RESUMO
Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the "3-bases-in, 3-bases-out" conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123-139, which may serve as a valuable epitope for surveillance and diagnostics.
Assuntos
Vírus Nipah/ultraestrutura , Proteínas do Nucleocapsídeo/ultraestrutura , Nucleocapsídeo/ultraestrutura , Microscopia Crioeletrônica , Modelos Moleculares , Conformação Molecular , Vírus Nipah/química , Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/química , RNA Viral/química , RNA Viral/ultraestruturaRESUMO
Understanding virus assembly mechanisms is important for developing therapeutic interventions. Nipah virus (NiV) is of interest because of its high mortality rate and efficient human-human transmissions. The current model for most enveloped viruses suggests that matrix proteins (M) recruit attachment glycoproteins (G) and fusion glycoproteins (F) to the assembly site at the plasma membrane. Here we report an assembly model that differs in many aspects from the current one. Examining NiV proteins on the cell plasma membrane using super-resolution microscopy reveals that clusters of F and G are randomly distributed on the plasma membrane regardless of the presence or absence of M. Our data suggests a model in which the M molecules assemble at the plasma membrane to form virus-like particles (VLPs), while the incorporation of F and G into the nascent VLPs is stochastic.
Assuntos
Microscopia/métodos , Vírus Nipah/fisiologia , Vírus Nipah/ultraestrutura , Montagem de Vírus , Animais , Linhagem Celular , Membrana Celular/virologia , Glicoproteínas/metabolismo , Células HeLa , Humanos , Transfecção , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/ultraestrutura , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/ultraestrutura , Vírion/metabolismoRESUMO
UNLABELLED: Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on NiV strain Malaysia. Using an enhanced green fluorescent protein (eGFP)-expressing M protein-deleted NiV, we observed a slightly increased cell-cell fusion, slow replication kinetics, and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV but is necessary for proper assembly and release of stable infectious NiV particles. IMPORTANCE: Henipaviruses cause a severe disease with high mortality in human patients. Therefore, these viruses can be studied only in biosafety level 4 (BSL-4) laboratories, making it more challenging to characterize their life cycle. Here we investigated the role of the Nipah virus matrix protein in virus-mediated cell-cell fusion and in the formation and release of newly produced particles. We found that even though low levels of infectious viruses are produced in the absence of the matrix protein, it is required for the release of highly infectious and stable particles. Fusogenicity of matrixless viruses was slightly enhanced, further demonstrating the critical role of this protein in different steps of Nipah virus spread.
Assuntos
Vírus Nipah/fisiologia , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Liberação de Vírus , Animais , Linhagem Celular , Deleção de Genes , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Vírus Nipah/genética , Vírus Nipah/efeitos da radiação , Vírus Nipah/ultraestrutura , Genética Reversa , Temperatura , Carga Viral , Proteínas da Matriz Viral/genética , Vírion/ultraestrutura , Cultura de Vírus , Replicação ViralRESUMO
Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease.
Assuntos
Vírus Nipah/imunologia , Vacinas Virais/imunologia , Vírion/imunologia , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Gigantes/virologia , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos BALB C , Vírus Nipah/ultraestrutura , Plasmídeos/genética , Reação em Cadeia da Polimerase , Transdução de Sinais/genética , Especificidade da Espécie , Transcrição Gênica , Transfecção , Proteínas Virais/metabolismo , Vírion/ultraestruturaRESUMO
The nucleocapsid protein of Nipah virus produced in Escherichia coli assembled into herringbone-like particles. The amino- and carboxy-termini of the N protein were shortened progressively to define the minimum contiguous sequence involved in capsid assembly. The first 29 aa residues of the N protein are dispensable for capsid formation. The 128 carboxy-terminal residues do not play a role in the assembly of the herringbone-like particles. A region with amino acid residues 30-32 plays a crucial role in the formation of the capsid particle. Deletion of any of the four conserved hydrophobic regions in the N protein impaired capsid formation. Replacement of the central conserved regions with the respective sequences from the Newcastle disease virus restored capsid formation.
Assuntos
Capsídeo/fisiologia , Vírus Nipah/genética , Proteínas do Nucleocapsídeo/genética , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Primers do DNA , Escherichia coli/virologia , Dados de Sequência Molecular , Mutagênese , Vírus Nipah/ultraestrutura , Proteínas do Nucleocapsídeo/ultraestrutura , Deleção de SequênciaRESUMO
In 1998, a novel paramyxovirus (order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae, genus Henipavirus) emerged in peninsular Malaysia causing fatal encephalitis in humans and severe respiratory illness with encephalitis in pigs. The virus was successfully isolated in cultured mammalian cells. Transmission electron microscopy of infected tissue culture cells played a crucial role in the early preliminary identification of the causative agent of the outbreak. This in turn was pivotal to determine the correct direction of control measures that subsequently brought the epidemic under control. In light of this investigation, and indeed identification of infectious agents associated with other disease episodes, electron microscopy will remain an important frontline method for rapid diagnostic virology and investigation of any future outbreak of new and unusual cases of illness suspected of an infectious aetiology.
Assuntos
Surtos de Doenças , Infecções por Henipavirus/epidemiologia , Vírus Nipah/isolamento & purificação , Animais , Chlorocebus aethiops , Infecções por Henipavirus/diagnóstico , Humanos , Microscopia Eletrônica , Vírus Nipah/ultraestrutura , Células VeroRESUMO
Matrix (M) proteins reportedly direct the budding of paramyxoviruses from infected cells. In order to begin to characterize the assembly process for the highly lethal, emerging paramyxovirus Nipah virus (NiV), we have examined the budding of NiV M. We demonstrated that expression of the NiV M protein is sufficient to produce budding virus-like particles (VLPs) that are physically and morphologically similar to NiV. We identified in NiV M a sequence, YMYL, with similarity to the YPDL late domain found in the equine infectious anemia virus Gag protein. When the YMYL within NiV M was mutated, VLP release was abolished and M was relocalized to the nucleus, but the mutant M proteins retained oligomerization activity. When YMYL was fused to a late-domain mutant of the Ebola virus VP40 matrix protein, VP40 budding was restored. These results suggest that the YMYL sequence may act as a trafficking signal and a late domain for NiV M.