RESUMEN
To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.
Asunto(s)
Productos del Gen gag , Virus de la Inmunodeficiencia Felina , Virus de la Inmunodeficiencia de los Simios , Ensamble de Virus , Dedos de Zinc , Animales , Virus de la Inmunodeficiencia Felina/genética , Virus de la Inmunodeficiencia Felina/metabolismo , Virus de la Inmunodeficiencia Felina/fisiología , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Productos del Gen gag/química , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Gatos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Línea Celular , Nucleocápside/metabolismo , Nucleocápside/genética , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , FenotipoRESUMEN
Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4' is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation.
Asunto(s)
Virus del Dengue , Proteínas de la Cápside/genética , Virus del Dengue/genética , Humanos , Nucleocápside/metabolismo , Oligonucleótidos/metabolismo , ARN/metabolismo , Ensamble de VirusRESUMEN
Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.
Asunto(s)
Proteínas de la Cápside/metabolismo , Citrus/virología , Enfermedades de las Plantas/virología , Proteínas de Movimiento Viral en Plantas/metabolismo , Virus de Plantas/metabolismo , Animales , Ácaros/virología , Nucleocápside/metabolismo , Virus de Plantas/patogenicidad , Protoplastos/metabolismo , Protoplastos/virologíaRESUMEN
UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses. IMPORTANCE: Alphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Encefalitis Equina Venezolana/fisiología , Nucleocápside/metabolismo , Virión/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/genética , Línea Celular , Cricetinae , Análisis Mutacional de ADN , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/ultraestructura , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ensayo de Placa Viral , Virión/ultraestructuraRESUMEN
UNLABELLED: The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE: The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be determined. In this report, novel findings are provided on critical interactions between the viral ribonucleoprotein components. We identify several amino acid residues in both the N-terminal and C-terminal domains of TCRV NP that differentially contribute to NP-NP and NP-RNA interactions and analyze their relevance for binding of NP to the L polymerase and for nucleocapsid activity. Our results provide insight into the contribution of NP self-interaction to RNP assembly and activity and reveal the involvement of the NP C-terminal domain in RNA binding.
Asunto(s)
Arenavirus del Nuevo Mundo/metabolismo , Regulación Viral de la Expresión Génica/genética , Modelos Moleculares , Nucleocápside/fisiología , Nucleoproteínas/metabolismo , ARN Viral/metabolismo , Ensamble de Virus/fisiología , Arenavirus del Nuevo Mundo/genética , Secuencia de Bases , Northern Blotting , Western Blotting , Biología Computacional , ARN Polimerasas Dirigidas por ADN/metabolismo , Inmunoprecipitación , Datos de Secuencia Molecular , Mutagénesis , Nucleocápside/metabolismo , Nucleoproteínas/genética , Plásmidos/genética , ARN Viral/biosíntesis , Análisis de Secuencia de ADN , Ensamble de Virus/genéticaRESUMEN
To gain a better understanding of the assembly process in simian immunodeficiency virus (SIV), we first established the conditions under which recombinant SIV Gag lacking the C-terminal p6 domain (SIV GagΔp6) assembled in vitro into spherical particles. Based on the full multimerization capacity of SIV GagΔp6, and to identify the Gag sequences involved in homotypic interactions, we next developed a pull-down assay in which a panel of histidine-tagged SIV Gag truncation mutants was tested for its ability to associate in vitro with GST-SIVGagΔp6. Removal of the nucleocapsid (NC) domain from Gag impaired its ability to interact with GST-SIVGagΔp6. However, this Gag mutant consisting of the matrix (MA) and capsid (CA) domains still retained 50% of the wild-type binding activity. Truncation of SIV Gag from its N-terminus yielded markedly different results. The Gag region consisting of the CA and NC was significantly more efficient than wild-type Gag at interacting in vitro with GST-SIVGagΔp6. Notably, a small Gag subdomain containing the C-terminal third of the CA and the entire NC not only bound to GST-SIVGagΔp6 in vitro at wild-type levels, but also associated in vivo with full-length Gag and was recruited into extracellular particles. Interestingly, when the mature Gag products were analyzed, the MA and NC interacted with GST-SIVGagΔp6 with efficiencies representing 20% and 40%, respectively, of the wild-type value, whereas the CA failed to bind to GST-SIVGagΔp6, despite being capable of self-associating into multimeric complexes.
Asunto(s)
Proteínas de la Cápside , Productos del Gen gag , Nucleocápside , Virus de la Inmunodeficiencia de los Simios/genética , Ensamble de Virus/genética , Animales , Western Blotting , Células COS , Proteínas de la Cápside/análisis , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Chlorocebus aethiops , Mapeo Cromosómico , Ensayo de Inmunoadsorción Enzimática , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Mutación , Nucleocápside/química , Nucleocápside/genética , Nucleocápside/metabolismo , Unión Proteica/genética , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/genética , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas de la Matriz Viral/análisis , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome that encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L), and a RING finger protein (Z), which is the driving force of arenavirus budding. We have established a plasmid-based system which allowed the successful packaging of TacV-like nucleocapsids along with Z and GP of JunV into infectious virus-like particles (VLPs). By coexpressing different combinations of the system components, followed by biochemical analysis of the VLPs, the requirements for the assembly of both N and GP into particles were defined. We found that coexpression of N with Z protein in the absence of minigenome and other viral proteins was sufficient to recruit N within lipid-enveloped Z-containing VLPs. In addition, whereas GP was not required for the incorporation of N, coexpression of N substantially enhanced the ratio of GP to Z into VLPs. Disruption of the RING structure or mutation of residue L79 to alanine within Z protein, although it had no effect on Z self-budding, severely impaired VLP infectivity. These mutations drastically altered intracellular Z-N interactions and the incorporation of both N and GP into VLPs. Our results support the conclusion that the interaction between Z and N is required for assembly of both the nucleocapsids and the glycoproteins into infectious arenavirus budding particles.
Asunto(s)
Infecciones por Arenaviridae/virología , Arenavirus del Nuevo Mundo/fisiología , Glicoproteínas/metabolismo , Nucleocápside/metabolismo , Proteínas Virales/química , Ensamble de Virus , Secuencia de Aminoácidos , Animales , Infecciones por Arenaviridae/metabolismo , Arenavirus del Nuevo Mundo/química , Arenavirus del Nuevo Mundo/genética , Línea Celular , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Datos de Secuencia Molecular , Nucleocápside/química , Nucleocápside/genética , Dominios RING Finger , Alineación de Secuencia , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.
Asunto(s)
Núcleo Celular/metabolismo , Hepacivirus/metabolismo , Nucleocápside/metabolismo , Pichia/metabolismo , Núcleo Celular/ultraestructura , Hepacivirus/ultraestructura , Microscopía Inmunoelectrónica , Nucleocápside/ultraestructura , Pichia/ultraestructuraRESUMEN
Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.
Asunto(s)
Núcleo Celular/metabolismo , Hepacivirus/metabolismo , Hepatitis C Crónica/metabolismo , Hepatocitos/metabolismo , Nucleocápside/metabolismo , Proteínas del Núcleo Viral/metabolismo , Núcleo Celular/ultraestructura , Humanos , Microscopía ElectrónicaRESUMEN
Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV.
Asunto(s)
Arenavirus del Nuevo Mundo/inmunología , Glicoproteínas/inmunología , Fiebre Hemorrágica Americana/prevención & control , Virus Junin , Proteínas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Antígenos Virales/inmunología , Arenavirus del Nuevo Mundo/genética , Arenavirus del Nuevo Mundo/metabolismo , Línea Celular , Reacciones Cruzadas , Glicoproteínas/genética , Glicoproteínas/metabolismo , Cobayas , Fiebre Hemorrágica Americana/virología , Inmunización , Virus Junin/genética , Virus Junin/inmunología , Virus Junin/metabolismo , Masculino , Pruebas de Neutralización , Nucleocápside/genética , Nucleocápside/inmunología , Nucleocápside/metabolismo , Pruebas de Precipitina , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus Vaccinia/metabolismoRESUMEN
A nucleocapsid protein of an RNA virus was characterised using computational methods. Similarity searches using standard algorithms and more sensitive methods based on profiles were performed. Also, secondary structure prediction and statistical methods were used. The results show that the protein belongs to a unique well-characterised family, with three regions with potential RNA binding capacity. The amino-terminal region is found to contain a mixed-charge segment similar to proteins that bear nucleic acid-protein interaction capacity. The middle-region has a slight homology to the nucleolar protein Fibrillarin containing an atypical RNP-1 conserved octamer. Finally, the carboxyl-terminal region has a putative zinc-finger.