There is compelling evidence that enveloped virions carry multiple host proteins both internally and externally during infection . To date, no studies have been carried out on the incorporation of cellular proteins in NDV virions. In this study, we obtained highly purified NDV particles by sucrose gradients ultracentrifugation. Virion-associated proteins were identified by 2-DE/MS proteomic analysis followed by Western blot and electron microscopy. A total of five viral proteins and thirty host proteins were successfully detected. Our study provided strong evidence that cellular proteins were incorporated into the enveloped viruses.
The present study identified all the structural constitutes of NDV virions except L protein. F and HN are two major glycosidoproteins located on the surface of membrane, which are easy to detect in intact virions. M protein is also easy to identify because it is the most abundant structural protein produced throughout the process of the virus infection. The RNP complex of NDV particle contains three inner protein components, the major structural subunit (NP) and two associated proteins (P and L) binding together to RNA genome. Both P and NP protein were successfully obtained due to their comparatively higher expression level and smaller molecular weight; whereas the identification of L protein by MS was a difficult task, possibly due to its low-abundant in the virion and large molecular weight (about 220 kD).
Of the thirty host cellular proteins associated with purified NDV particles we have identified, a significant number of proteins have also been reported to be present in virions of other virus families, such as herpes viruses, poxviruses and retroviruses [14–16]. Considering that these studies were performed independently using different cell types and different mass spectrometry methods, this similarity is probably not an issue of contamination. The most likely explanation is that these viruses all share some fundamental feature and that these host proteins are involved in the processes associated with that common trait. Enveloped viruses enter the cell via a membrane fusion manner and exit by budding. Therefore, one hypothesis would be that these common incorporated host proteins play a role in the entry and release stages of the virus life cycle.
Enveloped viruses acquire their membranes through budding from the host cell, thus cytoskeleton proteins may be integrated inside the virions because of their propinquity to viral assembly and budding sites. Our virion proteomics identified 9 host cytoskeleton system proteins in purified NDV particles, which were the most abundant group of cellular proteins, including Tropomyosin 1 alpha, actin, actin -related protein 3 ARP3, tubulin alpha-1, annexin A2, ezrin, CAP-GLY domain containing linker protein 2, capping protein (Actin filament) muscle Z-line and KIAA0174. Numerous viral proteins interact with cytoskeleton elements. Available evidences indicate that host cytoplasm cytoskeleton components are involved in virus transport in cells, especially in the stages of virus entry and release . Several studies have also indicated that cytoskeleton proteins such as Tubulin and Actin are required for viral gene expression [46, 47] and are involved in several virus budding processes . Interestingly, actin was originally thought as a cellular contaminant, but later demonstrated to be an internal component of the measles virus [49, 50]. In a number of viruses, such as HIV and moloney murine leukemia virus (MMLV), actin is important during their budding [51–53]. For influenza virus, actin plays indispensable roles during the endocytosis of the virus into polarized epithelia . An association of M with cytoskeleton elements has been reported , which indicates an essential function of actin in the replication cycle of coronavirus IBV. As for NDV, early studies have suggested that the cellular cytoskeletal framework actively participated in the structural and functional assembly of NDV transcriptive complex . Therefore, cytoplasm cytoskeleton-associated proteins might take part in the assembly and budding process of newly formed NDV virions, contribute to the transportation of the virus to the correct location of host cell, and also participate in assembling the RNP complex.
Annexins are a well-known multigene family of Ca2+ regulated phospholipid-binding and membrane binding proteins with diverse functions. The presence of annexin A2 is thought to support viral binding, fusion and replication [57–61]. In the present study, cellular annexin A2 was also identified in purified NDV virions, which has been found to be endogenously associated with HCMV, HIV, IV virions, and herpes simplex virus 1 [31, 35, 40, 62]. The exact role of annexin A2 in NDV life cycle needs to be further investigated.
Heat-shock proteins (HSP), known as molecular chaperones, has been identified in a number of envelope viruses. Several viruses require host molecular chaperones for entry, replication, and assembly, as well as other steps in viral production [63, 64]. In this study, we identified two molecular chaperones incorporated into purified NDV particles, HSP70 and HSP90. HSP70 interacts with various viral proteins and may be involved in the assembly of adenovirus , enterovirus , vaccinia virus  and hantaan virus . Virion-associated HSP70 might participate in early events of infection, uncoating the viral capsid in a manner similar to its role in the uncoating of clathrin cages. HSP70 and HSP90 have been shown to interact with hepatitis B virus reverse transcriptase and to facilitate the initiation of viral DNA synthesis from hepatitis B virus pregenomic RNA [66, 67]. HSP90, which can cooperate with other proteins such as p23 and HSP70, has 2–4 copies existing internally in a duck liver virus particle, and might be related to interaction between virus polymerase . Besides, it has been proposed that HSP90 is a major host factor for viral replication of many RNA viruses , implying a important role of HSP90 in NDV replication.
In this study, NDV virion proteomic analysis revealed four chromatin modifying proteins, including chromatin modifying protein 2A, 4B, 4 C and 5. It was reported that they can be expressed in chicken bursal lymphocytes, and may be associated with regulating a variety of gene expression in lymphocytes . Chromatin modifying proteins have also been found in KSHV by virion proteomics , providing a number of clues and potential links to understanding the mechanisms regulating the replication, transcription, and genome maintenance of KSHV. Therefore, NDV virion might modulate the gene expression of host cells through binding with chromatin modifying proteins for better propagation.
According to our investigation, three enzymes-binding proteins were identified, including tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; epsilon polypeptide and theta polypeptide, which have also been found expressed in chicken bursal lymphocytes , and may be related to the metabolic pathways during embryo development . Meanwhile, two calcium-binding proteins, calcium-binding protein A6 and A11 were associated with the purified NDV particles. The calcium-binding proteins play a vital role in the regulation of cellular growth and signal transduction pathways; however, their effect on virus infection remains to be investigated [31, 35, 38].
Among the indentified cellular proteins in our study, three have not yet been reported in other viruses, including two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52), which have not been described to be present in other virions of quite diverse virus families. Previous work has identified syntenin of the shrimp Penaeus monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection through its interaction with alpha-2-macroglobulin (alpha2M), which plays an important role in the immune defense mechanisms of viral infections of shrimps . Ras small GTPase is a very important host signaling mediator, regulating the replication of viruses [73, 74]. The molecular mechanisms of these two signaling mediators are largely unknown.