Mutagenesis scanning uncovers evolutionary constraints on tobacco etch potyvirus membrane-associated 6K2 protein

Abstract

RNA virus highmutation rate is a double-edged sword. At the one side,mostmutations jeopardize proteins functions; at the other side,mutations are needed to fuel adaptation. The relevant question then is the ratio between beneficial and deleterious mutations. To evaluate this ratio, we created a mutant library of the 6K2 gene of tobacco etch potyvirus that contains every possible singlenucleotide substitution. 6K2proteinanchors the virus replicationcomplex tothenetwork of endoplasmic reticulummembranes. The librarywas inoculated into the natural hostNicotiana tabacum, allowing competition among all thesemutants and selection of those that are potentially viable. We identified 11 nonsynonymousmutations that remain in the viral population atmeasurable frequencies and evaluated their fitness. Some had fitness values higher than the wild-type and some were deleterious. The effect of these mutations in the structure, transmembrane properties, and function of 6K2 was evaluated in silico. In parallel, the effect of these mutations in infectivity, virus accumulation, symptoms development, and subcellular localizationwas evaluated in the natural host. The α-helix H1 in the N-terminal part of 6K2 turned out to be under purifying selection,whilemost observedmutations affect the link betweentransmembrane α-helices H2 and H3, fusing themintoa longerhelix andincreasingits rigidity. Ingeneral, these changes are associatedwith higherwithin-host fitness and development ofmilder or no symptoms. This finding suggests that in nature selection upon 6K2 may result from a tradeoff between within-host accumulation and severity of symptoms.