Requirements for the Assembly and Release of Newcastle Disease Virus-Like Particles

ABSTRACT Paramyxoviruses, such as Newcastle disease virus (NDV), assemble in and bud from plasma membranes of infected cells. To explore the role of each of the NDV structural proteins in virion assembly and release, virus-like particles (VLPs) released from avian cells expressing all possible combinations of the nucleoprotein (NP), membrane or matrix protein (M), an uncleaved fusion protein (F-K115Q), and hemagglutinin-neuraminidase (HN) protein were characterized for densities, protein content, and efficiencies of release. Coexpression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3 and 83.8% ± 1.1%, respectively) similar to those of authentic virions. Expression of M protein alone, but not NP, F-K115Q, or HN protein individually, resulted in efficient VLP release, and expression of all different combinations of proteins in the absence of M protein did not result in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F, and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by coimmunoprecipitation. The colocalization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M-HN and M-NP interactions are responsible for incorporation of HN and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein.

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Solution Structure, Self-Assembly, and Membrane Interactions of the Matrix Protein from Newcastle Disease Virus at Neutral and Acidic pH

Journal of Virology ◽

10.1128/jvi.01450-18 ◽

2018 ◽

Vol 93 (6) ◽

Cited By ~ 1

Author(s):

E. V. Shtykova ◽

M. V. Petoukhov ◽

L. A. Dadinova ◽

N. V. Fedorova ◽

V. Yu Tashkin ◽

...

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Solution Structure ◽

Matrix Protein ◽

Viral Proteins ◽

Acidic Ph ◽

Binding Properties ◽

The Matrix ◽

Ph Conditions

ABSTRACTNewcastle 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.IMPORTANCEThe matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to theAvulavirussubfamily of theParamyxoviridaefamily ofMononegaviralesthat include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here, we investigate the solution structure and membrane binding properties of this protein at both acidic and neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals for the possible use of the virus as an adequate template for future drug or vaccine delivery.

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Chicken bromodomain-containing protein 2 interacts with the Newcastle disease virus matrix protein and promotes viral replication

Veterinary Research ◽

10.1186/s13567-020-00846-1 ◽

2020 ◽

Vol 51 (1) ◽

Author(s):

Zhiqiang Duan ◽

Yifan Han ◽

Lei Zhou ◽

Chao Yuan ◽

Yanbi Wang ◽

...

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Transcriptional Control ◽

Rna Synthesis ◽

Matrix Protein ◽

Viral Rna ◽

M Protein ◽

Dependent Manner ◽

Binding Studies

Abstract Bromodomain-containing protein 2 (BRD2) is a nucleus-localized serine-threonine kinase that plays pivotal roles in the transcriptional control of diverse genes. In our previous study, the chicken BRD2 (chBRD2) protein was found to interact with the Newcastle disease virus (NDV) matrix (M) protein using a yeast two-hybrid screening system, but the role of the chBRD2 protein in the replication of NDV remains unclear. In this study, we first confirmed the interaction between the M protein and chBRD2 protein using fluorescence co-localization, co-immunoprecipitation and pull-down assays. Intracellular binding studies indicated that the C-terminus (aa 264–313) of the M protein and the extra-terminal (ET) domain (aa 619–683) of the chBRD2 protein were responsible for interactions with each other. Interestingly, although two amino acids (T621 and S649) found in the chBRD2/ET domain were different from those in the human BRD2/ET domain and in that of other mammals, they did not disrupt the BRD2-M interaction or the chBRD2-M interaction. In addition, we found that the transcription of the chBRD2 gene was obviously decreased in both NDV-infected cells and pEGFP-M-transfected cells in a dose-dependent manner. Moreover, small interfering RNA-mediated knockdown of chBRD2 or overexpression of chBRD2 remarkably enhanced or reduced NDV replication by upregulating or downregulating viral RNA synthesis and transcription, respectively. Overall, we demonstrate for the first time that the interaction of the M protein with the chBRD2 protein in the nucleus promotes NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. These results will provide further insight into the biological functions of the M protein in the replication of NDV.

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A single amino acid mutation, R42A, in the Newcastle disease virus matrix protein abrogates its nuclear localization and attenuates viral replication and pathogenicity

Journal of General Virology ◽

10.1099/vir.0.062992-0 ◽

2014 ◽

Vol 95 (5) ◽

pp. 1067-1073 ◽

Cited By ~ 9

Author(s):

Zhiqiang Duan ◽

Juan Li ◽

Jie Zhu ◽

Jian Chen ◽

Haixu Xu ◽

...

Keyword(s):

Viral Replication ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Nuclear Localization ◽

Newcastle Disease ◽

Matrix Protein ◽

M Protein ◽

Single Amino Acid ◽

Basic Residue

The Newcastle disease virus (NDV) matrix (M) protein is a highly basic and nucleocytoplasmic shuttling viral protein. Previous study has demonstrated that the N-terminal 100 aa of NDV M protein are somewhat acidic overall, but the remainder of the polypeptide is strongly basic. In this study, we investigated the role of the N-terminal basic residues in the subcellular localization of M protein and in the replication and pathogenicity of NDV. We found that mutation of the basic residue arginine (R) to alanine (A) at position 42 disrupted M’s nuclear localization. Moreover, a recombinant virus with R42A mutation in the M protein reduced viral replication in DF-1 cells and attenuated the virulence and pathogenicity of the virus in chickens. This is the first report to show that a basic residue mutation in the NDV M protein abrogates its nuclear localization and attenuates viral replication and pathogenicity.

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The association of ribosomal protein L18 with Newcastle disease virus matrix protein enhances viral translation and replication

Avian Pathology ◽

10.1080/03079457.2021.2013435 ◽

2021 ◽

pp. 1-23

Author(s):

Zhiqiang Duan ◽

Hong Tang ◽

Yanbi Wang ◽

Caiqin Zhao ◽

Lei Zhou ◽

...

Keyword(s):

Newcastle Disease Virus ◽

Ribosomal Protein ◽

Disease Virus ◽

Newcastle Disease ◽

Matrix Protein ◽

Viral Translation

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Ubiquitination on Lysine 247 of Newcastle Disease Virus Matrix Protein Enhances Viral Replication and Virulence by Driving Nuclear-Cytoplasmic Trafficking

Journal of Virology ◽

10.1128/jvi.01629-21 ◽

2021 ◽

Author(s):

Tingyu Peng ◽

Xusheng Qiu ◽

Lei Tan ◽

Shengqing Yu ◽

Binghuan Yang ◽

...

Keyword(s):

Viral Replication ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Nuclear Export ◽

Viral Assembly ◽

M Protein ◽

Primary Function ◽

The World ◽

Export Signal

The Newcastle disease virus (NDV) matrix (M) protein is the pivotal element for viral assembly, budding and proliferation. It traffics through the cellular nucleus but performs its primary function in the cytoplasm. To investigate the biological importance of M’s nuclear–cytoplasmic trafficking and the mechanism involved, the regulatory motif nuclear export signal (NES) and nuclear localization signal (NLS) were deeply analyzed. Here, two types of combined NLS and NES signals were identified within NDV-M. The Herts/33-type M was found to mediate efficient nuclear export and stable virus-like particle (VLP) release, while the LaSota-type M was mostly retained in the nuclei and showed retarded VLP production. Two critical residues, 247 and 263, within the motif were identified and associated with nuclear export efficiency. We identified, for the first time, residue 247 as an important monoubiquitination site, the modification of which regulates the nuclear–cytoplasmic trafficking of NDV-M. Subsequently, mutant LaSota strains were rescued via reverse genetics, which contained either single or double amino acid substitutions that were similar to the M of Herts/33. The rescued rLaSota strains rLaSota-R247K, -S263R, and -DM (double mutation) showed about twofold higher HA titers and 10-fold higher EID 50 titers than wild-type (wt) rLaSota. Further, the MDT and ICPI values of those recombinant viruses were slightly higher than that of wt rLaSota probably due to their higher proliferation rates. Our findings contribute to a better understanding of the molecular mechanism of the replication and pathogenicity of NDV, and even those of all other paramyxoviruses. It is beneficial for the development of vaccines and therapies for paramyxoviruses. Importance Newcastle disease virus (NDV) is a pathogen that is lethal to birds and causes heavy losses in the poultry industry worldwide. The World Organization for Animal Health (OIE) ranked ND as the third most significant poultry disease and the eighth most important wildlife disease in the World Livestock Disease Atlas in 2011. The matrix (M) protein of NDV is very important for viral assembly and maturation. It is interesting that M proteins enter the cellular nucleus before performing their primary function in the cytoplasm. We found that NDV-M has a combined nuclear import and export signal. The ubiquitin modification of a lysine residue within this signal is critical for quick, efficient nuclear export and subsequent viral production. Our findings shed new light on viral replication and opens up new possibilities for therapeutics against NDV and other paramyxoviruses; furthermore, we demonstrate a novel approach to improving paramyxovirus vaccines.

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