Adaptor complex-mediated trafficking of Newcastle disease virus fusion protein is regulated by the YLMY motif of its cytoplasmic tail.

Previously, we reported that the mediation of Newcastle disease virus (NDV) pathogenicity by the 524YLMY527 motif depends mainly on the regulation of F protein transport to the cell surface. The virus and host determinants that govern this intracellular trafficking remain unknown. Here, we confirmed that host adaptor protein (AP) complexes are involved in NDV infection using small interfering RNA. The transport of viral F protein to the cell surface depends on host transport proteins. We observed that the trends for host expression of AP complexes AP1M1 and AP2M1 were similar to those of mutated F proteins, especially in the membrane protein. NDV F protein interacted with AP1M1 and AP2M1, and the YLMY motif influenced this interaction. Knockdown of AP1M1 or AP2M1 suppressed the intracellular and extracellular virus titre of mutated-YLMY-motif NDVs, especially rSG10*-F/Y527A and rSG10*-F/Y524AY527A, to varying degrees. Therefore, the YLMY motif regulates AP-mediated viral F protein transportation from the cytoplasm to the cell surface and subsequently affects viral titer. We further found that the YLMY-motif mutants were differently associated with the process of AAK1 and GAK kinase-mediated AP - viral F protein interaction. These data demonstrate that the essential YLMY motif located in the NDV F protein cytoplasmic tail recruits AP to direct the F protein to the cell surface, which is necessary for its ability to affect virus budding. This study provides support for a deeper understanding of virus and host determinants that facilitate virus trafficking, which can be exploited in the design of novel antiviral therapies.

YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity.

Newcastle disease virus (NDV) fusion protein mediates the virus's fusion activity, which is a determinant of NDV pathogenicity. The ectodomain of the F protein is known to have a major impact on fusion, and several reports have also indicated the role of the cytoplasmic tail (CT) in viral entry, F protein cleavage, and fusion, which are regulated by specific motifs. We found a highly conserved tyrosine residue located in the YLMY motif. The tyrosine residues at positions 524 and 527 have different roles in viral replication and pathogenicity and are associated with F protein intracellular processing. Tyrosine residues mutants affect the transportation of the F protein from the endoplasmic reticulum to the Golgi apparatus, resulting in different cleavage efficiencies. F protein is subsequently transported to the cell surface where it participates in viral budding, a process closely related to the distinctions in pathogenicity caused by the tyrosine residues. In addition, the different mutations all led to a hypofusogenic phenotype. We believe that the highly conserved tyrosine residue of the YLMY motif uses a similar mechanism to the tyrosine-based motif (YXXΦ) to regulate F protein transport and thus affect viral replication and pathogenicity. IMPORTANCE The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity. This study has increased our understanding of how NDV virulence is mediated by the F protein and provides a fresh perspective on the role of CT in the virus's life cycle. This information may be useful in the development of NDV as an effective vaccine vector and oncolytic agent.

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-ß 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-ß 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.

Appropriate amount of W protein of avian avulavirus 1 benefits viral replication and W shows strain-dependent subcellular localization.

In order to confirm the existence of W protein in Avian avulavirus 1 (AAvV-1) infected cells, two monoclonal antibodies were prepared. The presence of W protein in cells infected with lentogenic genotype II strain La Sota or velogenic genotype VII strain SG10 was confirmed with immunofluorescence and western blotting assays. WSG10 localized to the cytoplasm, whereas WLa Sota localized to the nucleus. The influence of W protein was investigated in vitro and in vivo with two AAvV-1 strains defective in the W C-terminus. The growth kinetic curves and pathogenicity tests in 3-week-old SPF chickens both showed that the replication abilities of strains with C-terminally deleted W proteins were lower than that of the parental strain. Restoring the appropriate dose of W protein increased the viral titers of these strains. The expression validation and functional exploration of W protein will facilitate our understanding of pathogenic mechanism of AAvV-1.

NP protein and F protein of pigeon paramyxovirus type 1 are associated with its low pathogenicity in chickens.

In this study, we investigated which structural proteins of pigeon paramyxovirus type 1 (PPMV-1) are responsible for its low pathogenicity in chickens. The results revealed that the pathogenicity of the virus is determined by multiple genes. The NP protein and F protein were found to have the strongest individual effect on virulence, and this effect further enhanced when the two proteins were expressed in combination. Our study highlights the influence of the NP and F proteins on the pathogenicity of PPMV-1 in chickens.

Recombinant Newcastle disease virus (NDV) La Sota expressing the haemagglutinin-neuraminidase protein of genotype VII NDV shows improved protection efficacy against NDV challenge.

Intensive vaccination strategies against Newcastle disease (ND) have been implemented in many countries for a long time, but ND outbreaks still occur frequently, with most isolates belonging to genotype VII of Newcastle disease virus (NDV). Many researchers have revealed that vaccines closely matched to epidemic viruses provide better protection. Therefore, using a previously established reverse genetics system, we generated a recombinant NDV vaccine strain (rLa Sota-HN) based on the La Sota vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of genotype VII NDV. The pathogenicity of the recombinant virus was confirmed by the mean death time in 9-day-old specific-pathogen-free embryonated chicken eggs and the intracerebral pathogenicity index in 1-day-old specific-pathogen-free chickens. Subsequently, 1-day-old chickens were immunized with commercial vaccine La Sota and recombinant virus rLa Sota-HN and then challenged with virulent genotype VII NDV strain. The results indicated that recombinant virus rLa Sota-HN provided increased protection of vaccinated chickens from morbidity and mortality, and inhibited the shedding of virulent virus after challenging with genotype VII virus, compared with the conventional vaccine La Sota. Our findings indicated that rLa Sota-HN is a promising vaccine candidate to improve the protection efficiency against ND in chickens, thereby preventing frequent outbreaks of this disease.

Hemagglutinin-Neuraminidase and fusion genes are determinants of NDV thermostability.

Newcastle disease (ND) caused by infections with virulent strains of Newcastle disease virus (NDV) continues to be a threat for poultry industry worldwide. The prospect of developing a thermostable and effective NDV vaccine is still highly desirable. To investigate the determinants of thermostability in NDV, we generated recombinant NDV strains by exchanging viral hemagglutinin-neuraminidase (HN) gene or by mutating the fusion (F) gene. The results showed that the HN and F protein were both determinants of NDV thermostability. With increased thermostability, the HN protein-chimeric virus showed significantly reduced neuraminidase and hemadsorption activities, but its hemolytic activity was retained. We also found that changing the amino acid in the F protein cleavage sites, affected the thermostability as well as the pathogenicity and fusogenic capacity of the virus. Taken together, our results suggest that HN and F proteins both contribute to the thermostability of NDV, and other viral biological activities change as the thermostability of the virus changes. These findings should be of benefit to the development of a thermostable and efficacious NDV vaccine.