West Nile Virus (WNV) is a single stranded positive sense RNA virus of the genus Flavivirus. The 11Kb RNA genome is translated in the cytoplasm as a polyprotein and processed to yield 3 structural (Capsid C, Premembrane prM/membrane M and Envelope E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins . Co-expression of prM and E proteins alone is sufficient for production of recombinant VLPs  that are similar to infectious virions in antigenic properties and have been commonly used to study virus assembly and budding. Although the field of Flavivirus assembly and release remains in its infancy, recent reports have identified certain residues in the prM that are important for WNV particle secretion [3, 4]. It is known that WNV genome replication occurs in the cytoplasm in the perinuclear region and virus particles assemble and bud into the Endoplasmic Reticulum (ER) lumen. Subsequently virions are transported to the plasma membrane (PM) via the cellular secretory pathway to be released from cells by exocytosis [5–8].
Following the synthesis of viral genome and proteins, enveloped viruses utilize cellular membranes to bud from infected cells. This is often facilitated by the presence of certain conserved motifs within viral proteins and their ability to interact with the cellular processes/machinery. The best known example of this process is the interaction of retroviral late domain motifs with components of the ESCRT (Endosomal Sorting Complex Required for Transport) sorting machinery to promote budding. Three types of consensus late domain motifs have been identified thus far: (i) the PT/SAP motif recruits the ESCRT-1 component Tsg101 (Tumor Susceptibility growth factor 101) [9, 10], (ii) the YXXL late domain motif interacts with the ESCRT associated protein Alix [11, 12] and the (iii) PPXY late domain motif binds to the Nedd4 family of E3 ubiquitin ligases that are involved in cargo recruitment during Multivesicular Body (MVB) formation [13, 14]. Besides retroviruses, late domain motifs have also been identified in other enveloped viruses like rhabdoviruses (vesicular stomatitis virus, rabies virus) [15–17], filoviruses (ebola, marburg) [18–22], arenaviruses (lymphocytic choriomeningitis virus, lassa virus) [23, 24], paramyxoviruses (Nipah virus, Sendai virus) [25, 26] and DNA viruses like hepatitis B virus, vaccinia virus, herpes simplex virus-1 and Epstein Barr virus [27–33]. Amongst flaviviruses, the NS3 of Japanese encephalitis virus (JEV) has been shown to associate with Tsg101  while the yellow fever virus (YFV) NS3 has been shown to interact with Alix  assisting in virus release. However, currently there is no information on the presence of late domains in WNV proteins.
The process of WNV budding into the lumen of the ER is topologically similar to the process of MVB biogenesis in that both occur in a direction that is away from the cytosol. MVB biogenesis is mediated by the family of ESCRT proteins namely ESCRT-0, -I, -II and -III and other associated proteins like Alix/AIP1. The membrane associated ESCRT-III complexes are finally disassembled and recycled by the ATPase Vps4. A number of enveloped viruses via the conserved late (L) domain motifs that mimic similar motifs in cellular proteins are able to recruit the ESCRT machinery to the site of virus budding . Disruption of L domain motifs or their function leads to defects in the final (late) stages of virus budding characterized by the tethering of virions to the cell surface [9, 14, 36, 37]. Most data on the role of ESCRT proteins and viral late domain motifs has come from research on retroviruses that primarily bud from the plasma membrane. Although there are reports that NS3 of other Flaviviruses can interact with ESCRT components [34, 35] there are no such reports for WNV. Furthermore, it is not known whether any late domain like motifs are present in WNV structural proteins especially E protein that is essential for assembly into virus like particles .