West Nile virus (WNV) follows an endemic sylvatic cycle between birds, its reservoir, and mosquitoes. Humans are infected by mosquito bite and represent a dead-end host for WNV. Vector entry introduces WNV to dendritic cells and epithelial cells (1) and attachment of WNV in polarized epithelial cells occurs primarily at the apical cell surface (2). Cell tropism, the entry pathway and the spread of the virus, is mediated by calcium-dependent (C-type) lectins DC-SIGN and DC-SIGNR in dendritic and epithelial cells, respectively. DC-SIGN/R are carbohydrate recognition transmembrane proteins on the surface of host cells that recognize and bind to carbohydrates on ligands. Data from Davis et al suggest that WNV propagated in mosquito cell line C6/36 has an increased binding affinity for the attachment factor C-type lectin DC-SIGN, attributed to the N-glycoslyated mannose rich site on C6/36 WNV envelope (E) protein (1). After attachment, virus internalization is mediated by the interaction of WNV E domain 3 proteins with ï¡Vï¢3 integrin host cell receptors (3). Studies suggested that binding of WNV E with ï¡Vï¢3 integrin starts an inter-cellular signaling cascade in which autophosphorylation of FAK triggers co-localization of vinculin, a cytoskeleton membrane cell adhesion protein, creating patches on the host cell surface where an endosomal pit will form (3). Recent data argues that although ï¡Vï¢3 might act as a receptor it is not necessary and that other receptors might play a role in WNV binding (4).
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After initial binding WNV is encased in a clathrin-coated pit, transversed across the plasma membrane, and a pH change in domain II of WNV E protein induces viral-endosomal membrane fusion releasing the genetic information into the cytoplasm (5). In dengue virus, a flavivirus similar to WNV, envelope (E) proteins on the virus capsid mediate episome fusion. Intercellular protons are pumped into the episome inducing conformational changes to E protein, the E protein dimmer trimerizes as it erects vertically, projecting outwards into the episome and lodges into the episome membrane. Further conformational changes to E protein pull the episome membrane towards the virus and fusion between membranes occurs. A funnel is formed and RNA (+) moves into the cytoplasm (6). Evidence suggests that the WNV membrane fusion mechanism is similar to dengue fusion (7) with an optimal pH fusion of 6.3 and cleavage of prM to M might also plays a critical role in uncoating (8).
Once RNA (+) is in the cytoplasm immediate translation occurs. WNV lacks a 3' poly-A tail and therefore must find another means of cyclization to promote translation (9). Data suggests that NS5, RNA-RNA interactions, and other host proteins play a role in facilitation cyclization of WNV (+). Host ribosomes translate WNV (+) into a single polyprotein, which is cleaved by the serine protease complex NS2B-NS3, and mature viral proteins are released. Compartmentalization of translation and transcription is initiated when nonstructural proteins involved in translation, NS1, NS2A, NS3, and NS4A associate with the perinuclear vesicle membranes and NS5 and NS2B, proteins associated with replication, colocalize to the convoluted membranes. Interaction between host protein translation elongation factor Ef-Tu, EF-Ts, and S-1 with NS5, the viral RNA-dependent RNA polymerase, is necessary to synthesize negative WNV strands from WNV (+), which is then transcribed into many positive, single-stranded RNAs simultaneously. Host cell proteins TIA-1 and TIAR bind to the 3' stem-loop of WNV minus strand, which acts as a promoter region for replication (10). Interaction of NS1 and NS4A was shown to play a role in initiation of WNV (-) synthesis while NS5 may play a role in freeing the newly synthesized WNV (+) from WNV (-) (9). Capping nascent RNA (+) is accomplished by NS3 (RNA triphosphatase), a guanylytransferase, and the cap is methylated by NS5 (methyltransferase).
Little is known about WNV capsid assembly and the mechanism by which capsid proteins recruit virus genome for encapsidation. Efficient genome encapsidation may be mediated by the capsid protein (11) and endoplasmic reticulum rearrangements (12). The transformation of cellular membranes such as the endoplasmic reticulum into pores and building platforms may coordinate the construction of a virion via a factory-like assembly line (12). This theory is supported by data showing that viral RNA is shuffled first from the rough endoplasmic reticulum to the smooth endoplasmic reticulum where it is then encapsulated (9). WNV buds at the endoplasmic reticulum and acquires its membrane in the process. Host secretory pathways transport the immature virions to the trans-Golgi network where E and prM viral capsid proteins are modified by cellular furin. Mature virions are transported to the plasma membrane in vesicles along microtubules where they are separated by polarization and egress by exocytosis with the aide of NS1 at the apical of the host cell (9, 2). In addition to secreting mature virions, infected cells also secret slowly sedimenting hemagglutinin (SHA) particles that consist of antigenic subviral particles lodged in the plasma membrane.
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In some individuals systemic WNV infection persists into the nervous system. Inoculation of WNV on two different mice strains yielded equal mortality and unique cell tropism at the same tissues in each strain (13). The data suggest that WNV invades the nervous system with specific cell tropism. It is unclear exactly how the virus transverses the blood-brain-barrier (BBB) but evidence suggests that innate immune response facilitates neuroinvasiveness many ways (14, 15 - 21). During initial infection cytolytic pathways such as Fas and perforin-dependent granule exocytosis are activated and increase inflammation (15). Inflammation loosens the barrier holding out larger particles such as WNV. In addition to inflammation, evidence suggest that cytokine trafficking signals, circulating monocytes, and a Trojan horse mechanism involving T-cells are all involved in transporting WNV across the BBB where it is introduced to neurons (15 - 21). Mice deficient in multiple types of chemokine receptors, including CCR5 and CCL2, show significantly decreased mortality rates (19, 21). However, mice deficient the Ccr2, a chemokine receptor on the surface of many leukocytes, showed increased mortality rates (17). These data suggest that WNV gains access to the nervous system through trafficking monocytes but it is thwarted by other leukocytes.