RESUMEN
Capsid-like poxvirus scaffold proteins self-assemble into semi-regular lattice that govern the formation of spherical immature virus particles. The scaffolding is a critical step in virus morphogenesis as exemplified by the drug rifampicin that impairs the recruitment of scaffold onto the viral membrane in vaccinia virus (VACV). Here we report cryo-electron microscopy structure of scaffolding protein Orfv075 of orf virus (ORFV) that causes smallpox-like diseases in sheep, goats and occasionally humans via zoonotic infection. We demonstrate that the regions that are involved in intertrimeric interactions for scaffold assembly are largely conserved in comparison to its VACV orthologue protein D13 whose intermediate assembly structures have been previously characterized. By contrast, less conserved regions are located away from these interfaces, indicating both viruses share similar assembly mechanisms. We also show that the phenylalanine-rich binding site of rifampicin in D13 is conserved in Orfv075, and molecular docking simulation confirms similar binding modes. Our study provides structural basis of scaffolding protein as a target for anti-poxvirus treatment across wide range of poxvirus genera.
Asunto(s)
Microscopía por Crioelectrón , Virus del Orf , Virus del Orf/química , Virus del Orf/ultraestructura , Simulación del Acoplamiento Molecular , Animales , Sitios de Unión , Conformación Proteica , Modelos Moleculares , Secuencia de Aminoácidos , Proteínas Virales/química , Proteínas Virales/ultraestructura , Proteínas Virales/metabolismo , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/ultraestructura , Proteínas Estructurales Virales/metabolismo , Rifampin/química , Rifampin/farmacologíaRESUMEN
Infectious bursal disease virus (IBDV) is a highly contagious virus with a dsRNA genome, predominantly infecting chickens and causing significant economic losses due to high mortality rates. The emergence of recombinant, novel variant, and highly virulent strains that evade current vaccines has led to frequent epidemics and outbreaks in the poultry industry. The lack of targeted antivirals for IBDV underscores the pressing requirement to develop potent therapeutic options. Within this framework, our research investigated the effectiveness of picroside II, a naturally derived iridoid glycoside, against viruses in DF-1 cells. Our findings demonstrate that picroside II significantly inhibits viral replication, with its efficacy increasing proportionally to the dosage administered. Through time-addition and antiviral duration analysis, we determined that picroside II therapeutically blocks IBDV replication, with its effects persisting for over 72â¯hours. Further investigation revealed that picroside II specifically inhibits the cellular replication stage of IBDV's lifecycle. Additionally, our findings indicate that picroside II impairs VP1 polymerase activity by binding to the active pocket, which significantly disrupts the interaction between VP1 and VP3. Mutations at three critical binding sites on VP1 not only impair virus replication but also hinder polymerase function and disrupt VP1-VP3 interactions. Collectively, these results demonstrate that picroside II, by inhibiting viral polymerase activity, represents a promising antiviral agent against IBDV.
Asunto(s)
Antivirales , Pollos , Cinamatos , Virus de la Enfermedad Infecciosa de la Bolsa , Glucósidos Iridoides , Replicación Viral , Replicación Viral/efectos de los fármacos , Virus de la Enfermedad Infecciosa de la Bolsa/efectos de los fármacos , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Animales , Cinamatos/farmacología , Glucósidos Iridoides/farmacología , Antivirales/farmacología , Línea Celular , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/tratamiento farmacológicoRESUMEN
Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-ß and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-ß promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2â¯C, 3â¯C and 3D inhibited the activation of IFN-ß promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNß induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses.
Asunto(s)
Factor 3 Regulador del Interferón , Interferón beta , Interferón beta/genética , Interferón beta/inmunología , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Animales , Humanos , Línea Celular , Transducción de Señal/efectos de los fármacos , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Inmunidad Innata , Bovinos , Búfalos/virología , FN-kappa B/metabolismoRESUMEN
The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Virus de la Hepatitis del Pato , Sitios Internos de Entrada al Ribosoma , Replicación Viral , Virus de la Hepatitis del Pato/fisiología , Virus de la Hepatitis del Pato/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Animales , Proteínas Estructurales Virales/metabolismo , Proteínas Estructurales Virales/genética , Patos , Enfermedades de las Aves de Corral/virología , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/virología , Infecciones por Picornaviridae/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Hepatitis Viral Animal/virología , Hepatitis Viral Animal/metabolismo , Biosíntesis de ProteínasRESUMEN
During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.
Asunto(s)
Bombyx , Nucleopoliedrovirus , Proteínas de la Matriz de Cuerpos de Oclusión , Nucleopoliedrovirus/genética , Animales , Proteínas de la Matriz de Cuerpos de Oclusión/genética , Bombyx/virología , Bombyx/genética , Nucleótidos/genética , Nucleótidos/metabolismo , Regiones Promotoras Genéticas , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Codón/genética , Regulación Viral de la Expresión Génica , Línea CelularRESUMEN
Infectious bursal disease (IBD) is an acute and fatal immunosuppressive disease caused by infectious bursal disease virus (IBDV). As an obligate intracellular parasite, IBDV infection is strictly regulated by host factors. Knowledge on the antiviral activity and possible mechanism of host factors might provide the theoretical basis for the prevention and control of IBD. In this study, RNA-sequencing results indicated that many host factors were induced by IBDV infection, among which the expression levels of OASL (2´,5´-oligadenylate synthetase-like protein) was significantly upregulated. OASL overexpression significantly inhibited IBDV replication, whereas OASL knockdown promoted IBDV replication. Interestingly, the antiviral ability of OASL was independent of its canonical enzymatic activity, i.e., OASL targeted viral protein VP2 for degradation, depending on the autophagy receptor p62/SQSTM1 in the autophagy pathway. Additionally, the 316 lysine (K) of VP2 was the key site for autophagy degradation, and its replacement with arginine disrupted VP2 degradation induced by OASL and enhanced IBDV replication. Importantly, our results for the first time indicate a unique and potent defense mechanism of OASL against double-stranded RNA virus by interaction with viral proteins, which leads to their degradation. IMPORTANCE: OASL (2´,5´-oligadenylate synthetase-like protein) exhibits broad-spectrum antiviral effects against single-stranded RNA viruses in mammals, potentially serving as a promising target for novel antiviral strategies. However, its role in inhibiting the replication of double-stranded RNA viruses (dsRNA viruses), such as infectious bursal disease virus (IBDV), in avian species remains unclear. Our findings indicated a unique and potent defense mechanism of OASL against dsRNA viruses. It has been previously shown in mammals that OASL inhibits virus replication through increasing interferon production. The groundbreaking aspect of our study is the finding that OASL has the ability to interact with IBDV viral protein VP2 and target it for degradation and thus exerts its antiviral effect. Our results reveal the interaction between avian natural antiviral immune response and IBDV infection. Our study not only enhances our understanding of bird defenses against viral infections but can also inform strategies for poultry disease management.
Asunto(s)
2',5'-Oligoadenilato Sintetasa , Autofagia , Infecciones por Birnaviridae , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa , Proteínas Estructurales Virales , Replicación Viral , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Animales , Infecciones por Birnaviridae/virología , Infecciones por Birnaviridae/metabolismo , Proteínas Estructurales Virales/metabolismo , Proteínas Estructurales Virales/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/metabolismo , Interacciones Huésped-Patógeno , Células HEK293 , Humanos , Línea CelularRESUMEN
The foot-and-mouth disease virus is a highly contagious and economically devastating virus of cloven-hooved animals, including cattle, buffalo, sheep, and goats, causing reduced animal productivity and posing international trade restrictions. For decades, chemically inactivated vaccines have been serving as the most effective strategy for the management of foot-and-mouth disease. Inactivated vaccines are commercially produced in cell culture systems, which require successful propagation and adaptation of field isolates, demanding a high cost and laborious time. Cell culture adaptation is chiefly indebted to amino acid substitutions in surface-exposed capsid proteins, altering the necessity of RGD-dependent receptors to heparan sulfate macromolecules for virus binding. Several amino acid substations in VP1, VP2, and VP3 capsid proteins of FMDV, both at structural and functional levels, have been characterized previously. This literature review combines frequently reported amino acid substitutions in virus capsid proteins, their critical roles in virus adaptation, and functional characterization of the substitutions. Furthermore, this data can facilitate molecular virologists to develop new vaccine strains against the foot-and-mouth disease virus, revolutionizing vaccinology via reverse genetic engineering and synthetic biology.
Asunto(s)
Sustitución de Aminoácidos , Proteínas de la Cápside , Virus de la Fiebre Aftosa , Tropismo Viral , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/química , Técnicas de Cultivo de Célula , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/metabolismo , Receptores Virales/metabolismo , Receptores Virales/genética , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismoRESUMEN
This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.
Asunto(s)
Transporte Activo de Núcleo Celular , Virus de la Fiebre Efímera Bovina , Lamina Tipo A , Señales de Localización Nuclear , Animales , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromosomas/metabolismo , Citoplasma/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Virus de la Fiebre Efímera Bovina/metabolismo , Proteínas Estructurales Virales/metabolismoRESUMEN
African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs. The causative agent of ASF is African swine fever virus (ASFV), a large double-stranded DNA virus with a complex virion structure. Among the various proteins encoded by ASFV, A137R is a crucial structural protein associated with its virulence. However, the structure and molecular mechanisms underlying the functions of A137R remain largely unknown. In this study, we present the structure of A137R determined by cryogenic electron microscopy single-particle reconstruction, which reveals that A137R self-oligomerizes to form a dodecahedron-shaped cage composed of 60 polymers. The dodecahedron is literally equivalent to a T = 1 icosahedron where the icosahedral vertexes are located in the center of each dodecahedral facet. Within each facet, five A137R protomers are arranged in a head-to-tail orientation with a long N-terminal helix forming the edge through which adjacent facets stitch together to form the dodecahedral cage. Combining structural analysis and biochemical evidence, we demonstrate that the N-terminal domain of A137R is crucial and sufficient for mediating the assembly of the dodecahedron. These findings imply the role of A137R cage as a core component in the icosahedral ASFV virion and suggest a promising molecular scaffold for nanotechnology applications. IMPORTANCE: African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. A137R is a structural protein of ASFV that is associated with its virulence. The discovery of the dodecahedron-shaped cage structure of A137R in this study is of great importance in understanding ASFV pathogenicity. This finding sheds light on the molecular mechanisms underlying the functions of A137R. Furthermore, the dodecahedral cage formed by A137R shows promise as a molecular scaffold for nanoparticle vectors. Overall, this study provides valuable insights into the structure and function of A137R, contributing to our understanding of ASFV and potentially opening up new avenues for the development of vaccines or treatments for ASF.
Asunto(s)
Virus de la Fiebre Porcina Africana , Porcinos , Proteínas Estructurales Virales , Animales , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/química , Virus de la Fiebre Porcina Africana/crecimiento & desarrollo , Virus de la Fiebre Porcina Africana/patogenicidad , Virus de la Fiebre Porcina Africana/ultraestructura , Microscopía por Crioelectrón , Relación Estructura-Actividad , Porcinos/virología , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismo , Proteínas Estructurales Virales/ultraestructura , Virión/química , Virión/metabolismo , Virión/ultraestructura , VirulenciaRESUMEN
Herpesviruses are enveloped and have an amorphous protein layer surrounding the capsid, which is termed the tegument. Tegument proteins perform critical functions throughout the viral life cycle. This review provides a comprehensive and comparative analysis of the roles of specific tegument proteins in capsid transport and virion morphogenesis of selected, well-studied prototypes of each of the three subfamilies of Herpesviridae i.e., human herpesvirus-1/herpes simplex virus-1 (Alphaherpesvirinae), human herpesvirus-5/cytomegalovirus (Betaherpesvirinae) and human herpesvirus -8/Kaposi's sarcomavirus (Gammaherpesvirinae). Most of the current knowledge is based on alpha herpesviruses, in particular HSV-1. While some tegument proteins are released into the cytoplasm after virus entry, several tegument proteins remain associated with the capsid and are responsible for transport to and docking at the nucleus. After replication and capsid formation, the capsid is enveloped at the nuclear membrane, which is referred to as primary envelopment, followed by de-envelopment and release into the cytoplasm. This requires involvement of at least three tegument proteins. Subsequently, multiple interactions between tegument proteins and capsid proteins, other tegument proteins and glycoproteins are required for assembly of the virus particles and envelopment at the Golgi, with certain tegument proteins acting as the central hub for these interactions. Some redundancy in these interactions ensures appropriate morphogenesis.
Asunto(s)
Herpesviridae , Herpesvirus Humano 1 , Herpesvirus Humano 8 , Humanos , Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Ensamble de Virus , Herpesviridae/metabolismo , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 8/metabolismo , Morfogénesis , Virión/metabolismo , Proteínas Estructurales Virales/metabolismoRESUMEN
IMPORTANCE: The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.
Asunto(s)
Avibirnavirus , Proteínas de Ciclo Celular , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa , Proteínas Serina-Treonina Quinasas , Proteínas Estructurales Virales , Replicación Viral , Animales , Avibirnavirus/química , Avibirnavirus/crecimiento & desarrollo , Avibirnavirus/metabolismo , Infecciones por Birnaviridae/enzimología , Infecciones por Birnaviridae/metabolismo , Infecciones por Birnaviridae/veterinaria , Infecciones por Birnaviridae/virología , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteínas de Ciclo Celular/metabolismo , Pollos/virología , Virus de la Enfermedad Infecciosa de la Bolsa/química , Virus de la Enfermedad Infecciosa de la Bolsa/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismoRESUMEN
IMPORTANCE: Alphaviruses threaten public health continuously, and Getah virus (GETV) is a re-emerging alphavirus that can potentially infect humans. Approved antiviral drugs and vaccines against alphaviruses are few available, but several host antiviral factors have been reported. Here, we used GETV as a model of alphaviruses to screen for additional host factors. Tetrachlorodibenzo-p-dioxin-inducible poly(ADP ribose) polymerase was identified to inhibit GETV replication by inducing ubiquitination of the glycoprotein E2, causing its degradation by recruiting the E3 ubiquitin ligase membrane-associated RING-CH8 (MARCH8). Using GETV as a model virus, focusing on the relationship between viral structural proteins and host factors to screen antiviral host factors provides new insights for antiviral studies on alphaviruses.
Asunto(s)
Alphavirus , Interacciones Microbiota-Huesped , Proteínas de Transporte de Nucleósidos , Poli(ADP-Ribosa) Polimerasas , Transcriptoma , Humanos , Alphavirus/crecimiento & desarrollo , Alphavirus/inmunología , Glicoproteínas/metabolismo , Proteínas de Transporte de Nucleósidos/genética , Proteínas de Transporte de Nucleósidos/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ubiquitinación , Proteínas Estructurales Virales/metabolismo , Replicación ViralRESUMEN
During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.
Asunto(s)
Enfermedades de los Peces , Factores Reguladores del Interferón , Proteínas Quinasas Activadas por Mitógenos , Infecciones por Rhabdoviridae , Ubiquitinación , Proteínas Estructurales Virales , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Rhabdoviridae/genética , Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/virología , Pez Cebra/genética , Pez Cebra/inmunología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Estabilidad Proteica , Proteolisis , Proteínas Estructurales Virales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Regulación hacia ArribaRESUMEN
The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid-liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes.
Asunto(s)
Birnaviridae , Virus de la Enfermedad Infecciosa de la Bolsa , Birnaviridae/metabolismo , Compartimentos de Replicación Viral , Línea Celular , Replicación Viral , Proteínas Virales/genética , Proteínas Virales/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas Estructurales Virales/metabolismoRESUMEN
Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".
Asunto(s)
Nucleoproteínas , Virus Sincitial Respiratorio Humano , Compartimentos de Replicación Viral , Proteínas Estructurales Virales , ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleoproteínas/metabolismo , Virus Sincitial Respiratorio Humano/metabolismo , Virus Sincitial Respiratorio Humano/fisiología , Compartimentos de Replicación Viral/metabolismo , Replicación Viral , Proteínas Estructurales Virales/metabolismo , HumanosRESUMEN
The infectious bursal diseases virus (IBDV) polymerase, VP1 protein, is responsible for transcription, initial translation and viral genomic replication. Knowledge about the new kind of post-translational modification of VP1 supports identification of novel drugs against the virus. Because the arginine residue is known to be methylated by protein arginine methyltransferase (PRMT) enzyme, we investigated whether IBDV VP1 is a substrate for known PRMTs. In this study, we show that VP1 is specifically associated with and methylated by PRMT5 at the arginine 426 (R426) residue. IBDV infection causes the accumulation of PRMT5 in the cytoplasm, which colocalizes with VP1 as a punctate structure. In addition, ectopic expression of PRMT5 significantly enhances the viral replication. In the presence of PMRT5, enzyme inhibitor and knockout of PRMT5 remarkably decreased viral replication. The polymerase activity of VP1 was severely damaged when R426 mutated to alanine, resulting in impaired viral replication. Our study reports a novel form of post-translational modification of VP1, which supports its polymerase function to facilitate the viral replication. IMPORTANCE Post-translational modification of infectious bursal disease virus (IBDV) VP1 is important for the regulation of its polymerase activity. Investigation of the significance of specific modification of VP1 can lead to better understanding of viral replication and can probably also help in identifying novel targets for antiviral compounds. Our work demonstrates the molecular mechanism of VP1 methylation mediated by PRMT5, which is critical for viral polymerase activity, as well as viral replication. Our study expands a novel insight into the function of arginine methylation of VP1, which might be useful for limiting the replication of IBDV.
Asunto(s)
Virus de la Enfermedad Infecciosa de la Bolsa , Proteína-Arginina N-Metiltransferasas , Replicación Viral , Animales , Línea Celular , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa/enzimología , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Metilación , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Replicación Viral/genética , MutaciónRESUMEN
Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid (aa) protein encoded by ORF51. UL11 is modified by acylation including myristoylation and palmitoylation. Myristoylation of EHV-1 UL11 is assumed to occur on the N-terminal glycine, while palmitoylation is assumed to occur on the seventh and ninth cysteines. ORF51, which encodes the first 24 aa, overlaps ORF50 encoding UL12. We previously demonstrated that UL11 was essential for EHV-1 replication in cultured cells and that UL11 was localized at the Golgi apparatus where herpesviruses obtain their final envelope. It is unclear whether the acylation is related to the localization of EHV-1 UL11 and viral replication. In this study, we investigated the role of UL11 acylation in the intracellular localization and viral growth and replication of EHV-1. We constructed seven UL11 acylation mutant plasmids and seven UL11 acylation mutant BAC DNAs; then, we analysed the localizations of the mutant UL11s and attempted virus rescue. We found that both the N-terminal glycine and the seventh or ninth cysteine, especially N-terminal glycine, were involved in the localization of UL11 and viral replication. Taken together, these results suggest that EHV-1 viral growth requires that UL11 is modified by myristoylation of an N-terminal glycine and by palmitoylation of at least one of the cysteines, and that UL11 is localized at the Golgi apparatus. This study shows that a single amino acid in EHV-1 can determine the fate of viral replication.
Asunto(s)
Herpesvirus Équido 1 , Animales , Caballos , Herpesvirus Équido 1/genética , Glicina/metabolismo , Proteínas Estructurales Virales/metabolismo , Replicación Viral , Línea Celular , Aminoácidos/metabolismo , CisteínaRESUMEN
Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Avibirnavirus in the family Birnaviridae. It can cause serious failure of vaccination in young poultry birds with impaired immune systems. Post-translational modifications of the VP1 protein are essential for viral RNA transcription, genome replication, and viral multiplication. Little information is available so far regarding the exact mechanism of phosphorylation of IBDV VP1 and its significance in the viral life cycle. Here, we provide several lines of evidence that the cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex phosphorylates VP1, which facilitates viral replication. We show that the CDK1-cyclin B1 specifically interacts with VP1 and phosphorylates VP1 on the serine 7 residue, located in the N-terminal 7SPAQ10 region, which follows the optimal phosphorylation motif of CDK1, p-S/T-P. Additionally, IBDV infection drives the cytoplasmic accumulation of CDK1-cyclin B1, which co-localizes with VP1, supporting the kinase activity of CDK1-cyclin B1. Treatment with CDK1 inhibitor RO3306 and knockdown of CDK1-cyclin B1 severely disrupts the polymerase activity of VP1, resulting in diminished viral replication. Moreover, the replication of S7A mutant recombinant IBDV was significantly decreased compared to that of wild-type (WT) IBDV. Thus, CDK1-cyclin B1 is a crucial enzyme which phosphorylates IBDV VP1 on serine 7, which is necessary both for the polymerase activity of VP1 and for viral replication. IMPORTANCE Infectious bursal disease virus still poses a great economic threat to the global poultry farming industry. Detailed information on the steps of viral genome replication is essential for the development of antiviral therapeutics. Phosphorylation is a common post-translational modification in several viral proteins. There is a lack of information regarding the significance of VP1 phosphorylation and its role in modulating the viral life cycle. In this study, we found that CDK1-cyclin B1 accumulates in the cytoplasm and phosphorylates VP1 on serine 7. The presence of a CDK1 inhibitor and the silencing of CDK1-cyclin B1 decrease IBDV replication. The mutation of VP1 serine 7 to alanine reduces VP1 polymerase activity, disrupting the viral life cycle, which suggests that this residue serves an essential function. Our study offers novel insights into the regulatory mechanism of VP1 phosphorylation.
Asunto(s)
Infecciones por Birnaviridae , Proteína Quinasa CDC2 , Ciclina B1 , Virus de la Enfermedad Infecciosa de la Bolsa , Animales , Infecciones por Birnaviridae/virología , Proteína Quinasa CDC2/metabolismo , Línea Celular , Pollos , Ciclina B1/metabolismo , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Fosforilación , Proteínas Estructurales Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.
Asunto(s)
Nucleocápside , Nucleoproteínas , ARN Viral , Virus Sincitial Respiratorio Humano , Empaquetamiento del Genoma Viral , Proteínas Estructurales Virales , Humanos , Nucleocápside/química , Nucleocápside/fisiología , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Fosfoproteínas/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/química , Virus Sincitial Respiratorio Humano/química , Virus Sincitial Respiratorio Humano/fisiología , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismoRESUMEN
Alphaviruses cause significant outbreaks of febrile illness and debilitating multi-joint arthritis for prolonged periods after initial infection. We have previously reported that several host hnRNP proteins bind to the Sindbis virus (SINV) RNAs, and disrupting the sites of these RNA-protein interactions results in decreased viral titers in tissue culture models of infection. Intriguingly, the primary molecular defect associated with the disruption of the hnRNP interactions is enhanced viral structural protein expression; however, the precise underlying mechanisms spurring the enhanced gene expression remain unknown. Moreover, our previous efforts were unable to functionally dissect whether the observed phenotypes were due to the loss of hnRNP binding or the incorporation of polymorphisms into the primary nucleotide sequence of SINV. To determine if the loss of hnRNP binding was the primary cause of attenuation or if the disruption of the RNA sequence itself was responsible for the observed phenotypes, we utilized an innovative protein tethering approach to restore the binding of the hnRNP proteins in the absence of the native interaction site. Specifically, we reconstituted the hnRNP I interaction by incorporating the 20nt bovine immunodeficiency virus transactivation RNA response (BIV-TAR) at the site of the native hnRNP I interaction sequence, which will bind with high specificity to proteins tagged with a TAT peptide. The reestablishment of the hnRNP I-vRNA interaction via the BIV-TAR/TAT tethering approach restored the phenotype back to wild-type levels. This included an apparent decrease in structural protein expression in the absence of the native primary nucleotide sequences corresponding to the hnRNP I interaction site. Collectively, the characterization of the hnRNP I interaction site elucidated the role of hnRNPs during viral infection.