Mechanisms and Consequences of Newcastle Disease Virus W Protein Subcellular Localization in the Nucleus or Mitochondria

Abstract

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.

Encoding of a transgene in-frame with a Newcastle disease virus protein increases transgene expression and stability

“…Fully functional RDRP activity also requires N and P proteins [9]. Due to a phenomenon of RNA editing, when the polymerase co-transcriptionally inserts additional nucleotides [10], the P gene in addition to the mRNA coding for P protein is transcribed into two other mRNAs coding for proteins V and W, the antagonists of the anti-viral response and the major determinants of NDV species restriction [11][12][13][14]. The viral polymerase transcribes NDV genes starting from the 3′ end of the genomic RNA so that it starts and stops the transcription of each gene individually.…”

The Influence of Newcastle Disease Virus Major Proteins on Virulence

“…After deletion of its NES, the W protein remains in the nucleus and cannot be sent back to the cytoplasm. We also found that the location of the W protein affects virulence [21] .…”

Development and Scalable Production of Newcastle Disease Virus-Vectored Vaccines for Human and Veterinary Use

“…The genome comprises six transcriptional units, encoding a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutinin-neuraminidase protein (HN) and a large polymerase protein (L) [ 1 ]. In addition, the RNA of the P gene can be edited to generate the V or W proteins, which are non-structural and generally associated with modulating the avian host’s immune response [ 44 ]. Techniques for generating recombinant NDV constructs are already well established, with protocols following the general steps of: antigenomic plasmid construction, transfection, rescue and amplification [ 45 ].…”