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1.
Nat Commun ; 11(1): 5043, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028863

RESUMO

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Integrases/ultraestrutura , Proteína Fosfatase 2/ultraestrutura , Vírus Linfotrópico T Tipo 1 de Símios/enzimologia , Proteínas Virais/ultraestrutura , Integração Viral , Motivos de Aminoácidos/genética , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Integrases/genética , Integrases/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Paraparesia Espástica Tropical/patologia , Paraparesia Espástica Tropical/virologia , Multimerização Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Vírus Linfotrópico T Tipo 1 de Símios/genética , Imagem Individual de Molécula , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
PLoS One ; 15(9): e0238291, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32870941

RESUMO

The establishment of link between high-risk human papillomavirus (HPV) infection and occurrence of cervical cancer has resulted in development of various HPV related control strategies for the prevention of cervical cancer. The objective of the present study was to assess the cost effectiveness of various screening strategies for cervical cancer and human papilloma virus (HPV) vaccination in India. A Markov model based on societal perspective was designed to estimate the lifetime costs and consequences of screening (with either visual inspect with acetic acid (VIA), Papanicolaou test or HPV DNA test at various time intervals) in a hypothetical cohort of 30-65 years age women or vaccination among adolescent girls. Diagnostic accuracy of the screening strategies, efficacy of HPV vaccination and data on transition probabilities was based on the results of the existing meta-analyses. Primary data was collected for assessing per person cost of screening, cost of treating cervical cancer and quality of life. We found that introduction of different screening strategies leads to reduction in lifetime occurrence of cervical cancer cases caused by HPV 16/18 from 20% to 61%, and cervical cancer deaths from 28% to 70%, as compared to no screening. Among various screening strategies, screening with both VIA 5 yearly and VIA 10 yearly came out to be cost effective at 1-time per capita GDP, with VIA every 5 years providing greater health benefits as compared to VIA 10 years. Hence, screening with VIA 5 years at an incremental cost of US$ 829 (INR 54,881) per QALY gained is the recommended strategy for India. Further, with regards to HPV vaccination, it leads to 60% reduction in cancer cases and mortality caused by HPV 16/18 as compared to no vaccination. Moreover, when this vaccinated cohort of adolescent girls is also screened later in their life (with VIA every 10 years and VIA 5 years), it leads to 69%-76% reduction in cancer cases and 71%-81% reduction in cancer deaths. As compared to no vaccination and no screening, both HPV vaccination alone and vaccination plus screening (with VIA every 5 yearly and VIA 10 yearly) appears to be cost effective with ICERs in the range of US$ 86 (INR 5,693) to US$ 476 (INR 31,511) per QALY gained. In the long run, when the cohort of adolescent girls, who were immunized for HPV, reach the age of 30 years, the screening frequency using VIA should be determined based on the coverage of HPV vaccination in that cohort.


Assuntos
Análise Custo-Benefício , Neoplasias do Colo do Útero/prevenção & controle , Adulto , Idoso , DNA Viral/análise , DNA Viral/metabolismo , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Índia/epidemiologia , Cadeias de Markov , Programas de Rastreamento/economia , Pessoa de Meia-Idade , Teste de Papanicolaou/economia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Qualidade de Vida , Neoplasias do Colo do Útero/economia , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/terapia , Vacinação , Adulto Jovem
4.
PLoS Pathog ; 16(8): e1008845, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866210

RESUMO

Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Vírus Vaccinia/fisiologia , Replicação Viral/fisiologia , Células A549 , Animais , Galinhas , Citoplasma/metabolismo , Citoplasma/virologia , DNA Viral/genética , DNA Viral/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Proteínas Repressoras/genética
5.
BMC Infect Dis ; 20(1): 600, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32795251

RESUMO

BACKGROUND: BK virus-associated nephropathy (BKVAN) is a relatively common cause of renal dysfunction in the first six months after renal transplantation. It arises from reactivation of the latent and usually harmless BK virus (BK virus) due to immunosuppression and other factors including some that are unique to renal transplantation such as allograft injury. BKVAN is much rarer in non-renal solid organ transplantation, where data regarding diagnosis and management are extremely limited. CASE PRESENTATION: We report a case of a 58-year-old man found to have worsening renal dysfunction nine months after bilateral sequential lung transplantation for chronic obstructive pulmonary disease (COPD). He had required methylprednisolone for acute allograft rejection but achieved good graft function. Urine microscopy and culture and renal ultrasound were normal. BK virus PCR was positive at high levels in urine and blood. Renal biopsy subsequently confirmed BKVAN. The patient progressed to end-stage renal failure requiring haemodialysis despite reduction in immunosuppression, including switching mycophenolate for everolimus, and the administration of intravenous immunoglobulin (IVIG). CONCLUSIONS: This very rare case highlights the challenges presented by BK virus in the non-renal solid organ transplant population. Diagnosis can be difficult, especially given the heterogeneity with which BKV disease has been reported to present in such patients, and the optimal approach to management is unknown. Balancing reduction in immunosuppression against prevention of allograft rejection is delicate. Improved therapeutic options are clearly required.


Assuntos
Transplante de Pulmão , Infecções por Polyomavirus/diagnóstico , Infecções Tumorais por Vírus/diagnóstico , Vírus BK/genética , Vírus BK/isolamento & purificação , DNA Viral/metabolismo , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Imunossupressores/uso terapêutico , Transplante de Rim , Transplante de Pulmão/efeitos adversos , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Ácido Micofenólico/uso terapêutico , Infecções por Polyomavirus/virologia , Doença Pulmonar Obstrutiva Crônica/terapia , Infecções Tumorais por Vírus/virologia
6.
Sci China Life Sci ; 63(8): 1103-1129, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32617827

RESUMO

Many years of fundamental studies on viral genome packaging motors have led to fruitful applications. The double-stranded DNA (dsDNA) viruses package their genomes into preformed protein shells via nanomotors including several elegant and meticulous coaxial modules. The motor is geared by the hexameric RNA ring. An open washer displayed as hexametric string of phi29 motor ATPase has been reported. The open washer linked into a filament as a queue with left-handed chirality along the dsDNA chain. It was found that a free 5'- and 3'-dsDNA end is not required for one gp16 dimer and four monomers to assemble into the hexametric washer on dsDNA. The above studies have inspired several applications in nanotechnology and nanomedicine. These applications include: (i) studies on the precision motor channels have led to their application in the single pore sensing; (ii) investigations into the hand-in-hand integration of the hexametric pRNA ring have resulted in the emergence of the new field of RNA nanotechnology; and (iii) the studies on the motor stoichiometry of homologous multi-subunits that subsequently have inspired the discovery of new methods in highly potent drug development. This review focuses on the structure and function of the viral DNA packaging motors and describes how fundamental studies inspired various applications. Given these advantages, more nanotechnological and biomedical applications using bacteriophage motor components are expected.


Assuntos
Adenosina Trifosfatases/metabolismo , Empacotamento do DNA , DNA Viral/metabolismo , Nanoporos , Montagem de Vírus , Sequência de Bases , Genoma Viral , Conformação Molecular , Nanomedicina , Nanotecnologia , RNA Viral/metabolismo , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(32): 19507-19516, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723814

RESUMO

Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis-the epidermal growth factor receptor (EGFR) kinase-regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB-EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.


Assuntos
Núcleo Celular/metabolismo , Citomegalovirus/fisiologia , Monócitos/virologia , Proteínas do Envelope Viral/metabolismo , Núcleo Celular/virologia , Células Cultivadas , DNA Viral/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Monócitos/metabolismo , Ligação Proteica , Transdução de Sinais , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia
8.
BMC Bioinformatics ; 21(1): 233, 2020 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513098

RESUMO

BACKGROUND: The detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods. RESULTS: We have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals. CONCLUSIONS: PVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Papillomaviridae/isolamento & purificação , Interface Usuário-Computador , DNA Viral/química , DNA Viral/metabolismo , Humanos , Papillomaviridae/genética , Fluxo de Trabalho
9.
BMC Infect Dis ; 20(1): 395, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503443

RESUMO

BACKGROUND: Tanzania has a high prevalence (7.17%) of chronic hepatitis B infection. Mother to Child transmission is very common, resulting in high rate of chronic infections. Currently, there is no screening program for HBV in pregnant women. This study investigated the prevalence and risk factors for chronic HBV infection in pregnant women in a tertiary hospital in Mwanza, Tanzania. METHODS: Seven hundred and forty-three women attending antenatal care and/or delivering at the Bugando Medical Centre were enrolled. All answered a questionnaire on sociodemographic and other risk factors and were tested for HBsAg using a rapid test. In HBsAg positive mothers, maternal blood and umbilical cord blood samples collected after delivery were analyzed for serological (HBsAg, HBeAg and anti-HBe) and virologic (HBV-DNA viral load and genotype) markers. All their babies were vaccinated within 24 h of delivery. The children were followed up at 3 years of age. Data was analyzed using the Mann-Whitney U-test, independent sample T-test and logistic regression. RESULTS: Of the 743 participants, 22 (3%) were positive for HBsAg, and 2 (9%) had detectable HBe-antigen. Low condom use was the only statistically significant risk factor for chronic HBV infection (OR = 3.514, 95%CI = 1.4-8.0). Of 14 maternal blood samples genotyped, 10 (71%) were genotype A and 4 (29%) were genotype D. HBV-DNA was detected in 21/22 samples, with a median of 241 IU/ml (range: 27.4-25.9 × 107 IU/ml). Five (33%) of 15 available cord blood samples were positive for HBsAg and 10 (67%) were negative. At follow-up, one child showed chronic HBV infection characteristics, one had anti-HBs level of 7 mIU/ml and 5/7(71%) had protective anti-HBs levels (> 10 mIU/ml). CONCLUSION: This cohort of pregnant women showed a lower-intermediate prevalence of HBV of 3%. In the 3 years follow-up only 1 out of 7 children showed evidence of chronic HBV infection. The child's mother with high viral load (25.9 × 107 IU/ml), was positive for HBeAg with a high degree of sequence similarity suggesting vertical transmission. These results highlight a need for improved diagnosis and treatment of HBV infection in pregnant women in Tanzania, in order to prevent vertical transmission.


Assuntos
Hepatite B Crônica/diagnóstico , Adolescente , Adulto , Estudos Transversais , DNA Viral/genética , DNA Viral/metabolismo , Feminino , Genótipo , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Cuidado Pré-Natal , Prevalência , Tanzânia/epidemiologia , Centros de Atenção Terciária , Carga Viral , Adulto Jovem
10.
PLoS Pathog ; 16(6): e1008588, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584886

RESUMO

The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment 'late virion accumulation compartment' (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , DNA Viral/metabolismo , Vírion/metabolismo , Replicação Viral , Células A549 , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/patologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , DNA Viral/genética , Humanos , Vírion/genética
11.
PLoS One ; 15(6): e0234773, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559248

RESUMO

Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death worldwide. Patients with hepatitis B virus (HBV) pre-S mutants in liver tissues or blood have been regarded as a high-risk population for HCC development and recurrence. Detection of pre-S mutants in clinical specimens is thus important for early diagnosis and prognosis of HCC to improve patient survival. Recently, we have developed a next-generation sequencing (NGS)-based platform that can quantitatively detect pre-S mutants in patient plasma with superior sensitivity and accuracy. In this study, we compared the pre-S genotyping results from plasma by the NGS-based analysis with those from liver tissues by the immunohistochemistry (IHC)-based analysis in 30 HBV-related HCC patients. We demonstrated that the detection rate of pre-S mutants was significantly higher by NGS- than by IHC-based analysis. There was a moderate to good agreement between both analyses in detection of pre-S mutants. Compared with the IHC, the NGS-based detection of pre-S mutants in patient plasma could determine the patterns of pre-S mutants in liver tissues more efficiently in a noninvasive manner. Our data suggest that the NGS-based platform may represent a promising approach for detection of pre-S mutants as biomarkers of HBV-related HCC in clinical practice.


Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Fígado/virologia , Precursores de Proteínas/genética , Adulto , Idoso , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , DNA Viral/sangue , DNA Viral/metabolismo , Feminino , Genótipo , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/complicações , Hepatite B Crônica/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Fígado/patologia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Mutação , Precursores de Proteínas/sangue , Análise de Sequência de DNA
12.
Nat Commun ; 11(1): 3121, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561747

RESUMO

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Šresolution. The structure together with functional analyses reveal that the B56γ (B'γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses.


Assuntos
Interações Hospedeiro-Patógeno/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Integrases/metabolismo , Proteína Fosfatase 2/genética , Proteínas Virais/metabolismo , Integração Viral/genética , Microscopia Crioeletrônica , DNA Viral/metabolismo , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Humanos , Integrases/ultraestrutura , Modelos Moleculares , Mutação Puntual , Ligação Proteica/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/ultraestrutura , Proteínas Virais/ultraestrutura
13.
Proc Natl Acad Sci U S A ; 117(22): 12368-12374, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32409608

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that establishes life-long infection and increases the risk for the development of several cancers and autoimmune diseases. The mechanisms by which chronic EBV infection leads to subsequent disease remain incompletely understood. Lytic reactivation plays a central role in the development of EBV-driven cancers and may contribute to other EBV-associated diseases. Thus, the clinical use of antivirals as suppressive therapy for EBV lytic reactivation may aid efforts aimed at disease prevention. Current antivirals for EBV have shown limited clinical utility due to low potency or high toxicity, leaving open the need for potent antivirals suitable for long-term prophylaxis. In the present study, we show that tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), drugs with excellent safety profiles used clinically for HIV prevention, inhibit EBV lytic DNA replication, with respective IC50 values of 0.30 µM and 84 nM. In a cell-based assay, TAF was 35- and 24-fold and TDF was 10- and 7-fold more potent than acyclovir and penciclovir, respectively, and TAF was also twice as potent as ganciclovir. The active metabolite of tenofovir prodrugs, tenofovir-diphosphate, inhibited the incorporation of dATP into a primed DNA template by the EBV DNA polymerase in vitro. In contrast to acyclovir, treatment of cells during latency for 24 h with TAF still inhibited EBV lytic DNA replication at 72 h after drug was removed. Our results suggest that tenofovir prodrugs may be particularly effective as inhibitors of EBV lytic reactivation, and that clinical studies to address critical questions about disease prevention are warranted.


Assuntos
Antivirais/farmacologia , Replicação do DNA/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Tenofovir/farmacologia , Proteínas Virais/antagonistas & inibidores , DNA Viral/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Pró-Fármacos/farmacologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
14.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461312

RESUMO

In this study, we describe seven vegetative phage genomes homologous to the historic phage B3 that infect Pseudomonas aeruginosa Like other phage groups, the B3-like group contains conserved (core) and variable (accessory) open reading frames (ORFs) grouped at fixed regions in their genomes; however, in either case, many ORFs remain without assigned functions. We constructed lysogens of the seven B3-like phages in strain Ps33 of P. aeruginosa, a novel clinical isolate, and assayed the exclusion phenotype against a variety of temperate and virulent superinfecting phages. In addition to the classic exclusion conferred by the phage immunity repressor, the phenotype observed in B3-like lysogens suggested the presence of other exclusion genes. We set out to identify the genes responsible for this exclusion phenotype. Phage Ps56 was chosen as the study subject since it excluded numerous temperate and virulent phages. Restriction of the Ps56 genome, cloning of several fragments, and resection of the fragments that retained the exclusion phenotype allowed us to identify two core ORFs, so far without any assigned function, as responsible for a type of exclusion. Neither gene expressed separately from plasmids showed activity, but the concurrent expression of both ORFs is needed for exclusion. Our data suggest that phage adsorption occurs but that phage genome translocation to the host's cytoplasm is defective. To our knowledge, this is the first report on this type of exclusion mediated by a prophage in P. aeruginosa IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium frequently isolated from infected immunocompromised patients, and the strains are resistant to a broad spectrum of antibiotics. Recently, the use of phages has been proposed as an alternative therapy against multidrug-resistant bacteria. However, this approach may present various hurdles. This work addresses the problem that pathogenic bacteria may be lysogenized by phages carrying genes encoding resistance against secondary infections, such as those used in phage therapy. Discovering phage genes that exclude superinfecting phages not only assigns novel functions to orphan genes in databases but also provides insight into selection of the proper phages for use in phage therapy.


Assuntos
DNA Viral , Genes Virais , Fases de Leitura Aberta , Prófagos , Fagos de Pseudomonas , Pseudomonas aeruginosa , DNA Viral/genética , DNA Viral/metabolismo , Prófagos/genética , Prófagos/metabolismo , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/virologia
15.
J Med Chem ; 63(11): 6066-6089, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32421339

RESUMO

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) have been suggested to be effective anti-HBV agents in both preclinical and clinical studies. In addition to blocking HBV replication, CAMs could reduce the formation of covalently closed circular DNA (cccDNA), which accounts for the persistence of HBV infection. Here, we describe the discovery of (1H-indazole-5-yl)sulfonamides and (1H-pyrazolo[3,4-c]pyridin-5-yl)sulfonamides as new CAM chemotypes by constraining the conformation of the sulfamoylbenzamide derivatives. Lead optimization resulted in compound 56 with an EC50 value of 0.034 µM and good metabolic stability in mouse liver microsomes. To increase the solubility, the amino acid prodrug (65) and its citric acid salt (67) were prepared. Compound 67 dose dependently inhibited HBV replication in a hydrodynamic injection-based mouse model of HBV infection, while 56 did not show in vivo anti-HBV activity, likely owing to its suboptimal solubility. This class of compounds may serve as a starting point to develop novel anti-HBV drugs.


Assuntos
Antivirais/química , Capsídeo/metabolismo , Vírus da Hepatite B/fisiologia , Sulfonamidas/química , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular , DNA Viral/metabolismo , Desenho de Fármacos , Farmacorresistência Viral/efeitos dos fármacos , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Terciária de Proteína , Pirazóis/química , Piridinas/química , Solubilidade , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
17.
PLoS One ; 15(4): e0231864, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302368

RESUMO

Metagenomics is a helpful tool for the analysis of unculturable organisms and viruses. Viruses that target bacteria and archaea play important roles in the microbial diversity of various ecosystems. Here we show that Methanosarcina virus MV (MetMV), the second Methanosarcina sp. virus with a completely determined genome, is characteristic of hydrocarbon pollution in environmental (soil and water) samples. It was highly abundant in Hungarian hydrocarbon polluted samples and its genome was also present in the NCBI SRA database containing reads from hydrocarbon polluted samples collected in Canada, indicating the stability of its niche and the marker feature of this virus. MetMV, as the only currently identified marker virus for pollution in environmental samples, could contribute to the understanding of the complicated network of prokaryotes and their viruses driving the decomposition of environmental pollutants.


Assuntos
Archaea/virologia , Vírus de Archaea/isolamento & purificação , Poluição Ambiental/análise , Hidrocarbonetos/análise , Archaea/isolamento & purificação , Vírus de Archaea/genética , Canadá , DNA Viral/química , DNA Viral/metabolismo , Genoma Viral , Hungria , Microbiologia do Solo , Microbiologia da Água
18.
BMC Infect Dis ; 20(1): 295, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32316920

RESUMO

BACKGROUND: Soft tissue or skin infections due to nontuberculous mycobacteria (NTM) have been reported frequently and are mostly associated with trauma or cosmetic interventions like plastic surgery. However, infection with NTM as a result of a dental procedure have rarely been described and the lack of clinical suspicion and a clear clinical manifestation makes diagnosis challenging. CASE PRESENTATION: We report on three patients with a facial cutaneous sinus tract of dental origin, due to an infection with respectively Mycobacterium fortuitum, M. abscessus and M. peregrinum. The infection source was the dental unit waterlines (DUWLs), which were colonized with NTM. CONCLUSIONS: Water of the DUWL can pose a health risk. This report emphasizes the need for quality control and certification of water flowing through DUWLs, including the absence of NTM. Our report also shows the need for a rapid recognition of NTM infections and accurate laboratory diagnosis in order to avoid long-term ineffective antibiotic treatment.


Assuntos
Face/microbiologia , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Adolescente , Criança , DNA Viral/metabolismo , Feminino , Fungos/isolamento & purificação , Humanos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium fortuitum/genética , Mycobacterium fortuitum/isolamento & purificação , Micobactérias não Tuberculosas/genética , Microbiologia da Água , Adulto Jovem
19.
PLoS Pathog ; 16(3): e1008459, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226051

RESUMO

Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs.


Assuntos
DNA Circular/metabolismo , DNA Viral/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Modelos Biológicos , Nucleocapsídeo/metabolismo , Desenvelopamento do Vírus , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , DNA Circular/genética , DNA Viral/genética , Células HEK293 , Células Hep G2 , Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Nucleocapsídeo/genética , Fosforilação , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
20.
Nucleic Acids Res ; 48(9): 5006-5015, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32255177

RESUMO

The assembly of double-stranded DNA viruses, from phages to herpesviruses, is strongly conserved. Terminase enzymes processively excise and package monomeric genomes from a concatemeric DNA substrate. The enzymes cycle between a stable maturation complex that introduces site-specific nicks into the duplex and a dynamic motor complex that rapidly translocates DNA into a procapsid shell, fueled by ATP hydrolysis. These tightly coupled reactions are catalyzed by terminase assembled into two functionally distinct nucleoprotein complexes; the maturation complex and the packaging motor complex, respectively. We describe the effects of nucleotides on the assembly of a catalytically competent maturation complex on viral DNA, their effect on maturation complex stability and their requirement for the transition to active packaging motor complex. ATP plays a major role in regulating all of these activities and may serve as a 'nucleotide switch' that mediates transitions between the two complexes during processive genome packaging. These biological processes are recapitulated in all of the dsDNA viruses that package monomeric genomes from concatemeric DNA substrates and the nucleotide switch mechanism may have broad biological implications with respect to virus assembly mechanisms.


Assuntos
Trifosfato de Adenosina/metabolismo , Genoma Viral , Montagem de Vírus , Nucleotídeos de Adenina/metabolismo , Bacteriófago lambda/enzimologia , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Capsídeo/metabolismo , DNA Viral/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/fisiologia , Fatores Hospedeiros de Integração/fisiologia
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