (DMSO). As compared to P0 and P40-control viruses, P40-CGP57380 virus replicated at significantly higher (∼10-fold) titers in presence of CGP57380, although a complete resistance could not be achieved. In a BPXV egg infection model, CGP57380 was found to prevent development of pock lesions on chorioallantoic membrane (CAM) as well as associated mortality of the embryonated chicken eggs. We for the first time demonstrated in vitro and in ovo antiviral efficacy of CGP57380 against BPXV and identified that ERK/MNK1 signaling is a prerequisite for synthesis of viral proteins. Our study also describes a rare report about generation of drug-resistant viral variants against a host-targeting antiviral agent.


Assuntos
Compostos de Anilina/farmacologia , Antivirais/farmacologia , DNA Viral/biossíntese , Inibidores de Proteases/farmacologia , Purinas/farmacologia , Vírus Vaccinia/efeitos dos fármacos , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacos , Animais , Embrião de Galinha , Galinhas , Membrana Corioalantoide/patologia , Farmacorresistência Viral , Óvulo/virologia , Inoculações Seriadas , Vírus Vaccinia/fisiologia , Carga Viral
17.
Artigo em Inglês | MEDLINE | ID: mdl-30224531

RESUMO

The capsid of the hepatitis B virus is an attractive antiviral target for developing therapies against chronic hepatitis B infection. Currently available core protein allosteric modulators (CpAMs) mainly affect one of the two major types of protein-protein interactions involved in the process of capsid assembly, namely, the interaction between the core dimers. Compounds targeting the interaction between two core monomers have not been rigorously screened due to the lack of screening models. We report here a cell-based assay in which the formation of core dimers is indicated by split luciferase complementation (SLC). Making use of this model, 2 compounds, Arbidol (umifenovir) and 20-deoxyingenol, were identified from a library containing 672 compounds as core dimerization regulators. Arbidol and 20-deoxyingenol inhibit the hepatitis B virus (HBV) DNA replication in vitro by decreasing and increasing the formation of core dimer and capsid, respectively. Our results provided a proof of concept for the cell model to be used to screen new agents targeting the step of core dimer and capsid formation.


Assuntos
Antivirais/farmacologia , Diterpenos/farmacologia , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/efeitos dos fármacos , Indóis/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas do Core Viral/antagonistas & inibidores , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , DNA Viral/biossíntese , DNA Viral/genética , Genes Reporter , Células HEK293 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Ensaios de Triagem em Larga Escala , Humanos , Luciferases/genética , Luciferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
18.
Antiviral Res ; 159: 55-62, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30266338

RESUMO

Pseudorabies virus (PRV) is an important viral pathogen of pigs that causes huge losses in pig herds worldwide. Ivermectin is a specific inhibitor of importin-α/ß-dependent nuclear transport and shows antiviral potential against several RNA viruses by blocking the nuclear localization of viral proteins. Since the replication of DNA viruses is in the nucleus, ivermectin may be functional against DNA virus infections if the DNA polymerase or other important viral proteins enter the nucleus via the importin-α/ß-mediated pathway. Here, we determined whether ivermectin suppresses PRV replication in hamster kidney BHK-21 cells and investigated the effect of ivermectin on the subcellular localization of the PRV UL42 protein, the accessory subunit of PRV DNA polymerase. Also, an in vivo anti-PRV assay was conducted in mice. Our data demonstrate that ivermectin treatment inhibits PRV infection in cells in a dose-dependent manner. Treatment of PRV-infected cells with ivermectin significantly suppressed viral DNA synthesis and progeny virus production. Ivermectin disrupted the nuclear localization of UL42 by targeting the nuclear localization signal of the protein in transfected cells. Ivermectin treatment increased the survival rates of mice infected with PRV and relieved infection as indicated by lower clinical scores and fewer gross lesions in the brain. Together, our results suggest that ivermectin may be a therapeutic or preventative agent against PRV infection.


Assuntos
Núcleo Celular/virologia , Herpesvirus Suídeo 1/efeitos dos fármacos , Ivermectina/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Cricetinae , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Virais/genética
19.
J Gen Virol ; 99(10): 1432-1437, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30176164

RESUMO

Herpesviruses, including herpes simplex virus-1, encode and express a DNA polymerase that is required for replication of their dsDNA genomes. The catalytic subunit of this enzyme contains a 3'-5' exonuclease that is involved in proofreading during replication. Although certain mutations that severely impair exonuclease activity are not lethal to the virus, it was reported that virus containing the substitution of alanine for aspartate 368 (D368A), which ablates exonuclease activity, could not be recovered, raising the possibility that this activity is essential for viral replication. To investigate this issue, we produced virus containing this mutation (D368A Pol) using a complementing cell line. D368A Pol virus was unable to form plaques on non-complementing cells. Viral DNA synthesis and polymerase activity were severely inhibited in D368A-infected cells, as was expression of the enzyme, suggesting that effects on polymerase expression rather than on exonuclease activity per se largely explain the lethal phenotype of this mutation.


Assuntos
DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/deficiência , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Proteínas Mutantes/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Substituição de Aminoácidos , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Expressão Gênica , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Ensaio de Placa Viral , Proteínas Virais/genética
20.
J Virol ; 92(21)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30111572

RESUMO

Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT, prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in wild-type hepatitis B virus (HBV WT)-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient-mutant-transfected cells and 1.3-mer HBV WT-transfected and reverse transcriptase inhibitor (entecavir or lamivudine)-treated cells, but all HBV proteins were expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase 3ß (GSK-3ß) and increased ß-catenin levels; however, downregulation of Sirt2 in HBV-nonreplicating cells impaired AKT/GSK-3ß/ß-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activated AKT and consequently increased ß-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by short hairpin RNAs (shRNAs), inhibition by 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2), or dominant negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased ß-catenin levels. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates ß-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to the development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC.IMPORTANCE Even though Sirt2, a NAD+-dependent protein deacetylase, is overexpressed in many HCCs, and overexpressed Sirt2 promotes hepatic fibrosis and associates positively with vascular invasion by primary HCCs through AKT/GSK-3ß/ß-catenin signaling, the relationship between Sirt2, HBV, HBx, and/or HBV-associated hepatocarcinogenesis is unclear. Here, we show that HBV DNA replication, not HBV expression, correlates positively with Sirt2 upregulation and AKT activation. We demonstrate that overexpression of Sirt2 further increases HBV replication, increases AKT activation, downregulates GSK-3ß, and increases ß-catenin levels. Conversely, inhibiting Sirt2 decreases HBV replication, reduces AKT activation, and decreases ß-catenin levels. Although HBx activates AKT to upregulate ß-catenin, Sirt2-mediated effects were not dependent on HBx. The results also indicate that a Sirt2 inhibitor may control HBV infection and prevent the development of hepatic fibrosis and HCC.


Assuntos
DNA Viral/biossíntese , Glicogênio Sintase Quinase 3 beta/metabolismo , Vírus da Hepatite B/genética , Hepatite B/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Viral/genética , Sirtuína 2/metabolismo , beta Catenina/metabolismo , DNA Viral/genética , Glicogênio Sintase Quinase 3 beta/genética , Células HEK293 , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Humanos , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Sirtuína 2/genética , Transcrição Genética , Ativação Transcricional , Replicação Viral , beta Catenina/genética
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