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2.
J Virol ; 85(6): 2818-27, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21209112

RESUMO

Host signaling pathways play important roles in the replication of influenza virus, but their functional effects remain to be characterized at the molecular level. Here we identify two receptor tyrosine kinase inhibitors (RTKIs) of the tyrphostin class that exhibit robust antiviral activity against influenza A virus replication in cultured cells. One of these (AG879) is a selective inhibitor of the nerve growth factor receptor and human epidermal growth factor receptor 2 (TrkA/HER2) signaling; the other, tyrphostin A9 (A9), inhibits the platelet-derived growth factor receptor (PDGFR) pathway. We find that each inhibits at least three postentry steps of the influenza virus life cycle: AG879 and A9 both strongly inhibit the synthesis of all three influenza virus RNA species, block Crm1-dependent nuclear export, and also prevent the release of viral particles through a pathway that is modulated by the lipid biosynthesis enzyme farnesyl diphosphate synthase (FPPS). Tests of short hairpin RNA (shRNA) knockdown and additional small-molecule inhibitors confirmed that interventions targeting TrkA can suppress influenza virus replication. Our study suggests that host cell receptor tyrosine kinase signaling is required for maximal influenza virus RNA synthesis, viral ribonucleoprotein (vRNP) nuclear export, and virus release and that specific RTKIs hold promise as novel anti-influenza virus therapeutics.


Assuntos
Antivirais/metabolismo , Inibidores Enzimáticos/metabolismo , Vírus da Influenza A/fisiologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tirfostinas/metabolismo , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Humanos
3.
Antimicrob Agents Chemother ; 55(12): 5553-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21930873

RESUMO

We have previously reported that two receptor tyrosine kinase inhibitors (RTKIs), called AG879 and tyrphostin A9 (A9), can each block the replication of influenza A virus in cultured cells. In this study, we further characterized the in vitro antiviral efficacies and specificities of these agents. The 50% effective concentration (EC(50)) of each against influenza A was found to be in the high nanomolar range, and the selectivity index (SI = 50% cytotoxic concentration [CC(50)]/EC(50)) was determined to be >324 for AG879 and 50 for A9, indicating that therapeutically useful concentrations of each drug produce only low levels of cytotoxicity. Each compound showed efficacy against representative laboratory strains of both human influenza A (H1N1 or H3N2) and influenza B viruses. Importantly, no drug-resistant influenza virus strains emerged even after 25 viral passages in the presence of AG879, whereas viruses resistant to amantadine appeared after only 3 passages. AG879 and A9 each also exhibited potent inhibitory activity against a variety of other RNA and DNA viruses, including Sendai virus (Paramyxoviridae), herpes simplex virus (Herpesviridae), mouse hepatitis virus (Coronaviridae), and rhesus rotavirus (Reoviridae), but not against Pichinde virus (Arenaviridae). These results together suggest that RTKIs may be useful as therapeutics against viral pathogens, including but not limited to influenza, due to their high selectivity indices, low frequency of drug resistance, and broad-spectrum antiviral activities.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tirfostinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/uso terapêutico , Linhagem Celular , Embrião de Galinha , Cricetinae , Vírus de DNA/efeitos dos fármacos , Vírus de DNA/fisiologia , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Camundongos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/fisiologia , Ratos , Resultado do Tratamento , Tirfostinas/química , Tirfostinas/uso terapêutico , Células Vero
4.
J Virol ; 82(1): 229-36, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17959657

RESUMO

The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3' and 5' termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5' sites in the PA or PB1 coding regions but extended across the entire (80-base) 5' coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.


Assuntos
Genoma Viral/genética , Vírus da Influenza A/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Montagem de Vírus , Substituição de Aminoácidos , Animais , Linhagem Celular , Análise Mutacional de DNA , Cães , Etilaminas/farmacologia , Humanos , Vírus da Influenza A/genética , Mutagênese , Mutagênicos/farmacologia , Mutação de Sentido Incorreto , Mutação Puntual , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Replicação Viral
5.
Nucleic Acids Res ; 35(6): 2026-34, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17341460

RESUMO

Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7 at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA-protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.


Assuntos
Proteínas do Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , HIV-1/genética , RNA Viral/química , Proteínas Virais/metabolismo , Sequência de Bases , Dimerização , Eletroforese em Gel de Poliacrilamida , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Espectrofotometria Ultravioleta , Temperatura , Produtos do Gene gag do Vírus da Imunodeficiência Humana
6.
Acad Pathol ; 5: 2374289518765435, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29637086

RESUMO

American hospitals are increasingly turning to service outsourcing to reduce costs, including laboratory services. Studies of this practice have largely focused on nonacademic medical centers. In contrast, academic medical centers have unique practice environments and unique mission considerations. We sought to elucidate and analyze clinical laboratory outsourcing experiences in US academic medical centers. Seventeen chairs of pathology with relevant experience were willing to participate in in-depth interviews about their experiences. Anticipated financial benefits from joint venture arrangements often eroded after the initial years of the agreement, due to increased test pricing, management fees, duplication of services in support of inpatients, and lack of incentive for utilization control on the part of the for-profit partner. Outsourcing can preclude development of lucrative outreach programs; such programs were successfully launched in several cases after joint ventures were either avoided or terminated. Common complaints included poor test turnaround time and problems with test quality (especially in molecular pathology, microbiology, and flow cytometry), leading to clinician dissatisfaction. Joint ventures adversely affected retention of academically oriented clinical pathology faculty, with adverse effects on research and education, which further exacerbated clinician dissatisfaction due to lack of available consultative expertise. Resident education in pathology and in other disciplines (especially infectious disease) suffered both from lack of on-site laboratory capabilities and from lack of teaching faculty. Most joint ventures were initiated with little or no input from pathology leadership, and input from pathology leadership was seen to have been critical in those cases where such arrangements were declined or terminated.

7.
Acad Pathol ; 5: 2374289518798556, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30327790

RESUMO

Assessment of physician workloads has become increasingly important in modern academic physician practice, where it is commonly used to allocate resources among departments, to determine staffing, and to set the compensation of individual physicians. The physician work relative value unit system is a frequently used metric in this regard. However, the application of this system to the practice of pathology has proven problematic. One area of uncertainty is the validity of using work relative value unit norms that were derived from general surgical pathology practice to assess the various subspecialties within anatomic pathology. Here, we used data from the 2017 Association of Pathology Chairs practice survey to assess salary and work relative value unit data for single-subspecialty practitioners in US academic pathology departments in the prior year (2016). Five subspecialties were evaluated: dermatopathology, gastrointestinal pathology, hematopathology/hematology, renal pathology, and neuropathology. Data for general surgical pathologists and cytopathologists were included for comparison. For this analysis, survey data were available for 168 practitioners in 43 US academic departments of pathology. Salary ranges varied little among subspecialties, with the exception of dermatopathology, where salaries were higher. In contrast, work relative value unit productivity varied widely among different subspecialties, with median values differing as much as 4- to 7-fold between subspecialties. These results suggest that the use of a single overall work relative value unit standard is not appropriate for specialty- or subspecialty-based anatomic pathology practice, and that either the benchmark norms should be tailored to individual practice patterns, or an alternative system of workload measurement should be developed.

8.
Mol Cell Biol ; 23(19): 6849-56, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12972604

RESUMO

Telomerase is a cellular reverse transcriptase that uses part of its integral RNA (called TER) as the template to synthesize telomeric DNA repeats. Vertebrate TERs are thought to share a conserved, highly structured core domain that includes the templating sequence and a pseudoknot, but not all features of the predicted core structure have been verified directly or shown to affect telomerase enzymatic activity. Here, we report a systematic mutational analysis of the core domain (residues 1 to 210) of human telomerase RNA (hTER). Our data confirm that optimal hTER activity requires the integrity of four short helices (P2a.1, P2a, P2b, and P3) which create the proposed pseudoknot and that features of both the primary sequence and secondary structure in P2b and P3 contribute to optimal function. At least part of the long-range P1 pairing is also required, despite the lack of a known P1 counterpart in rodent TERs. Among the predicted single-stranded regions, we found that J2b/3, portions of J2a/3, and residues in and around the template make sequence-specific contributions to telomerase function. Additionally, we provide evidence that naturally occurring hTER sequence polymorphisms found in some patients with aplastic anemia can inhibit telomerase activity by disrupting critical structures within the hTER core domain.


Assuntos
RNA/química , RNA/metabolismo , Telomerase/química , Telomerase/metabolismo , Anemia Aplástica/enzimologia , Anemia Aplástica/genética , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Consenso , Humanos , Mutação , Polimorfismo Genético , Estrutura Terciária de Proteína , RNA/genética , Relação Estrutura-Atividade , Telomerase/genética , Moldes Genéticos
9.
Science ; 354(6309): 197-202, 2016 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-27738167

RESUMO

Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the α4ß7 integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Imunização Passiva/métodos , Integrina alfa4/imunologia , Cadeias beta de Integrinas/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Viremia/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Terapia Combinada , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Trato Gastrointestinal/imunologia , Infusões Intravenosas , Células Matadoras Naturais/imunologia , Macaca mulatta , Masculino , Glicoproteínas de Membrana/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/isolamento & purificação , Subpopulações de Linfócitos T/imunologia , Tretinoína/sangue , Proteínas do Envelope Viral/imunologia , Carga Viral/imunologia , Viremia/sangue , Viremia/tratamento farmacológico , Viremia/virologia
10.
Nat Med ; 20(12): 1397-400, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25419708

RESUMO

α4ß7 integrin-expressing CD4(+) T cells preferentially traffic to gut-associated lymphoid tissue (GALT) and have a key role in HIV and simian immunodeficiency virus (SIV) pathogenesis. We show here that the administration of an anti-α4ß7 monoclonal antibody just prior to and during acute infection protects rhesus macaques from transmission following repeated low-dose intravaginal challenges with SIVmac251. In treated animals that became infected, the GALT was significantly protected from infection and CD4(+) T cell numbers were maintained in both the blood and the GALT. Thus, targeting α4ß7 reduces mucosal transmission of SIV in macaques.


Assuntos
Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , DNA Viral/análise , Integrinas/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Tecido Linfoide/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vagina/efeitos dos fármacos , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Colo do Útero/virologia , Colo/virologia , Feminino , Íleo/virologia , Integrinas/imunologia , Mucosa Intestinal/imunologia , Jejuno/virologia , Tecido Linfoide/imunologia , Macaca mulatta , Mucosa/efeitos dos fármacos , Mucosa/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Vagina/imunologia , Carga Viral
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