Novel antiviral drug targets for screening potential FDA-approved compounds to counter Flavivirus infection
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2025
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Abstract
Dengue virus (DENV) and West Nile virus (WNV) are major global health concerns, with WNV prevalent in the U.S. and DENV cases increasing in the U.S. with cases detected in Hawaii, Florida and Texas. Native Hawaiians, Other Pacific Islanders (NHOPIs), and Filipinos have an elevated risk of severe WNV and DENV disease due to high rates of chronic conditions. Currently, no antiviral treatments exist for flavivirus infections, highlighting the need for new drug targets. NS3, a key enzyme in flavivirus replication, must localize to the replication organelle (RO), composed of convoluted membranes (CMs) and vesicle packets (VPs). This study explores the role of NS4A, NS4B, and NS1 in facilitating NS3's movement and stabilization to the rough endoplasmic reticulum (ER), and in the RO, respectively. HEK293 cells were transfected with NS3 in combination with NS4A, NS4B, or NS4AB, with Sec61β-RFP (an ER marker), under WNV-infected and uninfected conditions. High resolution confocal imaging and Pearson Correlation Coefficient (PCC) analysis demonstrated that neither NS4A, NS4B, or NS4AB is involved in the ER localization of NS3. However, NS3 localizes to the NS4A-, NS4B-, and NS4AB-induced RO (which appears as fluorescent aggregates), indicating a random recruitment event, where cytoplasmic NS3 was enwrapped during RO formation. In infected cells, NS3 in combination with NS4A, NS4B, and NS4AB was additionally recruited to the ER before reaching the RO, as expected due to the presence of other native viral proteins. This observation also suggests that other viral proteins besides NS4A and NS4B play a role in NS3 ER localization and stabilization in the RO. NS1 in combination with NS3 and NS4A, NS4B, NS4AB, or Sec61β-RFP, were assayed in non-permeabilized and permeabilized conditions. NS1 is partially colocalized with NS4A, NS4B, or NS4AB in both the ER and the foci in non-permeabilized conditions. Additionally, NS1 is visually evident on the plasma membrane (PM) as expected for hexameric NS1. NS1 association with these proteins increased in permeabilized conditions, as expected, since this enables lumen-localized NS1 to be more accessible to the antibody staining, but the PM associated hexameric NS1 is visually decreasing. When NS3 was added to these combinations, it was partially visualized in the ER and the RO in both non-permeabilized and permeabilized conditions. These findings suggest two possible mechanisms for NS3 recruitment: (1) random encapsulation during RO formation and (2) a sequential process requiring additional viral factors. NS3 reaches the ER either through random encapsulation by the RO or via a stepwise mechanism involving NS2B. NS3 is possibly retained in the RO with mechanism (1), but it may be partially stabilized in the ER and the RO by NS1, but not NS4A, NS4B, or NS4AB with mechanism (2). Understanding these interactions may help identify new antiviral drug targets for further development of safe and effective anti-flaviviral therapies.
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Virology
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92 pages
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