A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

Abstract

West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

“…Notably, deletions in the N termini of DENV C proteins have been shown to affect virus particle formation (13). In addition, it has recently been shown that amino acid substitutions in the capsid protein of West Nile virus (WNV) can influence the virus' sensitivity to acidotropic compounds, potentially influencing virus infectivity (83). Our data suggest that interactions of NCL with DENV C may also affect the virus particle.…”

The Role of Deoxycytidine Kinase (dCK) in Radiation-Induced Cell Death

“…Besides that, autophagy induced by IR increased by 397% in pSUPER cells, and only by 134% in dCK knock-down cells, suggesting that dCK could increase IR-induced autophagy (Figure 3B). Ammonium chloride (NH4Cl) is a lysosomal inhibitor which can block organelle acidification and enable assessment of autophagic flux [27]. Western blotting revealed that LC3-II increased in a time-dependent manner (Figure 3C), reaching a peak in 72 h with NH4Cl+IR treatment in pSUPER cells, but there was low expression of LC3-II in dCK knock-down cells.…”

Flavivirus reverse genetic systems, construction techniques and applications: A historical perspective

“…This provides the possibility to investigate the consequences on replicative fitness of the introduction of a single mutation. Accordingly, a large number of genetically defined viral mutants have been constructed to study a variety of flaviviruses, including YFV (Muylaert et al, 1997), DENV (Leardkamolkarn et al, 2010;Matusan et al, 2001), JEV Sumiyoshi et al, 1995;Ye et al, 2012;Zhao et al, 2005), WNV (Arroyo et al, 2004;Martin-Acebes et al, 2013;Van Slyke et al, 2012) or TBEV (Gritsun et al, 2001;Mandl et al, 2000;Yoshii et al, 2004).…”