RESUMO
Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes. We previously identified double membrane vesicles (DMVs) in the cytoplasm of cells infected with Berne virus (BEV), the prototype member of the Torovirus genus (Nidovirales Order). Our previous analysis by transmission electron microscopy suggested that the DMVs form a reticulovesicular network (RVN) analogous those described for the related severe acute respiratory syndrome coronavirus (SARS-CoV-1). Here, we used serial sectioning and electron tomography to characterize the architecture of torovirus replication organelles, and to learn about their biogenesis and dynamics during the infection. The formation of a RVN in BEV infected cells was confirmed, where the outer membranes of the DMVs are interconnected with each other and with the ER. Paired or zippered ER membranes connected with the DMVs were also observed, and likely represent early structures that evolve to give rise to DMVs. Also, paired membranes forming small spherule-like invaginations were observed at late time post-infection. Although resembling in size, the tomographic analysis show that these structures are clearly different from the true spherules described previously for coronaviruses. Hence, BEV shows important similarities, but also some differences, in the architecture of the replication organelles with other nidoviruses.
Assuntos
Torovirus , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático , Replicação ViralRESUMO
Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or antivirals to treat ZIKV infections, which underscores an urgent medical need for the development of disease intervention strategies to treat ZIKV infection and associated disease. Drug repurposing offers various advantages over developing an entirely new drug by significantly reducing the timeline and resources required to advance a candidate antiviral into the clinic. Screening the ReFRAME library, we identified ten compounds with antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV). Moreover, we showed the ability of these ten compounds to inhibit influenza A and B virus infections, supporting their broad-spectrum antiviral activity. In this study, we further evaluated the broad-spectrum antiviral activity of the ten identified compounds by testing their activity against ZIKV. Among the ten compounds, Azaribine (SI-MTT = 146.29), AVN-944 (SI-MTT = 278.16), and Brequinar (SI-MTT = 157.42) showed potent anti-ZIKV activity in post-treatment therapeutic conditions. We also observed potent anti-ZIKV activity for Mycophenolate mofetil (SI-MTT = 20.51), Mycophenolic acid (SI-MTT = 36.33), and AVN-944 (SI-MTT = 24.51) in pre-treatment prophylactic conditions and potent co-treatment inhibitory activity for Obatoclax (SI-MTT = 60.58), Azaribine (SI-MTT = 91.51), and Mycophenolate mofetil (SI-MTT = 73.26) in co-treatment conditions. Importantly, the inhibitory effect of these compounds was strain independent, as they similarly inhibited ZIKV strains from both African and Asian/American lineages. Our results support the broad-spectrum antiviral activity of these ten compounds and suggest their use for the development of antiviral treatment options of ZIKV infection.
Assuntos
Antivirais/farmacologia , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Apoptose/efeitos dos fármacos , Azauridina/análogos & derivados , Azauridina/farmacologia , Compostos de Bifenilo/farmacologia , Carbamatos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Reposicionamento de Medicamentos , Síndrome de Guillain-Barré , Humanos , Microcefalia , Compostos de Fenilureia/farmacologia , Uganda , Células Vero , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/virologiaRESUMO
Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC50) = 3.80 nM to 1.73 µM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC50 = 0.14 µM to 0.55 µM; SI-MTT = 70.12 to >357.14) or cotreatment (EC50 = 34.69 nM to 7.52 µM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections.IMPORTANCE Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Células A549 , Animais , Proliferação de Células , Cães , Avaliação Pré-Clínica de Medicamentos , Genoma Viral , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos da radiação , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus da Influenza B/fisiologia , Células Madin Darby de Rim Canino , Replicação Viral/efeitos dos fármacosRESUMO
Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has been known to circulate for decades causing mild febrile illness. The more recent ZIKV outbreaks in the Americas and the Caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. In this study we demonstrate that direct delivery in mice of an infectious ZIKV cDNA clone allows the rescue of recombinant (r)ZIKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. The rZIKV recovered from these mice remained fully virulent in a second passage in mice. Interestingly, infectious rZIKV was also recovered after intraperitoneal inoculation of the rZIKV cDNA in the absence of transfection reagent. Further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDNA clone encoding an attenuated rZIKV was safe, highly immunogenic, and provided full protection against lethal ZIKV challenge. This novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and live-attenuated ZIKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.
Assuntos
Cromossomos Artificiais Bacterianos/genética , DNA Complementar/genética , Vetores Genéticos/administração & dosagem , Viremia/prevenção & controle , Infecção por Zika virus/prevenção & controle , Zika virus/genética , Animais , Chlorocebus aethiops , DNA Complementar/imunologia , DNA Viral/genética , DNA Viral/imunologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Receptor de Interferon alfa e beta/genética , Genética Reversa , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Viremia/genética , Viremia/imunologia , Zika virus/imunologia , Infecção por Zika virus/genética , Infecção por Zika virus/imunologiaRESUMO
Zika virus (ZIKV) infection is currently one of the major concerns in human public health due to its association with neurological disorders. Intensive effort has been implemented for the treatment of ZIKV, however there are not currently approved vaccines or antivirals available to combat ZIKV infection. In this sense, the identification of virulence factors associated with changes in ZIKV virulence could help to develop safe and effective countermeasures to treat ZIKV or to prevent future outbreaks. Here, we have compared the virulence of two related ZIKV strains from the recent outbreak in Brazil (2015), Rio Grande do Norte Natal (RGN) and Paraiba. In spite of both viruses being identified in the same period of time and region, significant differences in virulence and replication were observed using a validated mouse model of ZIKV infection. While ZIKV-RGN has a 50% mouse lethal dose (MLD50) of ~105 focus forming units (FFUs), ZIKV-Paraiba infection resulted in 100% of lethality with less than 10 FFUs. Combining deep-sequencing analysis and our previously described infectious ZIKV-RGN cDNA clone, we identified a natural polymorphism in the non-structural protein 2 A (NS2A) that increase the virulence of ZIKV. Moreover, results demonstrate that the single amino acid alanine to valine substitution at position 117 (A117V) in the NS2A was sufficient to convert the attenuated rZIKV-RGN in a virulent Paraiba-like virus (MLD50 < 10 FFU). The mechanism of action was also evaluated and data indicate that substitution A117V in ZIKV NS2A protein reduces host innate immune responses and viral-induced apoptosis in vitro. Therefore, amino acid substitution A117V in ZIKV NS2A could be used as a genetic risk-assessment marker for future ZIKV outbreaks.
Assuntos
Polimorfismo Genético , Proteínas não Estruturais Virais/genética , Infecção por Zika virus/virologia , Zika virus/fisiologia , Substituição de Aminoácidos , Animais , Apoptose , Linhagem Celular , Modelos Animais de Doenças , Feminino , Genoma Viral , Genômica/métodos , Interações Hospedeiro-Patógeno , Imunidade Inata , Camundongos , Virulência/genética , Replicação Viral , Infecção por Zika virus/imunologiaRESUMO
Although their origin, nature and structure are not identical, a common feature of positive-strand RNA viruses is their ability to subvert host lipids and intracellular membranes to generate replication and assembly complexes. Recently, lipin1, a cellular enzyme that converts phosphatidic acid into diacylglycerol, has been implicated in the formation of the membranous web that hosts hepatitis C virus (HCV) replicase. In the liver, lipin1 cooperates with lipin2 to maintain glycerolipid homeostasis. We extended our previous study of the lipin family on HCV infection, by determining the impact of the lipin2 silencing on viral replication. Our data reveal that lipin2 silencing interferes with HCV virion secretion at late stages of the infection, without significantly affecting viral replication or assembly. Moreover, uninfected lipin2-, but not lipin1-deficient cells display alterations in mitochondrial and Golgi apparatus morphology, suggesting that lipin2 contributes to the maintenance of the overall organelle architecture. Finally, our data suggest a broader function of lipin2 for replication of HCV and other RNA viruses, in contrast with the specific impact of lipin1 silencing on HCV replication. Overall, this study reveals distinctive functions of lipin1 and lipin2 in cells of hepatic origin, a context in which they are often considered functionally redundant.
Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Proteínas Nucleares/genética , Fosfatidato Fosfatase/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Glicerofosfolipídeos/metabolismo , Hepatite C/genética , Hepatite C/metabolismo , Humanos , Metabolismo dos Lipídeos , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Transporte Proteico , Replicação ViralRESUMO
Autophagy is a conserved eukaryotic process that mediates lysosomal degradation of cytoplasmic macromolecules and damaged organelles, also exerting an important role in the elimination of intracellular pathogens. Despite the antiviral role of autophagy, many studies suggest that some positive-stranded RNA viruses exploit this pathway to facilitate their own replication. In this study, we demonstrate that the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae Family, Nidovirales Order), induces autophagy at late times post-infection. Conversion of microtubule associated protein 1B light chain 3 (LC3) from cytosolic (LC3 I) to the membrane associated form (LC3 II), a canonical marker of autophagosome formation, is enhanced in BEV infected cells. However, neither autophagy induction, via starvation, nor pharmacological blockade significantly affect BEV replication. Similarly, BEV infection is not altered in autophagy deficient cells lacking either Beclin 1 or LC3B protein expression. Unexpectedly, the cargo receptor p62, a selective autophagy receptor, aggregates within the region where the BEV main protease (Mpro) localizes. This finding, coupled with observation that BEV replication also induces ER stress at the time when selective autophagy is taking place, suggests that the autophagy pathway is activated in response to the hefty accumulation of virus-encoded polypeptides during the late phase of BEV infection.
Assuntos
Autofagia , Infecções por Torovirus/virologia , Torovirus/fisiologia , Replicação Viral , Animais , Autofagossomos , Proteína Beclina-1/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Cavalos , Humanos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Transdução de Sinais , Infecções por Torovirus/fisiopatologiaRESUMO
The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial propagation.
Assuntos
Cromossomos Artificiais Bacterianos/genética , DNA Complementar/genética , Recombinação Genética/genética , Zika virus/genética , Animais , Chlorocebus aethiops , Células Clonais , Genoma Viral , Humanos , Células Vero , Vírion/genética , Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Zika virus (ZIKV) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the Western Hemisphere. Currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of ZIKV infections, and as of yet none are commercially available. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antimicrobial and antiviral activity. In this study, we evaluated ATA as a potential antiviral drug against ZIKV replication. The antiviral activity of ATA against ZIKV replication in vitro showed median inhibitory concentrations (IC50) of 13.87 ± 1.09 µM and 33.33 ± 1.13 µM in Vero and A549 cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (CC50) > 1,000 µM). Moreover, ATA protected both cell types from ZIKV-induced cytopathic effect (CPE) and apoptosis in a time- and concentration-dependent manner. In addition, pre-treatment of Vero cells with ATA for up to 72 h also resulted in effective suppression of ZIKV replication with similar IC50. Importantly, the inhibitory effect of ATA on ZIKV infection was effective against strains of the African and Asian/American lineages, indicating that this inhibitory effect was not strain dependent. Overall, these results demonstrate that ATA has potent inhibitory activity against ZIKV replication and may be considered as a potential anti-ZIKV therapy for future clinical evaluation.
RESUMO
Studying influenza A virus (IAV) requires the use of secondary approaches to detect the presence of virus in infected cells. To overcome this problem, we and others have generated recombinant IAV expressing fluorescent or luciferase reporter genes. These foreign reporter genes can be used as valid surrogates to track the presence of virus. However, the limited capacity for incorporating foreign sequences in the viral genome forced researchers to select a fluorescent or a luciferase reporter gene, depending on the type of study. To circumvent this limitation, we engineered a novel recombinant replication-competent bireporter IAV (BIRFLU) expressing both fluorescent and luciferase reporter genes. In cultured cells, BIRFLU displayed growth kinetics comparable to those of wild-type (WT) virus and was used to screen neutralizing antibodies or compounds with antiviral activity. The expression of two reporter genes allows monitoring of viral inhibition by fluorescence or bioluminescence, overcoming the limitations associated with the use of one reporter gene as a readout. In vivo, BIRFLU effectively infected mice, and both reporter genes were detected using in vivo imaging systems (IVIS). The ability to generate recombinant IAV harboring multiple foreign genes opens unique possibilities for studying virus-host interactions and for using IAV in high-throughput screenings (HTS) to identify novel antivirals that can be incorporated into the therapeutic armamentarium to control IAV infections. Moreover, the ability to genetically manipulate the viral genome to express two foreign genes offers the possibility of developing novel influenza vaccines and the feasibility for using recombinant IAV as vaccine vectors to treat other pathogen infections.IMPORTANCE Influenza A virus (IAV) causes a human respiratory disease that is associated with significant health and economic consequences. In recent years, the use of replication-competent IAV expressing an easily traceable fluorescent or luciferase reporter protein has significantly contributed to progress in influenza research. However, researchers have been forced to select a fluorescent or a luciferase reporter gene due to the restricted capacity of the influenza viral genome for including foreign sequences. To overcome this limitation, we generated, for the first time, a recombinant replication-competent bireporter IAV (BIRFLU) that stably expresses two reporter genes (one fluorescent and one luciferase) to track IAV infections in vitro and in vivo The combination of cutting-edge techniques from molecular biology, animal research, and imaging technologies brings researchers the unique opportunity to use this new generation of reporter-expressing IAV to study viral infection dynamics in both cultured cells and animal models of viral infection.
Assuntos
Genes Reporter/efeitos dos fármacos , Vírus da Influenza A/metabolismo , Medições Luminescentes/métodos , Animais , Antivirais/farmacologia , Cães , Feminino , Genes Reporter/genética , Engenharia Genética/métodos , Genoma Viral/efeitos dos fármacos , Células HEK293 , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Proteínas Luminescentes/genética , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Imagem Óptica/métodos , Infecções por Orthomyxoviridae/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Chronic hepatitis C virus (HCV) infection affects millions of humans throughout the globe, causing liver disease and hepatocellular carcinoma. Persistence of the virus in the infected host can last for decades as a result of a faulty immune response that fails to clear the virus while constituting a major player in viral pathogenesis. In addition to evading immune responses, HCV has evolved intracellular survival strategies that enable persistent replication without directly killing the host cell.After the generation of cell culture infection models for HCV, the knowledge about this virus and host-virus interactions acquired in the last decade has been greatly increased. Interestingly, persistent infection can also be established in cell culture. This model recapitulates persistent HCV RNA replication and viral protein expression as well as infectious progeny virus assembly and secretion and may be used to study not only these aspects of the virus replication cycle but also to study host-virus interactions in a model of prolonged HCV infection. In this chapter, we describe a methodology to generate persistently HCV-infected cultures and to monitor viral load and progeny virus production. Also, we provide generic protocols to study the impact of chemical compounds and host-targeting shRNAs to illustrate the applications of this model in the study of HCV infection in cell culture.
Assuntos
Técnicas de Cultura de Células/métodos , Hepacivirus/fisiologia , Hepatite C Crônica/metabolismo , Interações Hospedeiro-Patógeno , Antivirais/farmacologia , Linhagem Celular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , RNA Viral/genética , Carga Viral/métodos , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Influenza viruses are important pathogens that affect multiple animal species, including humans. There are four types of influenza viruses: A, B, C, and D (IAV, IBV, ICV, and IDV, respectively). IAV and IBV are currently circulating in humans and are responsible of seasonal epidemics (IAV and IBV) and occasional pandemics (IAV). ICV is known to cause mild infections in humans and pigs, while the recently identified IDV primarily affect cattle and pigs. Influenza non-structural protein 1 (NS1) is a multifunctional protein encoded by the NS segment in all influenza types. The main function of NS1 is to counteract the host antiviral defense, including the production of interferon (IFN) and IFN-stimulated genes (ISGs), and therefore is considered an important viral pathogenic factor. Despite of homologous functions, the NS1 protein from the diverse influenza types share little amino acid sequence identity, suggesting possible differences in their mechanism(s) of action, interaction(s) with host factors, and contribution to viral replication and/or pathogenesis. In addition, although the NS1 protein of IAV, IBV and, to some extent ICV, have been previously studied, it is unclear if IDV NS1 has similar properties. Using an approach that allow us to express NS1 independently of the nuclear export protein from the viral NS segment, we have generated recombinant IAV expressing IAV, IBV, ICV, and IDV NS1 proteins. Although recombinant viruses expressing heterotypic (IBV, ICV, and IDV) NS1 proteins were able to replicate similarly in canine MDCK cells, their viral fitness was impaired in human A549 cells and they were highly attenuated in vivo. Our data suggest that despite the similarities to effectively counteract innate immune responses in vitro, the NS1 proteins of IBV, ICV, or IDV do not fully complement the functions of IAV NS1, resulting in deficient viral replication and pathogenesis in vivo.
RESUMO
Zika virus (ZIKV) is an emergent mosquito-borne member of the Flaviviridae family that was responsible for a recent epidemic in the Americas. ZIKV has been associated with severe clinical complications, including neurological disorder such as Guillain-Barré syndrome in adults and severe fetal abnormalities and microcephaly in newborn infants. Given the significance of these clinical manifestations, the development of tools and reagents to study the pathogenesis of ZIKV and to develop new therapeutic options are urgently needed. In this respect, the implementation of reverse genetic techniques has allowed the direct manipulation of the viral genome to generate recombinant (r)ZIKVs, which have provided investigators with powerful systems to answer important questions about the biology of ZIKV, including virus-host interactions, the mechanism of transmission and pathogenesis or the function of viral proteins. In this review, we will summarize the different reverse genetic strategies that have been implemented, to date, for the generation of rZIKVs and the applications of these platforms for the development of replicon systems or reporter-expressing viruses.
Assuntos
Recombinação Genética , Genética Reversa , Infecção por Zika virus/virologia , Zika virus/genética , Animais , DNA Complementar , Genoma Viral , Humanos , Replicação Viral , Zika virus/fisiologiaRESUMO
The recent outbreaks of Zika virus (ZIKV), its association with Guillainâ»Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.
Assuntos
Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia , Zika virus/genética , Zika virus/patogenicidade , Células A549 , Substituição de Aminoácidos , Animais , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , DNA Complementar/genética , Feminino , Humanos , Camundongos , Camundongos Knockout , RNA Viral/genética , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Genética Reversa , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Replicação Viral , Zika virus/imunologia , Infecção por Zika virus/prevenção & controleRESUMO
Hepatitis C virus (HCV) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. HCV replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. We focused our attention on a phosphatidate phosphate (PAP) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. Lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. The best-characterized member of this family is lipin1, which cooperates with lipin2 to maintain glycerophospholipid homeostasis in the liver. Lipin1-deficient cell lines were generated by RNAi to study the role of this protein in different steps of HCV replication cycle. Using surrogate models that recapitulate different aspects of HCV infection, we concluded that lipin1 is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early HCV RNA replication. Infection studies in lipin1-deficient cells overexpressing wild type or phosphatase-defective lipin1 proteins suggest that lipin1 phosphatase activity is required to support HCV infection. Finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin1 may facilitate the generation of the membranous compartment that contains functional HCV replicase complexes.
Assuntos
Hepacivirus/fisiologia , Hepacivirus/patogenicidade , Hepatite C/metabolismo , Hepatite C/virologia , Fosfatidato Fosfatase/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Linhagem Celular , Hepacivirus/genética , Hepatite C/etiologia , Interações Hospedeiro-Patógeno , Humanos , Metabolismo dos Lipídeos , Fosfatidato Fosfatase/antagonistas & inibidores , Fosfatidato Fosfatase/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Viral/genética , RNA Viral/metabolismo , Replicação ViralRESUMO
Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double-membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV-infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double-stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.