Variable Fitness Impact of HIV-1 Escape Mutations to Cytotoxic T Lymphocyte (CTL) Response Ryan M. Troyer 1 , John McNevin 2 , Yi Liu 3 , Shao Chong Zhang 4 , Randall W. Krizan 1 , Awet Abraha 1 , Denis M. Tebit 1 , Hong Zhao 3 , Santiago Avila 1,5 , Michael A. Lobritz 6 , M. Juliana McElrath 2 , Sylvie Le Gall 4 , James I. Mullins 3 , Eric J. Arts 1 * 1 Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America, 2 Vaccine and Infectious Disease Institute, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Department of Microbiology, University of Washington, Seattle, Washington, United States of America, 4 Partners AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 5 National Institute of Respiratory Diseases, Center for Research in Infectious Diseases, Mexico City, Mexico, 6 Department of Molecular and Microbiology, Case Western Reserve University, Cleveland, Ohio, United States of America Abstract Human lymphocyte antigen (HLA)-restricted CD8 + cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs. Citation: Troyer RM, McNevin J, Liu Y, Zhang SC, Krizan RW, et al. (2009) Variable Fitness Impact of HIV-1 Escape Mutations to Cytotoxic T Lymphocyte (CTL) Response. PLoS Pathog 5(4): e1000365. doi:10.1371/journal.ppat.1000365 Editor: Christopher M. Walker, Nationwide Children’s Hospital, United States of America Received May 19, 2008; Accepted March 5, 2009; Published April 3, 2009 Copyright: ß 2009 Troyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research at Case Western Reserve University was supported by NIAID; NIH grants AI49170, AI57005, and AI058894; and at University of Washington by NIAID, NIH grants AI57005 and AI058894. RMT was supported by an American Foundation for AIDS Research (amfAR) fellowship 106532-35-RFGN. The funders had no role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, and in the preparation, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: eja3@case.edu Introduction It is well established that the CTL response plays a major role in control of both acute and chronic HIV-1 infection (reviewed in [1]). Thus, numerous vaccine strategies are being evaluated to elicit broad and potent CTL responses against HIV proteins [2]. However, a major obstacle is CTL-mediated selection leading to viral escape, which occurs frequently in acute/early [3–8] and chronic [9–11] infection. Recent studies have shown that CTL selection is a major force driving viral evolution within individual patients [12–14] and at the population level [15–18]. For some epitopes, escape mutations are often slow to emerge, or even absent despite strong specific CTL responses [13,19,20]. This rate of CTL escape during infection may be influenced by mutational pathways required for escape (e.g., requirements for transversion versus transition and single versus multiple nucleotide mutations), the killing efficiency (or ‘‘strength’’) of CTL responses [1] as well as stochastic processes [21]. However, another often cited factor for the slow appearance of CTL escape mutation may be the ‘‘cost’’ on replicative fitness of the infecting strain [22]. Several lines of experimental evidence have inferred fitness costs. First, specific HIV-1 and SIV escape variants can be maintained in viral populations after transmission to a host sharing the restricting HLA allele, and revert to the consensus, epitopic form when transmitted to an individual not sharing the donor restricting HLA allele [13,23–25]. Thus, in the absence of specific CTL responses, viruses with consensus amino acids are likely to have higher replicative fitness. Second, some escape mutations can be compensated by extra-epitopic mutations also under selection, implying a fitness cost for the escape mutation [26–29]. Lastly, introduction of CTL escape mutations into HIV-1 and SIV epitopes has been shown to reduce replication kinetics [26,30–34]. Martinez-Picado et al. have provided evidence that a specific T242N mutation in the TW10 Gag p24 epitope can reduce fitness when introduced into the autologous (patient-derived) HIV-1 sequence [32]. However, the collective assumption that CTL PLoS Pathogens | www.plospathogens.org 1 April 2009 | Volume 5 | Issue 4 | e1000365