West Nile Virus-Induced Activation of Mammalian Target of Rapamycin Complex 1 Supports Viral Growth and Viral Protein Expression

Abstract

Since its introduction in New York City, NY, in 1999, West Nile virus (WNV) has spread to all 48 contiguous states of the United States and is now the leading cause of epidemic encephalitis in North America. As a member of the family Flaviviridae, WNV is part of a group of clinically important human pathogens, including dengue virus and Japanese encephalitis virus. The members of this family of positive-sense, single-stranded RNA viruses have limited coding capacity and are therefore obligated to co-opt a significant amount of cellular factors to translate their genomes effectively. Our previous work has shown that WNV growth was independent of macroautophagy activation, but the role of the evolutionarily conserved mammalian target of rapamycin (mTOR) pathway during WNV infection was not well understood. mTOR is a serine/threonine kinase that acts as a central cellular censor of nutrient status and exercises control of vital anabolic and catabolic cellular responses such as protein synthesis and autophagy, respectively. We now show that WNV activates mTOR and cognate downstream activators of cap-dependent protein synthesis at early time points postinfection and that pharmacologic inhibition of mTOR (KU0063794) significantly reduced WNV growth. We used an inducible Raptor and Rictor knockout mouse embryonic fibroblast (MEF) system to further define the role of mTOR complexes 1 and 2 in WNV growth and viral protein synthesis. Following inducible genetic knockout of the major mTOR cofactors raptor (TOR complex 1 [TORC1]) and rictor (TORC2), we now show that TORC1 supports flavivirus protein synthesis via cap-dependent protein synthesis pathways and supports subsequent WNV growth. IMPORTANCE Since its introduction in New York West Nile virus (WNV) is an enveloped, single-stranded, positive-sense RNA virus in the genus Flavivirus, which includes multiple clinically important viral species such as dengue virus, yellow fever virus, and Japanese encephalitis virus. Since the first North American outbreak in New York City, NY, in 1999 (1), WNV has spread across the continent to become the leading cause of epidemic encephalitis (2). To date, there have been more than 37,000 confirmed cases of WNV disease, 16,000 cases of neuroinvasive disease, and 1,500 fatalities (www.cdc.gov/westnile). Currently, there is no licensed human vaccine or pharmacologic therapy for WNV. Owing to difficulties in predicting the location and timing of WNV outbreaks, insufficient enrollment of WNV-infected patients has complicated human clinical trial design for candidate vaccines and therapeutic interventions. A better understanding of the molecular pathogenesis of flaviviruses and the mechanisms behind how they successfully compete with host messages for access to translational components may reveal broad-spectrum antiflaviviral targets that can be evaluated and licensed for treatment of acute flaviviral infections.Due to the high mutation rates of RNA viral genomes and their subsequent ability to rapidly generate escape mutations, we ...

Alpha-Synuclein Expression Restricts RNA Viral Infections in the Brain

“…1). Primary striatal neurons were prepared and virus was inoculated as previously described (30). At 12 and 24 h postinoculation, the level of striatal neuron expression of the Asyn protein increased by 2-fold compared to that for the mock-infected control (P ϭ 0.086, analysis of variance [ANOVA]; Fig.…”

“…Since WNV infection was associated with increased Asyn protein expression in primary neurons and in the brains of humans with encephalitis, we investigated the effects of Asyn expression on WNV growth and pathogenesis in neurons and in a mouse model of WNV encephalitis as previously described (29,30,32). We obtained Snca Ϫ/Ϫ mice (The Jackson Laboratory) and bred them with wild-type C57BL/6 mice with an Snca ϩ/ϩ genotype to make F1 Snca Ϫ/ϩ mice.…”

“…To define the role of Asyn in virus-induced neuronal injury, we utilized primary cortical neuronal cultures as previously described (29,30). Additionally, we created a new model of WNV infection in primary mouse striatal neurons and compared viral growth, infectivity, and injury in these two neuronal subtypes in culture.…”

“…Whole-cell lysate preparation and Western blot (WB) analysis were performed as previously described (29,30). Cells were suspended in lysis buffer consisting of 1% Triton X-100, 10 mM triethanolamine-HCl, 150 mM NaCl, 5 mM EDTA, 1ϫ Halt protease, and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL) and disrupted using an ultrasonic processor (VCX130; Sonics & Materials).…”

Zika Virus

“…Diğer yapısal olmayan proteinlerin replikasyon aşamalarında önemli gö-revler üstlendiği düşünülmektedir (4)(5)(6) . Virüsün RNA genomu konak hücrenin mRNA'sına benzer yapıda olup, kolaylıkla yerine geçerek, kendi viral proteinleri ve RNA genomunu kodlayabilmektedir (7) . Virüs zarf glikoproteninin hücre reseptörüne tutunması (insanda: DIC-SIGN, Tyro3, AXL) sonucu hücreye endozom yardımıyla girer.…”