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1.
J Virol ; 96(3): e0171321, 2022 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-34787453

RESUMO

Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus, which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus, the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. IMPORTANCE The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus. Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.


Assuntos
Modelos Moleculares , Conformação Proteica , RNA Polimerase Dependente de RNA/química , Vírus da Febre do Vale do Rift/enzimologia , Motivos de Aminoácidos , Domínio Catalítico , Fenômenos Químicos , Sequência Conservada , Microscopia Crioeletrônica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão , Proteínas Virais/química
2.
Antiviral Res ; 127: 79-89, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26801627

RESUMO

Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target.


Assuntos
Antivirais/farmacologia , Replicação do DNA/fisiologia , Indóis/farmacologia , Proteína Fosfatase 1/metabolismo , Vírus da Febre do Vale do Rift/enzimologia , Vírus da Febre do Vale do Rift/fisiologia , Ureia/análogos & derivados , Replicação Viral/fisiologia , Animais , Antivirais/química , Antivirais/uso terapêutico , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Genoma Viral/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 1/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Viral/biossíntese , RNA Viral/efeitos dos fármacos , Febre do Vale de Rift/tratamento farmacológico , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/efeitos dos fármacos , Vírus da Febre do Vale do Rift/genética , Ureia/farmacologia , Células Vero , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Virulência , Replicação Viral/efeitos dos fármacos
3.
Antiviral Res ; 104: 84-92, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24486952

RESUMO

Rift Valley fever is a zoonotic, arthropod-borne disease that affects livestock and humans. The etiologic agent, Rift Valley fever virus (RVFV; Bunyaviridae, Phlebovirus) is primarily transmitted through mosquito bites, but can also be transmitted by exposure to infectious aerosols. There are presently no licensed vaccines or therapeutics to prevent or treat severe RVFV infection in humans. We have previously reported on the activity of favipiravir (T-705) against the MP-12 vaccine strain of RVFV and other bunyaviruses in cell culture. In addition, efficacy has also been documented in mouse and hamster models of infection with the related Punta Toro virus. Here, hamsters challenged with the highly pathogenic ZH501 strain of RVFV were used to evaluate the activity of favipiravir against lethal infection. Subcutaneous RVFV challenge resulted in substantial serum and tissue viral loads and caused severe disease and mortality within 2-3 days of infection. Oral favipiravir (200 mg/kg/day) prevented mortality in 60% or greater of hamsters challenged with RVFV when administered within 1 or 6h post-exposure and reduced RVFV titers in serum and tissues relative to the time of treatment initiation. In contrast, although ribavirin (75 mg/kg/day) was effective at protecting animals from the peracute RVFV disease, most ultimately succumbed from a delayed-onset neurologic disease associated with high RVFV burden observed in the brain in moribund animals. When combined, T-705 and ribavirin treatment started 24 h post-infection significantly improved survival outcome and reduced serum and tissue virus titers compared to monotherapy. Our findings demonstrate significant post-RVFV exposure efficacy with favipiravir against both peracute disease and delayed-onset neuroinvasion, and suggest added benefit when combined with ribavirin.


Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Viroses do Sistema Nervoso Central/virologia , Pirazinas/farmacologia , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/efeitos dos fármacos , Vírus da Febre do Vale do Rift/enzimologia , Amidas/uso terapêutico , Animais , Antivirais/uso terapêutico , Linhagem Celular , Viroses do Sistema Nervoso Central/tratamento farmacológico , Viroses do Sistema Nervoso Central/mortalidade , Viroses do Sistema Nervoso Central/patologia , Cricetinae , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Testes de Sensibilidade Microbiana , Pirazinas/uso terapêutico , Febre do Vale de Rift/tratamento farmacológico , Febre do Vale de Rift/mortalidade , Febre do Vale de Rift/patologia , Carga Viral
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