Mechanisms and consequences of Newcastle disease virus W protein subcellular localization in the nucleus or mitochondria

We previously demonstrated that W proteins from different Newcastle disease virus (NDV) strains localize in either the cytoplasm (e.g., NDV strain SG10) or the nucleus (e.g., NDV strain La Sota). To clarify the mechanism behind these cell localization differences, we overexpressed W protein derived from four different NDV strains or W protein associated with different cellular regions in Vero cells. This revealed that the key region for determining W protein localization is 180–227aa. Further experiments found that there is a nuclear export signal (NES) motif in W protein 211–224aa. W protein could be transported into the nucleus via interaction with KPNA1, KPNA2, and KPNA6 in a nuclear localization signal-dependent manner, and W protein containing an NES was transported back to the cytoplasm in a CRM1-independent manner. Interestingly, we observed that the cytoplasm-localized W protein colocalizes with mitochondria. We rescued the NES-deletion W protein NDV strain rSG10-ΔWC/WΔNES using an NDV reverse genetics system and found that the replication ability, virulence, and pathogenicity of an NDV strain were all higher when the W protein cellular localization was in the nucleus rather than the mitochondria. Further experiments revealed that W protein nuclear localization reduced the expression of IFN-β otherwise stimulated by NDV. Our research reveals the mechanism by which NDV W protein becomes localized to different parts of the cell and demonstrates the outcomes of nuclear or cytoplasmic localization both in vitro and in vivo, laying a foundation for subsequent functional studies of the W protein in NDV and other paramyxoviruses. IMPORTANCE In Newcastle disease virus (NDV), the W protein, like the V protein, is a nonstructural protein encoded by the P gene via RNA editing. Compared with V protein, W protein has a common N-terminal domain but a unique C-terminal domain. V protein is known as a key virulence factor and an important interferon antagonist across the family Paramyxoviridae. In contrast, very little is known about the function of NDV W protein, and this limited information is based on studies of the Nipah virus W protein. Here, we investigated the localization mechanism of NDV W protein and its subcellular distribution in mitochondria. We found that W protein localization differences impact IFN-β production, consequently affecting NDV virulence, replication, and pathogenicity. This work provides new insights on the differential localization mechanism of NDV W proteins, along with fundamental knowledge for understanding the functions of W proteins in NDV and other paramyxoviruses.

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The P gene of Newcastle disease virus does not encode an accessory X protein

Journal of General Virology ◽

10.1099/vir.0.80160-0 ◽

2004 ◽

Vol 85 (8) ◽

pp. 2375-2378 ◽

Cited By ~ 10

Author(s):

Ben Peeters ◽

Paul Verbruggen ◽

Frank Nelissen ◽

Olav de Leeuw

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Accessory Proteins ◽

X Protein ◽

V Protein ◽

Antiviral Responses ◽

P Gene ◽

Infected Cells ◽

Gene Mrna

Many paramyxoviruses encode non-essential accessory proteins that are involved in the regulation of virus replication and inhibition of cellular antiviral responses. It has been suggested that the P gene mRNA of Newcastle disease virus (NDV) encodes an accessory protein – the so-called X protein – by translation initiation at a conserved in-frame AUG codon at position 120. Using a monoclonal antibody that specifically detected the P and X proteins, it was shown that an accessory X protein was not expressed in NDV-infected cells. Recombinant NDV strains in which the AUG was changed into a GCC (Ala) or GUC (Val) codon were viable but showed a reduction in virulence, probably because the amino acid change affected the function of the P and/or V protein.

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Newcastle Disease Virus V Protein Is Associated with Viral Pathogenesis and Functions as an Alpha Interferon Antagonist

Journal of Virology ◽

10.1128/jvi.77.16.8676-8685.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8676-8685 ◽

Cited By ~ 152

Author(s):

Zhuhui Huang ◽

Sateesh Krishnamurthy ◽

Aruna Panda ◽

Siba K. Samal

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Antiviral Effect ◽

Vero Cells ◽

Parental Virus ◽

V Protein ◽

Inhibitory Function ◽

P Gene ◽

Carboxyl Terminal

ABSTRACT Newcastle disease virus (NDV) edits its P gene by inserting one or two G residues at the conserved editing site (UUUUUCCC, genome sense) and transcribes the P mRNA (unedited), the V mRNA (with a +1 frameshift), and the W mRNA (with a +2 frameshift). All three proteins are amino coterminal but vary at their carboxyl terminus in length and amino acid composition. Little is known about the role of the V and W proteins in NDV replication and pathogenesis. We have constructed and recovered two recombinant viruses in which the expression of the V or both the V and W proteins has been abolished. Compared to the parental virus, the mutant viruses showed impaired growth in cell cultures, except in Vero cells. However, transient expression of the carboxyl-terminal portion of the V protein enhanced the growth of the mutant viruses. In embryonated chicken eggs, the parental virus grew to high titers in embryos of different gestational ages, whereas the mutant viruses showed an age-dependent phenomenon, growing to lower titer in more-developed embryos. An interferon (IFN) sensitivity assay showed that the parental virus was more resistant to the antiviral effect of IFN than the mutant viruses. Moreover, infection with the parental virus resulted in STAT1 protein degradation, but not with the mutant viruses. These findings indicate that the V protein of NDV possesses the ability to inhibit alpha IFN and that the IFN inhibitory function lies in the carboxyl-terminal domain. Pathogenicity studies showed that the V protein of NDV significantly contributes to the virus virulence.

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Newcastle Disease Virus V protein interacts with hnRNP H1 to promote viral replication

Veterinary Microbiology ◽

10.1016/j.vetmic.2021.109093 ◽

2021 ◽

pp. 109093

Author(s):

Lina Tong ◽

Zhili Chu ◽

Xiaolong Gao ◽

Mengqing Yang ◽

Fathalrhman Eisa A. Adam ◽

...

Keyword(s):

Viral Replication ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

V Protein

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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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Production, characterization, and epitope mapping of a monoclonal antibody against genotype VII Newcastle disease virus V protein

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.07.009 ◽

2018 ◽

Vol 260 ◽

pp. 88-97 ◽

Cited By ~ 1

Author(s):

Jihong Li ◽

Chunchun Meng ◽

Tingting Ren ◽

Wei Wang ◽

Yaodan Zhang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Epitope Mapping ◽

V Protein ◽

Genotype Vii

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Type I Interferon-Sensitive Recombinant Newcastle Disease Virus for Oncolytic Virotherapy

Journal of Virology ◽

10.1128/jvi.01553-09 ◽

2010 ◽

Vol 84 (8) ◽

pp. 3835-3844 ◽

Cited By ~ 54

Author(s):

Subbiah Elankumaran ◽

Vrushali Chavan ◽

Dan Qiao ◽

Raghunath Shobana ◽

Gopakumar Moorkanat ◽

...

Keyword(s):

Newcastle Disease Virus ◽

Tumor Cells ◽

Disease Virus ◽

Newcastle Disease ◽

Human Cells ◽

Type I ◽

V Protein ◽

Normal Human ◽

Normal Human Cells ◽

Tumor Selective

ABSTRACT Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic because of its potent ability to induce apoptosis. Several studies have demonstrated that NDV is selectively cytotoxic to tumor cells but not normal cells due to defects in the interferon (IFN) antiviral responses of tumor cells. Many naturally occurring strains of NDV have an intact IFN-antagonistic function and can still replicate in normal human cells. To avoid potential toxicity issues with NDV, especially in cancer patients with immunosuppression, safe NDV-oncolytic vectors are needed. We compared the cell killing abilities of (i) a recombinant NDV (rNDV) strain, Beaudette C, containing an IFN-antagonistic, wild-type V protein (rBC), (ii) an isogenic recombinant virus with a mutant V protein (rBC-Edit virus) that induces increased IFN in infected cells and whose replication is restricted in normal human cells, and (iii) a recombinant LaSota virus with a virulent F protein cleavage site that is as interferon sensitive as rBC-Edit virus (LaSota V.F. virus). Our results indicated that the tumor-selective replication of rNDV is determined by the differential regulation of IFN-α and downstream antiviral genes induced by IFN-α, especially through the IRF-7 pathway. In a nude mouse model of human fibrosarcoma, we show that the IFN-sensitive NDV variants are as effective as IFN-resistant rBC virus in clearing the tumor burden. In addition, mice treated with rNDV exhibited no signs of toxicity to the viruses. These findings indicate that augmentation of innate immune responses by NDV results in selective oncolysis and offer a novel and safe virotherapy platform.

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Newcastle Disease Virus V Protein Is a Determinant of Host Range Restriction

Journal of Virology ◽

10.1128/jvi.77.17.9522-9532.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9522-9532 ◽

Cited By ~ 160

Author(s):

Man-Seong Park ◽

Adolfo García-Sastre ◽

Jerome F. Cros ◽

Christopher F. Basler ◽

Peter Palese

Keyword(s):

Newcastle Disease Virus ◽

Host Range ◽

Disease Virus ◽

Newcastle Disease ◽

Range Restriction ◽

V Protein ◽

Species Specific ◽

Chicken Eggs ◽

Host Range Restriction ◽

Embryonated Chicken

ABSTRACT It has been demonstrated that the V protein of Newcastle disease virus (NDV) functions as an alpha/beta interferon (IFN-α/β) antagonist (M. S. Park, M. L. Shaw, J. Muñoz-Jordan, J. F. Cros, T. Nakaya, N. Bouvier, P. Palese, A. García-Sastre, and C. F. Basler, J. Virol. 77:1501-1511, 2003). We now show that the NDV V protein plays an important role in host range restriction. In order to study V functions in vivo, recombinant NDV (rNDV) mutants, defective in the expression of the V protein, were generated. These rNDV mutants grow poorly in both embryonated chicken eggs and chicken embryo fibroblasts (CEFs) compared to the wild-type (wt) rNDV. However, insertion of the NS1 gene of influenza virus A/PR8/34 into the NDV V(−) genome [rNDV V(−)/NS1] restores impaired growth to wt levels in embryonated chicken eggs and CEFs. These data indicate that for viruses infecting avian cells, the NDV V protein and the influenza NS1 protein are functionally interchangeable, even though there are no sequence similarities between the two proteins. Interestingly, in human cells, the titer of wt rNDV is 10 times lower than that of rNDV V(−)/NS1. Correspondingly, the level of IFN secreted by human cells infected with wt rNDV is much higher than that secreted by cells infected with the NS1-expressing rNDV. This suggests that the IFN antagonist activity of the NDV V protein is species specific. Finally, the NDV V protein plays an important role in preventing apoptosis in a species-specific manner. The rNDV defective in V induces apoptotic cell death more rapidly in CEFs than does wt rNDV. Taken together, these data suggest that the host range of NDV is limited by the ability of its V protein to efficiently prevent innate host defenses, such as the IFN response and apoptosis.

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