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1.
J Virol ; 97(6): e0047523, 2023 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-37272800

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA (dsDNA) gammaherpesvirus with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5; however, ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus and cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production. IMPORTANCE Although the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the Kaposi's sarcoma-associated herpesvirus (KSHV) terminase complex is still largely uncharacterized. This complex presumably contains KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for terminal repeat (TR) cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the artificial intelligence (AI)-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.


Assuntos
Herpesvirus Humano 8 , Proteínas Virais , Humanos , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/enzimologia , Herpesvirus Humano 8/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
2.
J Virol ; 97(10): e0042623, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37830820

RESUMO

IMPORTANCE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.


Assuntos
Proteínas do Envelope de Coronavírus , SARS-CoV-2 , Humanos , COVID-19 , Lisossomos/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas do Envelope de Coronavírus/metabolismo , Motivos de Aminoácidos , Liberação de Vírus
3.
Med Mol Morphol ; 57(2): 124-135, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38393367

RESUMO

In this study, we analyzed the morphological structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells. We identified the two types of viral particles present within the vacuoles of infected cells. Using transmission electron microscopy, we observed that SARS-CoV-2 particles exhibited both low- and high-electron-density structures, which was further confirmed through three-dimensional reconstruction using electron tomography. The budding of these particles was exclusively observed within these vacuoles. Intriguingly, viral particles with low-electron-density structures were confined to vacuoles, whereas those with high-electron-density structures were found in vacuoles and on the cell membrane surface of infected cells. Notably, high-electron-density particles found within vacuoles exhibited the same morphology as those outside the infected cells. This observation suggests that the two types of viral particles identified in this study had different maturation status. Our findings provide valuable insights into the molecular details of SARS-CoV-2 particles, contributing to our understanding of the virus.


Assuntos
COVID-19 , Tomografia com Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , SARS-CoV-2 , Vacúolos , Vírion , Humanos , SARS-CoV-2/ultraestrutura , SARS-CoV-2/fisiologia , Vacúolos/ultraestrutura , Vacúolos/virologia , Vírion/ultraestrutura , COVID-19/virologia , COVID-19/patologia , Imageamento Tridimensional , Chlorocebus aethiops , Células Vero
4.
J Virol ; 96(18): e0068422, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36073924

RESUMO

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in alpha- and betaherpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring nonspherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Interestingly, ORF17 function was also necessary for TR cleavage. Thus, our data revealed ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCE In herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficient KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.


Assuntos
Capsídeo , Genoma Viral , Infecções por Herpesviridae , Herpesvirus Humano 8 , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/genética , Humanos , Dedos de Zinco
5.
Biochem Biophys Res Commun ; 637: 181-188, 2022 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-36403481

RESUMO

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular replication of the replicon genome. Through a chemical compound library screen using the Gluc reporter CHIKV replicon, we identified several compounds that suppressed CHIKV infection in Vero cells. Among the hits identified, CP-154,526, a non-peptide antagonist of the corticotropin-releasing factor receptor type-1 (CRF-R1), showed the strongest anti-CHIKV activity and inhibited CHIKV infection in Huh-7 cells. Interestingly, other CRF-R1 antagonists, R121919 and NGD 98-2, also exhibited inhibitory effects on CHIKV infection. Time-of-drug addition and virus entry assays indicated that CP-154,526 suppressed a post-entry step of infection, suggesting that CRF-R1 antagonists acted on a target in the intracellular replication process of CHIKV. Therefore, the Gluc reporter replicon system established in this study would greatly facilitate the development of antiviral drugs against CHIKV infection.


Assuntos
Arecaceae , Febre de Chikungunya , Vírus Chikungunya , Copépodes , Chlorocebus aethiops , Animais , Vírus Chikungunya/genética , Febre de Chikungunya/tratamento farmacológico , Células Vero , Hormônio Liberador da Corticotropina , Replicon/genética , Luciferases/genética , Replicação Viral
6.
Int J Mol Sci ; 23(20)2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36293480

RESUMO

Flaviviruses (the genus Flavivirus of the Flaviviridae family) include many arthropod-borne viruses, often causing life-threatening diseases in humans, such as hemorrhaging and encephalitis. Although the flaviviruses have a significant clinical impact, it has become apparent that flavivirus replication is restricted by cellular factors induced by the interferon (IFN) response, which are called IFN-stimulated genes (ISGs). SHFL (shiftless antiviral inhibitor of ribosomal frameshifting) is a novel ISG that inhibits dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) infections. Interestingly, SHFL functions as a broad-spectrum antiviral factor exhibiting suppressive activity against various types of RNA and DNA viruses. In this review, we summarize the current understanding of the molecular mechanisms by which SHFL inhibits flavivirus infection and discuss the molecular basis of the inhibitory mechanism using a predicted tertiary structure of SHFL generated by the program AlphaFold2.


Assuntos
Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Flavivirus , Infecção por Zika virus , Zika virus , Humanos , Flavivirus/fisiologia , Interferons/farmacologia , Antivirais/farmacologia , RNA , Replicação Viral
7.
Med Mol Morphol ; 55(1): 60-67, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34825978

RESUMO

SARS-CoV-2 is the cause of COVID-19. The three-dimensional morphology of viral particles existing and multiplying in infected cells has not been established by electron tomography, which is different from cryo-electron tomography using frozen samples. In this study, we establish the morphological structure of SARS-CoV-2 particles by three-dimensional reconstruction of images obtained by electron tomography and transmission electron microscopy of biological samples embedded in epoxy resin. The characteristic roots of spike structures were found to be arranged at the surface of a virion covered with an envelope. A high-electron-density structure that appears to be a nucleocapsid was observed inside the envelope of the virion on three-dimensional images reconstructed by electron tomography. The SARS-CoV-2 particles that budded in the vacuoles in the cytoplasm were morphologically identical to those found outside the cells, suggesting that mature and infectious SARS-CoV-2 particles were already produced in the vacuoles. Here, we show the three-dimensional morphological structure of SARS-CoV-2 particles reconstructed by electron tomography. To control infection, inhibition of viral release from vacuoles would be a new target in the development of prophylactic agents against SARS-CoV-2.


Assuntos
Tomografia com Microscopia Eletrônica , SARS-CoV-2 , COVID-19 , Humanos , Imageamento Tridimensional , SARS-CoV-2/ultraestrutura , Vírion/ultraestrutura
8.
Biochem Biophys Res Commun ; 581: 103-109, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34678685

RESUMO

The controlled release of medications using nanoparticle-based drug delivery carriers is a promising method to increase the efficacy of pharmacotherapy and gene therapy. One critical issue that needs to be overcome with these drug delivery carriers is their target specificity. We focused on the cell tropism of a virus to solve this issue, i.e., we attempted to apply hepatitis B virus-like particle (HBV-VLP) as a novel hepatic cell-selective carrier for medication and DNA. To prepare HBV-VLP, 293T cells were transfected with expression plasmids carrying HBV envelope surface proteins, large envelope protein (L), and small envelope protein (S). After 72 h post-transfection, VLP-containing culture supernatants were harvested, and HBV-VLP was labeled with red fluorescent dye (DiI) and was purified by sucrose gradient ultracentrifugation. An anticancer drugs (geldanamycin or doxorubicin) and GFP-expressing plasmid DNA were incorporated into HBV-VLP, and medication- and plasmid DNA-loaded VLPs were prepared. We evaluated their delivery capabilities into hepatocytes, other organ-derived cells, and hepatocytes expressing sodium taurocholate cotransporting polypeptide (NTCP), which functions as the cellular receptor for HBV by binding to HBV L protein. HBV-VLP selectively delivered both anticancer drugs and plasmid DNA not into HepG2, Huh7, and other organ cells but into HepG2 cells expressing NTCP. In summary, we developed a novel delivery nanocarrier using HBV-VLP that could be used as a hepatitis selective drug- and DNA-carrier for cancer treatment and gene therapy.


Assuntos
Partículas Artificiais Semelhantes a Vírus/metabolismo , Portadores de Fármacos , Técnicas de Transferência de Genes , Vírus da Hepatite B/química , Proteínas do Envelope Viral/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Partículas Artificiais Semelhantes a Vírus/química , Benzoquinonas/química , Benzoquinonas/farmacologia , Carbocianinas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacologia , Composição de Medicamentos/métodos , Corantes Fluorescentes/química , Expressão Gênica , Células HEK293 , Células HeLa , Células Hep G2 , Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Coloração e Rotulagem/métodos , Simportadores/genética , Simportadores/metabolismo , Proteínas do Envelope Viral/metabolismo
9.
Biochem Biophys Res Commun ; 575: 36-41, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34455219

RESUMO

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant.


Assuntos
Anti-Infecciosos/farmacologia , Desinfetantes/farmacologia , Desinfecção/métodos , SARS-CoV-2/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Água/farmacologia , Animais , Calicivirus Felino/efeitos dos fármacos , Calicivirus Felino/crescimento & desenvolvimento , Chlorocebus aethiops , Contagem de Colônia Microbiana , Eletrólise , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/crescimento & desenvolvimento , Legionella/efeitos dos fármacos , Legionella/crescimento & desenvolvimento , Camundongos , Parvovirus Canino/efeitos dos fármacos , Parvovirus Canino/crescimento & desenvolvimento , SARS-CoV-2/crescimento & desenvolvimento , Salmonella/efeitos dos fármacos , Salmonella/crescimento & desenvolvimento , Pele/efeitos dos fármacos , Células Vero , Carga Viral
10.
J Infect Chemother ; 27(7): 1068-1071, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34006453

RESUMO

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is a global pandemic caused by a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral load of SARS-CoV-2 is associated with mortality in COVID-19 patients. Measurement of viral load requires the use of reverse transcription quantitative PCR (RT-qPCR), which in turn requires advanced equipment and techniques. In this study, we aimed to evaluate the viral load measurement using reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is a simpler procedure compared to RT-qPCR. MATERIALS AND METHODS: RNA was extracted by using the QIAamp Viral RNA Mini Kit. The RT-LAMP assay was performed by using the Loopamp® 2019-SARS-CoV-2 detection reagent kit and 10-fold serial dilutions of known viral load RT-LAMP were used to measure Tt, which is the time until the turbidity exceeds the threshold. Based on the relationship between viral load and Tt, the linearity and detection sensitivity of the calibration curve were evaluated. In addition, 117 clinical specimens were measured, and RT-qPCR and RT-LAMP assay results were compared. RESULTS: The dilution linearity of the calibration curve was maintained at five orders of magnitude 1.0× 106 to 1.0 × 101 copies/µL, and was confirmed to be detectable down to 1.0 × 100 copies/µL. The limit of quantification of RNA extracted from clinical specimens using RT-LAMP correlated well with that obtained using RT-qPCR (r2 = 0.930). CONCLUSION: The findings indicate that RT-LAMP is an effective method to determine the viral load of SARS-CoV-2.


Assuntos
COVID-19 , RNA Viral , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Transcrição Reversa , SARS-CoV-2 , Sensibilidade e Especificidade
11.
Biochem Biophys Res Commun ; 530(4): 617-623, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32762941

RESUMO

cDNA expression cloning has been shown to be a powerful approach in the search for cellular factors that control virus replication. In this study, cDNA library screening using a pool of cDNA derived from interferon-treated human cells was combined with the MinION sequencer to identify cellular genes inhibiting Chikungunya virus (CHIKV) replication. Challenge infection of CHIKV to Vero cells transduced with the cDNA library produced virus-resistant cells. Then, the MinION sequence of cDNAs extracted from the surviving cells revealed that the open reading frames of TOM7, S100A16, N-terminally truncated form of ECI1 (ECI1ΔN59), and RPL29 were inserted in many of the cells. Importantly, the transient expression of TOM7, S100A16, and ECI1ΔN59 was found to inhibit the replication of CHIKV in Huh7 cells, indicating that these cellular factors were potentially anti-CHIKV molecules. Thus, our study demonstrated that cDNA expression cloning combined with the MinION sequencer allowed a rapid and comprehensive detection of cellular inhibitors against CHIKV.


Assuntos
Febre de Chikungunya/genética , Vírus Chikungunya/fisiologia , Dodecenoil-CoA Isomerase/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Proteínas S100/genética , Replicação Viral , Linhagem Celular , Febre de Chikungunya/virologia , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Mutação , Regulação para Cima
12.
Med Mol Morphol ; 52(4): 226-234, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31134430

RESUMO

Helicobacter pylori possesses intrabacterial nanotransportation system (ibNoTS) for transporting CagA, VacA, and urease within the bacterial cytoplasm, which is controlled by the extrabacterial environment. The route of ibNoTS for CagA is reported to be associated with the MreB filament, whereas the route of ibNoTS for urease is not yet known. In this study, we demonstrated by immunoelectron microscopy that urease along the route of ibNoTS localizes closely with the FtsZ filament in the bacterium. Supporting this, we found by enzyme immunoassay and co-immunoprecipitation analysis that urease interacted with FtsZ. These findings indicate that urease along the route of ibNoTS is closely associated with the FtsZ filament. Since these phenomena were not observed in ibNoTS for CagA, the route of ibNoTS for CagA is different from that of ibNoTS for urease. We propose that the route of ibNoTS for urease is associated with the FtsZ filament in H. pylori.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Helicobacter pylori/metabolismo , Humanos , Microscopia Imunoeletrônica/métodos , Urease/metabolismo
13.
PLoS Pathog ; 12(1): e1005357, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26735137

RESUMO

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.


Assuntos
Vírus da Dengue/fisiologia , Dengue/imunologia , Interferons/imunologia , Proteínas Virais/genética , Replicação Viral/imunologia , Linhagem Celular , Vírus da Dengue/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Reação em Cadeia da Polimerase , Transfecção
14.
Med Mol Morphol ; 50(2): 103-111, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28283804

RESUMO

It remains unclear why gastric disease does not develop in all cases of Helicobacter pylori infection. In this study, we analyzed whether simian virus 5 (SV5) enhanced adherence of H. pylori to adenocarcinoma epithelial cells (AGS). H. pylori in AGS (harboring SV5) and SV5-infected Vero cells, and an agglutination of H. pylori mixed with SV5 were observed by light microscopy, scanning and transmission electron microscopies. The adherent rate of H. pylori to SV5-infected Vero cells and treated with an anti-SV5 antibody was determined. H. pylori adhered to the surface of AGS cells near SV5 particles, as shown by scanning and transmission electron microscopies. The adherence of H. pylori to SV5-infected Vero cells was significantly enhanced compared with that to Vero cells. In contrast, the adherence of H. pylori to Vero cells was decreased by treatment with the anti-SV5 antibody. Agglutination of H. pylori mixed with SV5 was observed by scanning and transmission electron microscopies. Agglutination did not occur when SV5 was treated with the anti-SV5 antibody before mixing. These findings demonstrated that SV5 enhanced the adherence of H. pylori to host cells, suggesting that a persistently infected virus may be a factor enhancing the pathogenicity of H. pylori in humans.


Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Helicobacter pylori/efeitos dos fármacos , Interações Microbianas , Vírus da Parainfluenza 5/efeitos dos fármacos , Aglutinação/efeitos dos fármacos , Animais , Aderência Bacteriana/efeitos dos fármacos , Linhagem Celular Tumoral , Chlorocebus aethiops , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/patogenicidade , Helicobacter pylori/ultraestrutura , Humanos , Vírus da Parainfluenza 5/crescimento & desenvolvimento , Células Vero
15.
J Biol Chem ; 289(38): 26368-26382, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25107902

RESUMO

Integration, one of the hallmarks of retrovirus replication, is mediated by a nucleoprotein complex called the preintegration complex (PIC), in which viral DNA is associated with many protein components that are required for completion of the early phase of infection. A striking feature of the PIC is its powerful integration activity in vitro. The PICs from a freshly isolated cytoplasmic extract of infected cells are able to insert viral DNA into exogenously added target DNA in vitro. Therefore, a PIC-based in vitro assay is a reliable system for assessing protein factors influencing retroviral integration. In this study, we applied a microtiter plate-based in vitro assay to a screening study using a protein library that was produced by the wheat germ cell-free protein synthesis system. Using a library of human E3 ubiquitin ligases, we identified RFPL3 as a potential stimulator of human immunodeficiency virus, type 1 (HIV-1) PIC integration activity in vitro. This enhancement of PIC activity by RFPL3 was likely to be attributed to its N-terminal RING domain. To further understand the functional role of RFPL3 in HIV infection, we created a human cell line overexpressing RFPL3. Immunoprecipitation analysis revealed that RFPL3 was associated with the human immunodeficiency virus, type 1 PICs in infected cells. More importantly, single-round HIV-1 infection was enhanced significantly by RFPL3 expression. Our proteomic approach displays an advantage in the identification of new cellular proteins affecting the integration activity of the PIC and, therefore, contributes to the understanding of functional interaction between retroviral integration complexes and host factors.


Assuntos
Proteínas de Transporte/fisiologia , HIV-1/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Células HEK293 , Humanos , Vírus da Leucemia Murina de Moloney/fisiologia , Ligação Proteica , Titulometria , Integração Viral
16.
Biochem Biophys Res Commun ; 448(1): 95-100, 2014 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-24769207

RESUMO

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.


Assuntos
Citocinas/metabolismo , Vírus da Dengue/fisiologia , Interferon Tipo I/farmacologia , Ubiquitinas/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Cricetinae , Vírus da Dengue/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Interferência de RNA , Proteínas não Estruturais Virais/metabolismo
17.
Mar Drugs ; 12(1): 462-76, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24451189

RESUMO

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.


Assuntos
Hepacivirus/enzimologia , Naftalenos/química , Naftalenos/farmacologia , Poríferos/metabolismo , Inibidores de Serina Proteinase/farmacologia , Sesterterpenos/química , Sesterterpenos/farmacologia , Ésteres do Ácido Sulfúrico/química , Ésteres do Ácido Sulfúrico/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Elétrons , Naftalenos/isolamento & purificação , RNA Viral/metabolismo , Serina Proteases/química , Sesterterpenos/isolamento & purificação , Ésteres do Ácido Sulfúrico/isolamento & purificação
18.
Antiviral Res ; 210: 105487, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36657882

RESUMO

Chikungunya virus (CHIKV) is an enveloped RNA virus that causes Chikungunya fever (CHIKF), which is transmitted to humans through the bite of infected Aedes mosquitos. Although CHIKVF had been regarded as an endemic disease in limited regions of Africa and Asia, the recent global reemergence of CHIKV heightened awareness of this infectious disease, and CHIKV infection is currently considered an increasing threat to public health. However, no specific drug or licensed vaccine is available for CHIKV infection. As seen in other RNA virus infections, CHIKV triggers the interferon (IFN) response that plays a central role in host defense against pathogens. Experimental evidence has demonstrated that control of CHIVK replication by the IFN response is achieved by antiviral effector molecules called interferon-stimulated genes (ISGs), whose expressions are upregulated by IFN stimulation. This review details the molecular basis of the IFN-mediated suppression of CHIKV, particularly the ISGs restricting CHIKV replication.


Assuntos
Febre de Chikungunya , Vírus Chikungunya , Interferon Tipo I , Animais , Humanos , Vírus Chikungunya/genética , Antivirais/farmacologia , Antivirais/metabolismo , Replicação Viral
19.
RSC Adv ; 13(13): 8779-8793, 2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36950081

RESUMO

Membrane fusion is a critical and indispensable step in the replication cycles of viruses such as SARS-CoV-2 and human immunodeficiency virus type-1 (HIV-1). In this step, a trimer of the heptad repeat 1 (HR1) region interacts with the three HR2 regions and forms a 6-helix bundle (6-HB) structure to proceed with membrane fusion of the virus envelope and host cells. Recently, several researchers have developed potent peptidic SARS-CoV-2 fusion inhibitors based on the HR2 sequence and including some modifications. We have developed highly potent HIV-1 fusion inhibitors by dimerization of its HR2 peptides. Here, we report the development of dimerized HR2 peptides of SARS-CoV-2, which showed significantly higher antiviral activity than the corresponding monomers, suggesting that the dimerization strategy can facilitate the design of potent inhibitors of SARS-CoV-2.

20.
Front Microbiol ; 13: 866271, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35401487

RESUMO

Chikungunya virus (CHIKV), the causative agent of Chikungunya fever (CHIKVF) that is often characterized by fever, headache, rash, and arthralgia, is transmitted to humans by Aedes mosquito bites. Although the mortality rate associated with CHIKV infection is not very high, CHIKVF has been confirmed in more than 40 countries, not only in tropical but also in temperate areas. Therefore, CHIKV is a growing major threat to the public health of the world. However, a specific drug is not available for CHIKV infection. As demonstrated by many studies, the processes completing the replication of CHIKV are assisted by many host factors, whereas it has become clear that the host cell possesses some factors limiting the virus replication. This evidence will provide us with an important clue for the development of pharmacological treatment against CHIKVF. In this review, we briefly summarize cellular molecules participating in the CHIKV infection, particularly focusing on introducing recent genome-wide screen studies that enabled illuminating the virus-host interactions.

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