Solution Structure, Self-Assembly, and Membrane Interactions of the Matrix Protein from Newcastle Disease Virus at Neutral and Acidic pH
Eleonora V. ShtykovaMaxim V. PetoukhovLjubov A. DadinovaNatalia V. FedorovaV. Yu. TashkinТ. А. ТимофееваAlexander L. KsenofontovNikita A. LoshkarevLudmila A. BaratovaCy M. JeffriesDmitri I. SvergunOleg V. Batishchev
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Abstract:
Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.Keywords:
VP40
Mononegavirales
Newcastle Disease
Pneumovirinae
The matrix protein (M) of respiratory syncytial virus (RSV), the prototype viral member of the Pneumovirinae (family Paramyxoviridae, order Mononegavirales), has been crystallized and the structure determined to a resolution of 1.6 A. The structure comprises 2 compact beta-rich domains connected by a relatively unstructured linker region. Due to the high degree of side-chain order in the structure, an extensive contiguous area of positive surface charge covering approximately 600 A(2) can be resolved. This unusually large patch of positive surface potential spans both domains and the linker, and provides a mechanism for driving the interaction of the protein with a negatively-charged membrane surface or other virion components such as the nucleocapsid. This patch is complemented by regions of high hydrophobicity and a striking planar arrangement of tyrosine residues encircling the C-terminal domain. Comparison of the RSV M sequence with other members of the Pneumovirinae shows that regions of divergence correspond to surface exposed loops in the M structure, with the majority of viral species-specific differences occurring in the N-terminal domain.
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Respiratory syncytial virus (RSV) is the main cause of severe respiratory infection in young children worldwide, and no therapies have been approved for the treatment of RSV infection. Data from recent clinical trials of fusion or L polymerase inhibitors for the treatment of RSV-infected patients revealed the emergence of escape mutants, highlighting the need for the discovery of inhibitors with novel mechanisms of action. Here we describe stapled peptides derived from the N terminus of the phosphoprotein (P) that act as replication inhibitors. We demonstrate that these peptides inhibit RSV replication in vitro and in vivo by preventing the formation of the N0-P complex. The present strategy provides a novel means of targeting RSV replication with constrained macrocyclic peptides or small molecules and is broadly applicable to other viruses of the Mononegavirales order.
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Human Respiratory Syncytial Virus–the Type Species Pneumovirus Avian pneumovirus (APV)–the Type Species Metapneumovirus Classification Pneumovirus Proteins Metapneumovirus Proteins Polymorphisms among Pneumovirus and Metapneumovirus Virus Replication Virus Assembly Infection and Experimental Animal Models Disease Pathogenesis Diagnosis Management and Treatment Vaccine Development Immunology and Host Factors Immunopathology and the Response to RSV Proteins Epidemiology Perspective
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ABSTRACT We recently developed a system for the generation of infectious bovine respiratory syncytial virus (BRSV) from cDNA. Here, we report the recovery of fully viable chimeric recombinant BRSVs (rBRSVs) that carry human respiratory syncytial virus (HRSV) glycoproteins in place of their BRSV counterparts, thus combining the replication machinery of BRSV with the major antigenic determinants of HRSV. A cDNA encoding the BRSV antigenome was modified so that the complete G and F genes, including the gene start and gene end signals, were replaced by their HRSV A2 counterparts. Alternatively, the BRSV F gene alone was replaced by that of HRSV Long. Each antigenomic cDNA directed the successful recovery of recombinant virus, yielding rBRSV/A2 and rBRSV/LongF, respectively. The HRSV G and F proteins or the HRSV F in combination with BRSV G were expressed efficiently in cells infected with the appropriate chimeric virus and were efficiently incorporated into recombinant virions. Whereas BRSV and HRSV grew more efficiently in bovine and human cells, respectively, the chimeric rBRSV/A2 exhibited intermediate growth characteristics in a human cell line and grew better than either parent in a bovine line. The cytopathology induced by the chimera more closely resembled that of BRSV. BRSV was confirmed to be highly restricted for replication in the respiratory tract of chimpanzees, a host that is highly permissive for HRSV. Interestingly, the rBRSV/A2 chimeric virus was somewhat more competent than BRSV for replication in chimpanzees but remained highly restricted compared to HRSV. This showed that the substitution of the G and F glycoproteins alone was not sufficient to induce efficient replication in chimpanzees. Thus, the F and G proteins contribute to the host range restriction of BRSV but are not the major determinants of this phenotype. Although rBRSV/A2 expresses the major neutralization and protective antigens of HRSV, chimpanzees infected with this chimeric virus were not significantly protected against subsequent challenge with wild-type HRSV. This suggests that the growth restriction of rBRSV/A2 was too great to provide adequate antigen expression and that the capacity of this chimeric vaccine candidate for replication in primates will need to be increased by the importation of additional HRSV genes.
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Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout.
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To determine whether thrombocytosis, a platelet count of more than 500 x 10(9)/l, occurs at an early stage of respiratory tract viral infection.The medical records of 345 patients with respiratory syncytial virus (RSV), influenza, measles, adenovirus or human herpes virus 6 infections were retrospectively reviewed.The mean platelet count was significantly higher in RSV patients than in patients with other respiratory infections. Among the 29 patients with thrombocytosis, 24 (82.8%) had RSV infection.When thrombocytosis is positive at an early stage of respiratory tract infection, RSV should be taken into account as a causative agent.
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Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.
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Mononegavirales
Newcastle Disease
Pneumovirinae
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