scholarly journals A Recombinant Newcastle Disease Virus with Low-Level V Protein Expression Is Immunogenic and Lacks Pathogenicity for Chicken Embryos

2001 ◽  
Vol 75 (1) ◽  
pp. 420-428 ◽  
Author(s):  
Teshome Mebatsion ◽  
Stefan Verstegen ◽  
Leonarda T. C. De Vaan ◽  
Angela Römer-Oberdörfer ◽  
Carla C. Schrier
Keyword(s):  
Newcastle Disease Virus ◽  
Newcastle Disease ◽  
Editing Site ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
V Protein ◽  
P Gene ◽  
Chicken Eggs ◽  
Embryonated Chicken

ABSTRACT Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3′-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5,000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

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

2003 ◽  
Vol 77 (17) ◽  
pp. 9522-9532 ◽  
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.

scholarly journals Identification of a Mutation in Editing of Defective Newcastle Disease Virus Recombinants That Modulates P-Gene mRNA Editing and Restores Virus Replication and Pathogenicity in Chicken Embryos

2003 ◽  
Vol 77 (17) ◽  
pp. 9259-9265 ◽  
Author(s):  
Teshome Mebatsion ◽  
Leonie T. C. de Vaan ◽  
Niels de Haas ◽  
Angela Römer-Oberdörfer ◽  
Marian Braber
Keyword(s):  
Newcastle Disease Virus ◽  
Virus Replication ◽  
Newcastle Disease ◽  
Editing Site ◽  
Replication Capacity ◽  
Mrna Editing ◽  
V Protein ◽  
Chicken Embryos ◽  
P Gene ◽  
Gene Mrna

ABSTRACT Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5′-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5′-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5′-A5 stretch (5′-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5′-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5′-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants.

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

2004 ◽  
Vol 85 (8) ◽  
pp. 2375-2378 ◽  
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.

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

2021 ◽  
Author(s):  
Yanling Yang ◽  
Jia Xue ◽  
Qingyuan Teng ◽  
Xiao Li ◽  
Yawen Bu ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Nuclear Localization ◽  
Newcastle Disease ◽  
Nuclear Export ◽  
Protein Localization ◽  
Dependent Manner ◽  
V Protein ◽  
Terminal Domain ◽  
P Gene

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.

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

2003 ◽  
Vol 77 (16) ◽  
pp. 8676-8685 ◽  
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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