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The impact of these factors on protein sequence evolution is interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available data set of 32,163 sequenced isolates from drug-na\u00efve individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple-base mutations in HIV-1 protease is strongly influenced by mutational sampling.<\/jats:p>","DOI":"10.1093\/molbev\/msz022","type":"journal-article","created":{"date-parts":[[2019,2,3]],"date-time":"2019-02-03T15:06:36Z","timestamp":1549206396000},"page":"798-810","source":"Crossref","is-referenced-by-count":14,"title":["Constrained Mutational Sampling of Amino Acids in HIV-1 Protease Evolution"],"prefix":"10.1093","volume":"36","author":[{"given":"Jeffrey I","family":"Boucher","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Troy W","family":"Whitfield","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Massachusetts Medical School, Worcester, MA"},{"name":"Program in Bioinformatics and Integrative Biology, University of 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