Revealing Effective Antiviral Targets: Unraveling the Mechanisms Behind the Recruitment of West Nile Virus Components to the Replication Complexes

Tseng, Alanna
Kaufusi, Pakieli
Molecular Biosciences and Bioengineering
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West Nile virus (WNV) is a global mosquito-borne pathogen that poses a major threat to public health. WNV preferentially infects neuronal cells in the central nervous system and can cause encephalitis, meningitis or paralysis, which can lead to long-term neurocognitive impairment. Infection of WNV is characterized by the reorganization of the rough endoplasmic reticulum (RER) membranes to create unique intracellular compartments known as replication organelles (RO). These RO comprise the viral non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) for robust virus replication. The RO consists of vesicle packets (VP) and convoluted membranes (CM) compartments, which serve as the putative sites for RNA synthesis and proteolytic processing, respectively. Out of all of the NS proteins, only the two largest NS proteins, NS3 and NS5, have clearly defined functions and contain all of the enzymatic roles required for viral RNA replication. NS3 consists of protease and RNA helicase domains while NS5 harbors the methyltransferase and RNA-dependent RNA polymerase domains. Given that NS3 and NS5 are soluble proteins that must associate with the RO to promote efficient virus replication, how NS3 and NS5 are recruited from the cytoplasm and retained at the ER-derived RO compartments remains unclear. Previous studies have shown that other NS proteins may contribute to the functional activation of NS3 and NS5, but the mechanistic details of these interactions at the RO are unknown. Furthermore, how the RO structures are initially formed has not yet been elucidated. In this dissertation, we characterized the viral NS protein(s) that (1) promote the ER association and RO compartment localization of NS3 and NS5 and (2) induce RO formation in human embryonic kidney cells (HEK293T). Using high-resolution confocal microscopy (HRCM) and biochemical assays, we demonstrate that soluble NS3 appeared to be peripherally recruited to the ER and was proteolytically active when NS2B was provided in trans. Therefore, we reveal the potential dual role for NS2B in not only serving as the cofactor for NS3 enzymatic activity, but also in recruiting NS3 to the ER membrane for RO association. In addition, we observed that NS5 did not localize to the ER in the presence of other viral NS proteins using HRCM. However, NS1 or NS3 appeared to partially influence the membrane association of NS5 in our sucrose gradient centrifugation assay. For NS5 to completely associate with the ER, we therefore propose that other events of the viral life cycle, such as the enwrapment of NS5 by the RO compartments, are important for NS5 retention in the ER. Furthermore, the partial association of NS5 with NS1 led us to examine whether NS1 initiates the VP structures, where NS5 exerts its enzymatic functions. Using transmission electron microscopy (TEM) of transfected HEK293T cells, we clearly demonstrate that expression of NS1 alone remodeled the ER membrane to form vesicles that were reminiscent of those formed during WNV infection. To the extent of our knowledge, this is the first report showing that WNV NS1, in the absence of other viral NS proteins, initiates VP formation. Taken together, these studies provide valuable insight on the physical and enzymatic link between NS1, NS3, and NS5 in VP localization and biogenesis, which will aid in the identification of novel targets for antiviral drug therapy.
Molecular biology, Virology, Flaviviruses, NS1, NS3, NS5, Replication organelles, West Nile virus
171 pages
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