Mutagenesis Scanning Uncovers Evolutionary Constraints on Tobacco Etch Potyvirus Membrane-Associated 6K2 Protein

GBE Mutagenesis Scanning Uncovers Evolutionary Constraints on Tobacco Etch Potyvirus Membrane-Associated 6K2 Protein Rub en Gonz alez1,†, Beilei Wu2,5,†, Xianghua Li3,†, Fernando Martınez2,*, and Santiago F. Elena1,4,* 1 Instituto de Biologıa Integrativa de Sistemas (I2SysBio), CSIC-Universitat de València, València, Spain 2 Instituto de Biologıa Molecular y Celular de Plantas (IBMCP), CSIC-Universitat Politècnica de València, València, Spain 3  Geno mica (CRG), The Barcelona Institute of Science and Technology, PRBB, Barcelona, Spain Systems Biology Program, Centre de Regulacio The Santa Fe Institute, Santa Fe, New Mexico 5 Present address: Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China † These authors contributed equally to this work. *Corresponding authors: E-mails: ; . Accepted: March 26, 2019 Abstract RNA virus high mutation rate is a double-edged sword. At the one side, most mutations 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. 6K2 protein anchors the virus replication complex to the network of endoplasmic reticulum membranes. The library was inoculated into the natural host Nicotiana tabacum, allowing competition among all these mutants and selection of those that are potentially viable. We identified 11 nonsynonymous mutations that remain in the viral population at measurable 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 localization was evaluated in the natural host. The a-helix H1 in the N-terminal part of 6K2 turned out to be under purifying selection, while most observed mutations affect the link between transmembrane a-helices H2 and H3, fusing them into a longer helix and increasing its rigidity. In general, these changes are associated with higher within-host fitness and development of milder 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. Key words: bulk selection, mutagenesis, Potyvirus, TEV, virulence, virus fitness. Introduction Mutation, Selection, and Mutant Swarms in RNA Virus Populations RNA viruses exist as complex mutant swarms that result from the combination of three factors: high mutation rates, very short generation times, and very large population sizes (Domingo et al. 2012). These mutant swarms are commonly referred in the virological literature as viral quasispecies (Domingo et al. 2012). Quasispecies theory represent a twist of the classic mutation-selection balance concept from population genetics (Wilke 2005) in which high mutation rates ensure the coupling between genotypes and thus selection operates not on the individual genotype but on the cloud of mutants that are all linked by one or few mutational steps (Bull et al. 2005). The mutant swarm is dominated by a master sequence with the higher fitness surrounded by a cloud of mutants in frequencies that rank according to their fitness. One of the principal tenants of the quasispecies theory is that viral populations replicate close to the so-called error threshold, that is, the highest mutation rate compatible with maintaining genetic information and which is usually proportional to the inverse of the genome length (Bull et al. 2005; Domingo et al. 2012). Increases in mutation rate over the error threshold push the viral populations into a regime known as the error catastrophe in which the frequency of genotypes in the population is not proportional anymore to ß The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Genome Biol. Evol. 11(4):1207–1222. doi:10.1093/gbe/evz069 Advance Access publication March 27, 2019 1207 4 GBE Gonzalez et al. 1208 Garcıa 2015). An additional peptide translates from a small ORF that results from a þ 2 slippage within the P3 cistron (Chung et al. 2008). The 6K2, which receives its name from its molecular weight of 6 kDa, is a small protein of only 53 amino acids with a transmembrane region encompassing residues about 20–42 (Schaad et al. 1997). There is another protein with similar characteristics, named 6K1. Both proteins are indispensable for viral replication (Merits et al. 2002; Cui and Wang 2016). Potyvirus 6K2: An Essential Component of the Virus Replication Complex Positive-strand RNA viruses replicate on intracellular vesicles that often result from extensive rearrangements of the endoplasmic reticulum (ER) membrane network (Salonen et al. 2005; Miller and Krijnse-Locker 2008; Den Boon and Ahlquist 2010). The role of these membranous vesicles is manifold. First, they provide the scaffold for anchoring the so-called virus replication complex (VRC) to cellular membranes. Second, by restricting the replication process to a small and closed region of the cytoplasm, the concentration of relevant molecules can be high (e.g., replication factors, coopted host factors, and nucleotides). Third, these vesicles provide a safe environment to protect double-stranded RNA replication intermediates from the action of the DICER endonucleases that trigger the RNAi-mediated cellular antiviral system. Interestingly, some had argued that vesicles containing VRCs composed by viral proteins interacting with a large number of cellular factors, altogether dubbed as virus replication factories (Den Boon and Ahlquist 2010), should be considered as the real alive virus (the virion being just a transmission inert stage) (Claverie 2006) and that such virus factories may even be the remote ancestor of the eukaryotic nucleus (Bell 2001; Koonin and Dolja 2013). 6K2 is able to create vesicles by itself even though it needs to interact with other viral and cellular proteins to create functional VRC (Löhmus et al. 2016; Geng et al. 2017). Purifying high-molecular-weight complexes involving 6K2 and using proteomic techniques to identify components, Löhmus et al. (2016) found that 6K2 associates with the potyviral NIb replicase, CI helicase, HC-Pro suppressor of RNA silencing, and the VPg that attaches covalently to the 50 end of the genomic RNA. In addition, they identified (...truncated)