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1.
Curr Opin Virol ; 44: 139-144, 2020 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-32892072

RESUMO

Infectious bursal disease (IBD), which is caused by infectious bursal disease virus (IBDV) infection, leads to severe immunosuppression in young chickens and results in significant economic losses in the poultry industry. To date, vaccination with live-attenuated vaccine (LAV) is a convenient method to provide effective protection against IBDV infection. Classical attenuated viruses are usually obtained by either passaging virus in cultured cells or natural isolation. However, these empiric attenuation methods, which are time-consuming and not guaranteed, are not reliable for emergent antigenic variant and very virulent IBDV strains. The reverse genetics (RG) system opens a new avenue for the development of IBDV LAV. In this review, we summarize the current knowledge on the biological characteristics of IBDV structure and genome organization, as well as the established RG systems. We also describe the details for the strategies used to develop IBDV LAV based on the RG systems.

2.
Curr Opin Virol ; 44: 66-72, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32721864

RESUMO

Several mammarenaviruses can cause severe hemorrhagic fever disease with a very high case fatality rate, representing important threats to human health within the viruses' endemic regions. To date, there are no United States (US) Food and Drug Administration (FDA)-licensed vaccines available to combat mammarenavirus infections in humans, and current anti-mammarenavirus therapy is limited to off-label use of the guanosine analog ribavirin, which has limited efficacy and has been associated with significant side effects. Vaccination is one of the most effective ways to prevent viral diseases, and live-attenuated vaccines (LAVs) have been shown to often provide long-term protection against a subsequent natural infection by the corresponding virulent form of the virus. The development of mammarenavirus reverse genetics systems has provided investigators with a powerful approach for the investigation of the molecular and cell biology of mammarenaviruses and also for the generation of recombinant viruses containing predetermined mutations in their genome for their implementation as LAVs for the treatment of mammarenavirus infections. In this review, we summarize the current knowledge on the mammarenavirus molecular and cell biology, and the use of reverse genetic approaches for the generation of recombinant mammarenaviruses. Moreover, we briefly discus some novel LAV approaches for the treatment of mammarenavirus infections based on the use of reverse genetics approaches.

3.
J Virol ; 94(19)2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669332

RESUMO

The New World mammarenavirus Tacaribe virus (TCRV) has been isolated from fruit bats, mosquitoes, and ticks, whereas all other known New World mammarenaviruses are maintained in rodents. TCRV has not been linked to human disease, but it has been shown to protect against Argentine hemorrhagic fever-like disease in marmosets infected with the New World mammarenavirus Junín virus (JUNV), indicating the potential of TCRV as a live-attenuated vaccine for the treatment of Argentine hemorrhagic fever. Implementation of TCRV as a live-attenuated vaccine or a vaccine vector would be facilitated by the establishment of reverse genetics systems for the genetic manipulation of the TCRV genome. In this study, we developed, for the first time, reverse genetics approaches for the generation of recombinant TCRV (rTCRV). We successfully rescued a wild-type (WT) rTCRV (a trisegmented form of TCRV expressing two reporter genes [r3TCRV]) and a bisegmented TCRV expressing a single reporter gene from a bicistronic viral mRNA (rTCRV/GFP). These reverse genetics approaches represent an excellent tool to investigate the biology of TCRV and to explore its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of other viral infections. Notably, we identified a 39-nucleotide (nt) deletion (Δ39) in the noncoding intergenic region (IGR) of the viral large (L) segment that is required for optimal virus multiplication. Accordingly, an rTCRV containing this 39-nt deletion in the L-IGR (rTCRV/Δ39) exhibited decreased viral fitness in cultured cells, suggesting the feasibility of using this deletion in the L-IGR as an approach to attenuate TCRV, and potentially other mammarenaviruses, for their implementation as live-attenuated vaccines or vaccine vectors.IMPORTANCE To date, no Food and Drug Administration (FDA)-approved vaccines are available to combat hemorrhagic fever caused by mammarenavirus infections in humans. Treatment of mammarenavirus infections is limited to the off-label use of ribavirin, which is partially effective and associated with significant side effects. Tacaribe virus (TCRV), the prototype member of the New World mammarenaviruses, is nonpathogenic in humans but able to provide protection against Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever, demonstrating the feasibility of using TCRV as a live-attenuated vaccine vector for the treatment of JUNV and potentially other viral infections. Here, we describe for the first time the feasibility of generating recombinant TCRV (rTCRV) using reverse genetics approaches, which paves the way to study the biology of TCRV and also its potential use as a live-attenuated vaccine or a vaccine vector for the treatment of mammarenavirus and/or other viral infections in humans.

4.
mBio ; 11(2)2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209677

RESUMO

Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication.IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.

5.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098811

RESUMO

Lassa virus (LASV) is endemic in Western Africa and is estimated to infect hundreds of thousands of individuals annually. A considerable number of these infections result in Lassa fever (LF), which is associated with significant morbidity and a case-fatality rate as high as 69% among hospitalized confirmed patients. U.S. Food and Drug Administration-approved LF vaccines are not available. Current antiviral treatment is limited to off-label use of a nucleoside analogue, ribavirin, that is only partially effective and associated with significant side effects. We generated and characterized a recombinant LASV expressing a codon-deoptimized (CD) glycoprotein precursor gene (GPC), rLASV-GPC/CD. Comparison of growth kinetics and peak titers showed that rLASV-GPC/CD is slightly attenuated in cell culture compared to wild-type (WT) recombinant LASV (rLASV-WT). However, rLASV-GPC/CD is highly attenuated in strain 13 and Hartley guinea pigs, as reflected by the absence of detectable clinical signs in animals inoculated with rLASV-GPC/CD. Importantly, a single subcutaneous dose of rLASV-GPC/CD provides complete protection against an otherwise lethal exposure to LASV. Our results demonstrate the feasibility of implementing a CD approach for developing a safe and effective LASV live-attenuated vaccine candidate. Moreover, rLASV-GPC/CD might provide investigators with a tool to safely study LASV outside maximum (biosafety level 4) containment, which could accelerate the elucidation of basic aspects of the molecular and cell biology of LASV and the development of novel LASV medical countermeasures.IMPORTANCE Lassa virus (LASV) infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever (LF) cases. Licensed LF vaccines are not available, and anti-LF therapy is limited to off-label use of the nucleoside analog ribavirin with uncertain efficacy. We describe the generation of a novel live-attenuated LASV vaccine candidate. This vaccine candidate is based on mutating wild-type (WT) LASV in a key region of the viral genome, the glycoprotein precursor (GPC) gene. These mutations do not change the encoded GPC but interfere with its production in host cells. This mutated LASV (rLASV-GPC/CD) behaves like WT LASV (rLASV-WT) in cell culture, but in contrast to rLASV-WT, does not cause disease in inoculated guinea pigs. Guinea pigs immunized with rLASV-GPC/CD were protected against an otherwise lethal exposure to WT LASV. Our results support the testing of this candidate vaccine in nonhuman primate models ofLF.


Assuntos
Febre Lassa/prevenção & controle , Vírus Lassa/genética , Vírus Lassa/imunologia , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Células A549 , África Ocidental , Sequência de Aminoácidos , Animais , Arenaviridae , Arenavirus , Bunyaviridae , Chlorocebus aethiops , Códon , Modelos Animais de Doenças , Feminino , Genes Virais/genética , Genoma Viral , Glicoproteínas/genética , Cobaias , Humanos , Febre Lassa/imunologia , Febre Lassa/virologia , Masculino , Ribavirina , Vacinas Atenuadas/genética , Células Vero
6.
Viruses ; 12(2)2020 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-32024281

RESUMO

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift. Previously, we described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV) in vitro, and prophylactic and therapeutic activities in vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrated how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian-produced KPF1-HEK hMAb. KPF1-Antx hMAb showed broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, which was comparable to that observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrated, for the first time, a plant-produced influenza hMAb with in vitro and in vivo activity against influenza virus. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.

7.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31915284

RESUMO

Infectious bursal disease virus (IBDV) of the Birnaviridae family leads to immunosuppression of young chickens by destroying B cells in the bursa of Fabricius (BFs). Given the increasing number of variant IBDV strains, we urgently require a method to produce attenuated virus for vaccine development. To accomplish this goal, the dual-promoter plasmids in which the RNA polymerase II and RNA polymerase I (Pol I) promoters were placed upstream of the IBDV genomic sequence, which was followed by mouse Pol I terminator and a synthetic polyadenylation signal, were developed for rapid generation of IBDV. This approach did not require trans-supplementation of plasmids for the expression of VP1 and VP3, the main components of IBDV ribonucleoprotein (RNP). Based on the finding in this study that the IBDV RNP activity was partially retained by VP1-FLAG, we successfully rescued the replication-competent IBDV/1FLAG expressing VP1-FLAG. Compared with its parental counterpart, IBDV/1FLAG formed smaller size plaques in cultured cells and induced the same 100% immune protection in vivo However, neither retarded development nor severe BFs lesion was observed in the IBDV/1FLAG-inoculated chickens. Collectively, this is the first report that viral RNP activity was affected by the addition of an epitope tag on the componential viral proteins. Furthermore, this work demonstrates the rapid generation of attenuated IBDV from dual-promoter plasmids via reducing viral RNP activity by a fused FLAG tag on the C terminus of VP1. This would be a convenient strategy to attenuate epidemic variant IBDV strains for rapid and efficient vaccine development.IMPORTANCE Immunosuppression in chickens as a result of infectious bursal disease virus (IBDV) infection leads to significant economic losses in the poultry industry worldwide every year. Currently, vaccination is still the best way to prevent the prevalence of IBDV. However, with the occurrence of increasing numbers of variant IBDV strains, it is challenging to develop antigen-matched live attenuated vaccine. Here, we first developed a dual-promoter reverse-genetic system for the rapid generation of IBDV. Using this system, the attenuated IBDV/1FLAG expressing VP1-FLAG, which displays the decreased viral RNP activity, was rescued. Moreover, IBDV/1FLAG inoculation induced a similar level of neutralizing antibodies to that of its parental counterpart, protecting chickens against lethal challenge. Our study, for the first time, describes a dual-promoter reverse-genetic approach for the rapid generation of attenuated IBDV while maintaining entire parental antigenicity, suggesting a potential new method to attenuate epidemic variant IBDV strains for vaccine development.


Assuntos
Vírus da Doença Infecciosa da Bursa/genética , Ribonucleoproteínas/metabolismo , Vacinas Atenuadas , Vacinas Virais , Animais , Infecções por Birnaviridae/prevenção & controle , Infecções por Birnaviridae/veterinária , Linhagem Celular , Galinhas/virologia , Epitopos/química , Células HEK293 , Humanos , Camundongos , Plasmídeos/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/química , Poliadenilação , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Regiões Promotoras Genéticas , Domínios Proteicos , Proteínas Recombinantes/química , Genética Reversa , Proteínas Estruturais Virais/genética
8.
FASEB J ; 33(1): 286-300, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29979632

RESUMO

Infectious bursal disease virus (IBDV) infection triggers the induction of type I IFN, which is mediated by melanoma differentiation-associated protein 5 recognition of the viral genomic double-stranded RNA (dsRNA). However, the mechanism of IBDV overcoming the type I IFN antiviral response remains poorly characterized. Here, we show that IBDV genomic dsRNA selectively binds to the host cellular RNA binding protein Staufen1 (STAU1) in vitro and in vivo. The viral dsRNA binding region was mapped to the N-terminal moiety of STAU1 (residues 1-468). Down-regulation of STAU1 impaired IBDV replication and enhanced IFN-ß transcription in response to IBDV infection, while having little effect on the viral attachment to the host cells and cellular entry. Conversely, overexpression of STAU1 but not the IBDV dsRNA-binding deficient STAU1 mutant (469-702) led to a suppression of IBDV dsRNA-induced IFN-ß promoter activity. Moreover, we found that the binding of STAU1 to IBDV dsRNA decreased the association of melanoma differentiation-associated protein 5 but not VP3 with the IBDV dsRNA in vitro. Finally, we showed that STAU1 and VP3 suppressed IFN-ß gene transcription in response to IBDV infection in an additive manner. Collectively, these findings provide a novel insight into the evasive strategies used by IBDV to escape the host IFN antiviral response.-Ye, C., Yu, Z., Xiong, Y., Wang, Y., Ruan, Y., Guo, Y., Chen, M., Luan, S., Zhang, E., Liu, H. STAU1 binds to IBDV genomic double-stranded RNA and promotes viral replication via attenuation of MDA5-dependent ß interferon induction.


Assuntos
Infecções por Birnaviridae/virologia , Proteínas do Citoesqueleto/metabolismo , Vírus da Doença Infecciosa da Bursa/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Animais , Antivirais/metabolismo , Infecções por Birnaviridae/genética , Infecções por Birnaviridae/metabolismo , Galinhas , Proteínas do Citoesqueleto/genética , Genômica , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Interferon beta/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética
9.
J Virol ; 92(2)2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29093101

RESUMO

Infectious bursal disease virus (IBDV) is a bisegmented double-strand RNA (dsRNA) virus of the Birnaviridae family. While IBDV genomic dsRNA lacks a 5' cap, the means by which the uncapped IBDV genomic RNA is translated effectively is unknown. In this study, we describe a cap-independent pathway of translation initiation of IBDV uncapped RNA that relies on VP1 and VP3. We show that neither purified IBDV genomic dsRNA nor the uncapped viral plus-sense RNA transcripts were directly translated and rescued into infectious viruses in host cells. This defect in translation of the uncapped IBDV genomic dsRNA was rescued by trans-supplementation of the viral proteins VP1 and VP3 which was dependent on both the intact polymerase activity of VP1 and the dsRNA binding activity of VP3. Deletion analysis showed that both 5' and 3' untranslated regions (UTRs) of IBDV dsRNA were essential for VP1/VP3-dependent translation initiation. Significantly, VP1 and VP3 could also mediate the recovery of infectious IBDV from the authentic minus-sense strand of IBDV dsRNA. Moreover, downregulation or inhibition of the cap-binding protein eIF4E did not decrease but, rather, enhanced the VP1/VP3-mediated translation of the uncapped IBDV RNA. Collectively, our findings for the first time reveal that VP1 and VP3 compensate for the deficiency of the 5' cap and replace eIF4E to confer upon the uncapped IBDV RNA the ability to be translated and rescued into infectious viruses.IMPORTANCE A key point of control for virus replication is viral translation initiation. The current study shows that the uncapped IBDV RNA cannot be translated into viral proteins directly by host translation machinery and is thus noninfectious. Our results constitute the first direct experimental evidence that VP1 and VP3 are required and sufficient to initiate translation of uncapped IBDV genomic RNA by acting as a substitute for cap and replacing the cap-binding protein eIF4E. Significantly, VP1/VP3 mediate the recovery of infectious IBDV not only from the plus-sense strand but also from the minus-sense strand of the IBDV dsRNA. These findings provide not only new insights into the molecular mechanisms of the life cycle of IBDV but also a new tool for an alternative strategy for the recovery of IBDV from both the plus- and the minus-sense strands of the viral genomic dsRNA.


Assuntos
Infecções por Birnaviridae/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , RNA de Cadeia Dupla/genética , Proteínas Estruturais Virais/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Linhagem Celular , Galinhas , Células HEK293 , Humanos , Camundongos , Biossíntese de Proteínas , Replicação Viral
10.
J Immunol ; 199(12): 4142-4154, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29127148

RESUMO

Although the immune adaptor adhesion and degranulation-promoting adaptor protein (ADAP) acts as a key mediator of integrin inside-out signaling leading to T cell adhesion, the regulation of this adaptor during integrin activation and clustering remains unclear. We now identify Ubc9, the sole small ubiquitin-related modifier E2 conjugase, as an essential regulator of ADAP where it is required for TCR-induced membrane recruitment of the small GTPase Rap1 and its effector protein RapL and for activation of the small GTPase Rac1 in T cell adhesion. We show that Ubc9 interacted directly with ADAP in vitro and in vivo, and the association was increased in response to anti-CD3 stimulation. The Ubc9-binding domain on ADAP was mapped to a nuclear localization sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by short hairpin RNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCR-induced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, although it had little effect on the TCR proximal signaling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 and RapL, but not Rap1-interacting adaptor molecule. Taken together, our data demonstrate for the first time, to our knowledge, that Ubc9 acts as a functional binding partner of ADAP and plays a selective role in integrin-mediated T cell adhesion via modulation of Rap1-RapL membrane recruitment and Rac1 activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Linfócitos T/citologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Adesão Celular , Chlorocebus aethiops , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Integrinas/fisiologia , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/fisiologia , Camundongos , Modelos Imunológicos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/farmacologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética
11.
J Virol ; 91(3)2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27881656

RESUMO

While the entry of infectious bursal disease virus (IBDV) is initiated by the binding of the virus to the two major receptors integrin and HSP90, the signaling events after receptor binding and how they contribute to virus entry remain elusive. We show here that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src both in DF-1 chicken fibroblasts and in vivo in the bursa of Fabricius from specific-pathogen-free (SPF) chickens. Importantly, inactivated IBDV fails to stimulate c-Src Y416 phosphorylation, and a very virulent IBDV strain induces a much higher level of c-Src Y416 phosphorylation than does an attenuated strain. Inhibition of c-Src activation by an Src kinase inhibitor or expression of a c-Src dominant negative mutant results in a significant decrease in the internalization of IBDV but has little effect on virus adhesion. Furthermore, short hairpin RNA (shRNA) downregulation of integrin, either the α4 or ß1 subunit, but not HSP90 remarkably attenuates IBDV-induced c-Src Y416 phosphorylation, resulting in a decrease in IBDV internalization but not virus adhesion. Moreover, interestingly, inhibition of either c-Src downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt-RhoA signaling cascade or actin rearrangement leads to a significant decrease in IBDV internalization irrespective of the IBDV-induced high levels of c-Src phosphorylation. Cumulatively, our results suggest a novel feed-forward model whereby IBDV activates c-Src for benefiting its cell entry via an integrin-mediated pathway by the activation of downstream PI3K/Akt-RhoA signaling and cytoskeleton actin rearrangement. IMPORTANCE: While IBDV-caused immunosuppression is highly related to viral invasion, the molecular basis of the cellular entry of IBDV remains elusive. In this study, we demonstrate that IBDV activates c-Src by inducing the phosphorylation of the Y416 residue in c-Src to promote virus internalization but not virus adhesion. The ability to induce the level of c-Src Y416 phosphorylation correlates with the pathogenicity of an IBDV strain. IBDV-induced c-Src Y416 activation is α4ß1 integrin but not HSP90 dependent and involves the activation of the downstream PI3K/Akt-RhoA GTPase-actin rearrangement cascade. Thus, our findings provide new insights into the IBDV infection process and the potential for c-Src as a candidate target for the development of IBDV therapeutic drugs.


Assuntos
Infecções por Birnaviridae/metabolismo , Infecções por Birnaviridae/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , Integrina alfa4beta1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Internalização do Vírus , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Actinas/metabolismo , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular , Embrião de Galinha , Galinhas , Fibroblastos , Proteínas de Choque Térmico HSP90/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Fosforilação , Transdução de Sinais , Ligação Viral
12.
Autophagy ; 11(3): 503-15, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25714412

RESUMO

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.


Assuntos
Autofagia , Avibirnavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Membrana Celular/metabolismo , Galinhas , Citosol/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Fosforilação , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo
13.
Microbiol Immunol ; 58(10): 590-9, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25082455

RESUMO

Negri bodies (NBs) are formed in the cytoplasm of rabies virus (RABV)-infected cells and are accompanied by a number of host factors to NBs, in which replication and transcription occur. Here, it was found that chaperonin containing TCP-1 subunit alpha (CCTα) relocalizes to NBs in RABV-infected cells, and that cotransfection of nucleo- and phospho-proteins of RABV is sufficient to recruit CCTα to the NBs' structure. Inhibition of CCTα expression by specific short hairpin RNA knockdown inhibited the replication and transcription of RABV. Therefore, this study showed that the host factor CCTα is associated with RABV infection and is very likely required for efficient virus transcription and replication.


Assuntos
Chaperonina com TCP-1/metabolismo , Vírus da Raiva/genética , Raiva/enzimologia , Raiva/virologia , Transcrição Genética , Replicação Viral , Animais , Linhagem Celular , Chaperonina com TCP-1/genética , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão Viral/enzimologia , Corpos de Inclusão Viral/virologia , Camundongos , Transporte Proteico , Raiva/genética , Vírus da Raiva/fisiologia
14.
J Virol ; 88(19): 11154-65, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25031338

RESUMO

UNLABELLED: Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-ß) in DF-1 cells pretreated with IFN-α/ß. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-ß expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-ß expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5. IMPORTANCE: MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-ß did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-ß expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.


Assuntos
Proteínas Aviárias/genética , RNA Helicases DEAD-box/genética , Imunidade Inata/efeitos dos fármacos , Vírus da Doença Infecciosa da Bursa/imunologia , RNA Viral/antagonistas & inibidores , Proteínas Estruturais Virais/genética , Animais , Proteínas Aviárias/imunologia , Proteínas Aviárias/farmacologia , Ligação Competitiva , Linhagem Celular , Galinhas , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/farmacologia , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Vírus da Doença Infecciosa da Bursa/genética , Interferon beta/antagonistas & inibidores , Interferon beta/biossíntese , Interferon beta/imunologia , Ligação Proteica , RNA de Cadeia Dupla/antagonistas & inibidores , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA Viral/genética , RNA Viral/imunologia , Transdução de Sinais , Proteínas Estruturais Virais/imunologia , Proteínas Estruturais Virais/farmacologia
15.
Electrophoresis ; 35(8): 1130-43, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24519676

RESUMO

We carried out a proteomic analysis of THP-1-derived macrophages with and without Brucella abortus A19 (B. abortus A19) infection in order to study the cellular responses to B. abortus A19. The proteins were analyzed at different time points after infection with 2DE followed by MALDI-TOF/TOF identification. Comparative analysis of multiple 2DE gels revealed that the majority of changes in protein abundance appeared between 48 and 96 h after infection. MS identified 44 altered proteins, including 20 proteins increased in abundance and 24 proteins decreased in abundance, which were found to be involved in cytoskeleton, signal transduction, energy metabolism, host macromolecular biosynthesis, and stress response. Moreover, 22 genes corresponding to the altered proteins were quantified by real-time RT-PCR to examine the transcriptional profiles between infected and uninfected THP-1-derived macrophages. Finally, we mapped the altered pathways and networks using ingenuity pathway analysis, which suggested that the altered protein species were heavily favored germ cell-Sertoli cell junction signaling as the primary pathway. Furthermore, mechanisms of viral exit from host cell and macrophage stimulating protein-recepteur d'origine nantais signaling appeared to be major pathways modulated in infected cells. This study effectively provides useful dynamic protein-related information concerning B. abortus infection in macrophages.


Assuntos
Brucella abortus/fisiologia , Brucelose/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Proteínas/metabolismo , Humanos , Macrófagos/metabolismo , Mapas de Interação de Proteínas , Proteínas/análise , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
16.
J Virol ; 87(13): 7608-21, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23637400

RESUMO

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit.


Assuntos
Chaperonina com TCP-1/metabolismo , Corpos de Inclusão Viral/metabolismo , Neurônios/metabolismo , Vírus da Raiva/fisiologia , Raiva/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Primers do DNA/genética , Eletroforese em Gel Bidimensional , Imunofluorescência , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Camundongos , Microscopia Confocal , Chaperonas Moleculares , Neurônios/virologia , Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Estruturais Virais/metabolismo , Replicação Viral/fisiologia
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