Evolution of the human immunodeficiency virus type 1 protease: effects on viral replication capacity and protease robustness Elena Capel, Glo ` ria Martrus, Mariona Parera, Bonaventura Clotet and Miguel Angel Martı ´nez Correspondence Miguel Angel Martı ´nez mmartinez@irsicaixa.es Received 21 June 12 Accepted 27 August 12 Fundacio ´ irsiCaixa, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain The rapid spread of human immunodeficiency virus type 1 (HIV-1) in humans has been accompanied by continuous extensive genetic diversification of the virus. The aim of this study was to investigate the impact of HIV-1 diversification on HIV-1 replication capacity (RC) and mutational robustness. Thirty-three HIV-1 protease sequences were amplified from three groups of viruses: two naı ¨ve sample groups isolated 15 years apart plus a third group of protease inhibitor-(PI) resistant samples. The amplified proteases were recombined with an HXB2 infectious clone and RC was determined in MT-4 cells. RC was also measured in these three groups after random mutagenesis in vitro using error-prone PCR. No significant RC differences were observed between recombinant viruses from either early or recent naı ¨ve isolates (P50.5729), even though the proteases from the recent isolates had significantly lower sequence conservation scores compared with a subtype B ancestral sequence (P,0.0001). Randomly mutated recombinant viruses from the three groups exhibited significantly lower RC values than the corresponding wild-type viruses (P,0.0001). There was no significant difference regarding viral infectivity reduction between viruses carrying randomly mutated naı ¨ve proteases from early or recent sample isolates (P50.8035). Interestingly, a significantly greater loss of RC was observed in the PI-resistant protease group (P50.0400). These results demonstrate that protease sequence diversification has not affected HIV-1 RC or protease robustness and indicate that proteases carrying PI resistance substitutions are less robust than naı ¨ve proteases. INTRODUCTION Like other RNA viruses, human immunodeficiency virus type 1 (HIV-1) is highly mutable, highly adaptable and is capable of rapid evolution (Ma ´s et al., 2010). Since the introduction of the most recent common ancestor of the HIV-1 group M in humans (Sharp & Hahn, 2011), the viral population has become extraordinarily diverse (Hemelaar, 2012; Korber et al., 2000). The rate of evolution has been estimated to be 0.0024 substitutions per base pair per year for the HIV-1 gp160 envelope protein and 0.0019 for the HIV-1 gag protein (Korber et al., 2000) (reviewed in Vermund & Leigh-Brown, 2012). As a consequence, site- specific sequence conservation values have decreased over time at the population level. This raises the question: to what extent is viral diversification influencing HIV-1 fitness and virulence over the course of the epidemic? HIV-1 transmission is characterized by an acute genetic bottleneck in which a single transmitted founder virus frequently establishes infection (Derdeyn et al., 2004; Keele et al., 2008). Drastic ex vivo replication capacity (RC) losses are observed in HIV-1 upon serial plaque-to-plaque bottleneck events in MT-4 cells (Yuste et al., 1999). Analysis of the entire genomic nucleotide sequences of formerly bottlenecked viruses showed the accumulation of 4–28 mutations per genome (Yuste et al., 2000). These results confirmed the effect of Muller’s ratchet, which describes the tendency of populations of asexual organisms to lose fitness due to the accumulation of deleterious mutations that cannot be compensated for by sex or recombination (Muller, 1964). Another key issue is the potential impact of immune-mediated or drug-resistant mutations on HIV-1 RC at the population level (Chopera et al., 2011). Cytotoxic T lymphocyte (CTL) and antiretroviral drug escape mutations are detrimental to HIV-1 RC (Leslie et al., 2004; Martinez-Picado et al., 1999; Nijhuis et al., 1999), yet they may persist in new hosts in the absence of immune or drug pressure. Most of the RC- reducing escape mutations occur in conserved viral regions (Troyer et al., 2009). RC defects can, at least in part, be restored by secondary compensatory mutations. Trans- mission of low RC HIV-1 escape variants may provide a clinical benefit to recipients (Stoddart et al., 2001); The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are JQ846091–JQ846240. Journal of General Virology (2012), 93, 2625–2634 DOI 10.1099/vir.0.045492-0 045492 G 2012 Printed in Great Britain 2625