Mutation of Phenylalanine 23 of Newcastle Disease Virus Matrix Protein Inhibits Virus Release by Disrupting the Interaction between the FPIV L-Domain and Charged Multivesicular Body Protein 4B.

The matrix (M) protein FPIV L-domain is conserved among multiple paramyxoviruses; however, its function and the associated mechanism remain unclear. In this study, the paramyxovirus Newcastle disease virus (NDV) was employed to study the FPIV L-domain. Two recombinant NDV strains, each carrying a single amino acid mutation at the Phe (F23) or Pro (P24) site of 23FPIV/I26 L-domain, were rescued. Growth defects were observed in only the recombinant SG10-F23A (rSG10-F23A) strain. Subsequent studies focused on rSG10-F23A revealed that the virulence, pathogenicity, and replication ability of this strain were all weaker than those of wild-type strain rSG10 and that a budding deficiency contributed to those weaknesses. To uncover the molecular mechanism underlying the rSG10-F23A budding deficiency, the bridging proteins between the FPIV L-domain and endosomal sorting complex required for transported (ESCRT) machinery were explored. Among 17 candidate proteins, only the charged multivesicular body protein 4 (CHMP4) paralogues were found to interact more strongly with the NDV wild-type M protein (M-WT) than with the mutated M protein (M-F23A). Overexpression of M-WT, but not of M-F23A, changed the CHMP4 subcellular location to the NDV budding site. Furthermore, a knockdown of CHMP4B, the most abundant CHMP4 protein, inhibited the release of rSG10 but not that of rSG10-F23A. From these findings, we can reasonably infer that the F23A mutation of the FPIV L-domain blocks the interaction between the NDV M protein and CHMP4B and that this contributes to the budding deficiency and consequent growth defects of rSG10-F23A. This work lays the foundation for further study of the FPIV L-domain in NDV and other paramyxoviruses. IMPORTANCE Multiple viruses utilize a conserved motif, termed the L-domain, to act as a cellular adaptor for recruiting host ESCRT machinery to their budding site. Despite the FPIV type L-domain having been identified in some paramyxoviruses 2 decades ago, its function in virus life cycles and its method of recruiting the ESCRT machinery are poorly understood. In this study, a single amino acid mutation at the F23 site of the 23FPIV26 L-domain was found to block NDV budding at the late stage. Furthermore, CHMP4B, a core component of the ESCRT-III complex, was identified as a main factor that links the FPIV L-domain and ESCRT machinery together. These results extend previous understanding of the FPIV L-domain and, therefore, not only provide a new approach for attenuating NDV and other paramyxoviruses but also lay the foundation for further study of the FPIV L-domain.

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.

Role of Stress Granules in Suppressing Viral Replication by the Infectious Bronchitis Virus Endoribonuclease.

Infectious bronchitis virus (IBV), a γ-coronavirus, causes the economically important poultry disease infectious bronchitis. Cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation. Previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. Here, we aimed to delineate the molecular mechanisms regulating the SG response to pathogenic IBV strain infection. We found that most chicken embryo kidney (CEK) cells formed no SGs during IBV infection and IBV replication inhibited arsenite-induced SG formation. This inhibition was not caused by changes in the integrity or abundance of SG proteins during infection. IBV nonstructural protein 15 (Nsp15) endoribonuclease activity suppressed SG formation. Regardless of whether Nsp15 was expressed alone, with recombinant viral infection with Newcastle disease virus as a vector, or with EndoU-deficient IBV, the Nsp15 endoribonuclease activity was the main factor inhibiting SG formation. Importantly, uridine-specific endoribonuclease (EndoU)-deficient IBV infection induced colocalization of IBV N protein/dsRNA and SG-associated protein TIA1 in infected cells. Additionally, overexpressing TIA1 in CEK cells suppressed IBV replication and may be a potential antiviral factor for impairing viral replication. These data provide a novel foundation for future investigations of the mechanisms by which coronavirus endoribonuclease activity affects viral replication. IMPORTANCE Endoribonuclease is conserved in coronaviruses and affects viral replication and pathogenicity. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal, and reproductive systems, causing millions of dollars in lost revenue to the poultry industry worldwide annually. Mutating the viral endoribonuclease poly(U) resulted in SG formation, and TIA1 protein colocalized with the viral N protein and dsRNA, thus damaging IBV replication. These results suggest a new antiviral target design strategy for coronaviruses.

Development of a Nanoparticle Multiepitope DNA Vaccine against Virulent Infectious Bronchitis Virus Challenge.

To develop a safe and effective nanoparticle (NP) multiepitope DNA vaccine for controlling infectious bronchitis virus (IBV) infection, we inserted the multiepitope gene expression box SBNT into a eukaryotic expression vector pcDNA3.1(+) to construct a recombinant plasmid pcDNA/SBNT. The NP multiepitope DNA vaccine pcDNA/SBNT-NPs were prepared using chitosan to encapsulate the recombinant plasmid pcDNA/SBNT, with a high encapsulation efficiency of 94.90 ± 1.35%. These spherical pcDNA/SBNT-NPs were 140.9 ± 73.2 nm in diameter, with a mean ζ potential of +16.8 ± 4.3 mV. Our results showed that the chitosan NPs not only protected the plasmid DNA from DNase degradation but also mediated gene transfection in a slow-release manner. Immunization with pcDNA/SBNT-NPs induced a significant IBV-specific immune response and partially protected chickens against homologous IBV challenge. Therefore, the chitosan NPs could be a useful gene delivery system, and NP multiepitope DNA vaccines may be a potential alternative for use in the development of a novel, safe, and effective IBV vaccine.

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.

Identification of Novel T-Cell Epitopes on Infectious Bronchitis Virus N Protein and Development of a Multi-epitope Vaccine.

Cellular immune responses play a key role in the control of viral infection. The nucleocapsid (N) protein of infectious bronchitis virus (IBV) is a major immunogenic protein that can induce protective immunity. To screen for potential T-cell epitopes on IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. Four T-cell epitope peptides were identified by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot), intracellular cytokine staining, and carboxyfluorescein succinimidyl ester (CFSE) lymphocyte proliferation assays; among them, three peptides (N211-230, N271-290, and N381-400) were cytotoxic T lymphocyte (CTL) epitopes, and one peptide (N261-280) was a dual-specific T-cell epitope, which can be recognized by both CD8+ and CD4+ T cells. Multi-epitope gene transcription cassettes comprising four neutralizing epitope domains and four T-cell epitope peptides were synthesized and inserted into the genome of Newcastle disease virus strain La Sota between the P and M genes. Recombinant IBV multi-epitope vaccine candidate rLa Sota/SBNT was generated via reverse genetics, and its immune protection efficacy was evaluated in specific-pathogen-free chickens. Our results show that rLa Sota/SBNT induced IBV-specific neutralizing antibody and T-cell responses and provided significant protection against homologous and heterologous IBV challenge. Thus, the T-cell epitope peptides identified in this study could be good candidates for IBV vaccine development, and recombinant Newcastle disease virus-expressing IBV multi-epitope genes represent a safe and effective vaccine candidate for controlling infectious bronchitis. IMPORTANCE T-cell-mediated immune responses are critical for the elimination of IBV-infected cells. To screen conserved T-cell epitopes in the IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. By combining IFN-γ ELISpot, intracellular cytokine staining, and CFSE lymphocyte proliferation assays, we identified three CTL epitopes and one dual-specific T-cell epitope. The value of T-cell epitope peptides identified in the N protein was further verified by the design of an IBV multi-epitope vaccine. Results show that IBV multi-epitope vaccine candidate rLa Sota/SBNT provided cross protection against challenges with a QX-like or a TW-like IBV strain. So, T-cell-mediated immune responses play an important role in the control of viral infection, and conserved T-cell epitopes serve as promising candidates for use in multi-epitope vaccine construction. Our results provide a new perspective for the development of a safer and more effective IBV vaccine.

Coronavirus Endoribonuclease Ensures Efficient Viral Replication and Prevents Protein Kinase R Activation.

Coronavirus (CoV) nsp15 is an endoribonuclease conserved throughout the CoV family. The enzymatic activity and crystal structure of infectious bronchitis virus (IBV) nsp15 are undefined, and the protein's role in replication remains unclear. We verified the uridylate-specific endoribonuclease (EndoU) activity of IBV and found that the EndoU active sites were located in the C-terminus of nsp15 and included His223, His238, Lys278 and Tyr334. We further constructed an infectious clone of the IBV-rSD strain (rSD-wild-type [WT]) and EndoU-deficient IBVs by changing the codon for the EndoU catalytic residues to alanine. Both the rSD-WT and EndoU-deficient viruses propagated efficiently in embryonated chicken eggs. Conversely, EndoU-deficient viral propagation was severely impaired in chicken embryonic kidney cells, which was reflected in the lower viral mRNA accumulation and protein synthesis. After infecting chickens with the parental rSD-WT strain and EndoU-deficient viruses, the EndoU-deficient-virus-infected chickens presented reduced mortality, tissue injury and viral shedding.IMPORTANCE Coronaviruses can emerge from animal reservoirs into naive host species to cause pandemic respiratory and gastrointestinal diseases with significant mortality in humans and domestic animals. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal and reproductive systems, causing millions of dollars in lost revenue worldwide annually. Mutating the viral endoribonuclease resulted in an attenuated virus and prevented protein kinase R activation. Therefore, EndoU activity is a virulence factor in IBV infections, thus providing an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.