Journal of Clinical Virology 36 (2006) 264–271 Natural polymorphisms in the protease gene modulate the replicative capacity of non-B HIV-1 variants in the absence of drug pressure Africa Holgu´ ın a,∗ , Carlos Su ˜ ne b , Franc ¸ois Hamy c , Vincent Soriano a , Thomas Klimkait c,d a Service of Infectious Diseases, Hospital Carlos III, Madrid, Spain b Centro Nacional de Biotecnologia, Campus Universidad Autonoma, Cantoblanco, Madrid, Spain c InPheno AG, Basel, Switzerland d Basel Center of HIV Research (BCHR), Institute for Medical Microbiology, Basel, Switzerland Received 3 August 2005; received in revised form 18 April 2006; accepted 2 May 2006 Abstract Background: Genetic variation in the HIV-1 pol gene, which encodes the main targets for anti-HIV drugs, may favors different susceptibility and resistance pathways to antiretroviral agents. Several amino acid substitutions occur frequently in some non-B viruses at positions associated with drug resistance in clade B viruses. The clinical relevance of those polymorphisms is unclear. Objective: To evaluate the effect of two natural protease (PR) polymorphisms, K20I and M36I, which are frequently found in non-B subtypes, on the virus replicative capacity in the presence and absence of protease inhibitors (PI). Study design: Infectious HIV-1 clones carrying K20I, M36I or K20I/M36I were designed. Their replication kinetics were analyzed by viral competition in the absence of PI. Susceptibility to six different PI was phenotypically assessed in clones and in recombinant viruses carrying non-B proteases from 16 drug-naive individuals. Results: In the absence of drug, the M36I clone replicated more rapidly than wt (wild type) or the double mutant K20I/M36I. Natural polymorphisms 20I and/or 36I improved the virus replicative capacity under drug pressure, reducing the susceptibility to saquinavir and indinavir, with IC 50 values 2–3.5-fold higher than wt. All but one drug-naive individual carrying non-B viruses were fully susceptibility to all tested PI, suggesting that additional substitutions within the PR might compensate the reduced PI susceptibility caused by K20I and/or M36I. Conclusion: Natural PR polymorphisms in non-B HIV-1 variants can influence in vitro the virus replication capacity in the presence and/or absence or certain PI. Hypothetically, the improved viral replication of mutant 36I might favor a more rapid spreading of non-B subtypes of HIV-1. © 2006 Elsevier B.V. All rights reserved. Keywords: HIV-1; Non-B subtypes; Protease; Natural polymorphisms; Drug resistance; Protease inhibitors; Fitness 1. Introduction The genetic diversity of the HIV-1 population in the infected host (quasispecies) results from a high rate of HIV-1 replication paired with a low fidelity of the HIV reverse tran- scriptase (RT) enzyme, which lacks a proofreading function. Subtype variants, as well as circulating recombinant forms ∗ Corresponding author at: Hospital Carlos III, Calle Sinesio Delgado 10, 28029 Madrid, Spain. Tel.: +34 91 4532500; fax: +34 91 733 6614. E-mail address: aholguin.hciii@salud.madrid.org (A. Holgu´ ın). within HIV-1 group M, possess a broad genetic variability (Leitner et al., 2003; Robertson et al., 2000). Although HIV-1 subtype B is still the most prevalent subtype in North Amer- ica and Western Europe, a rapid spreading of other variants has been increasingly noticed in recent years. Drug resistance testing has become an important tool in the management of HIV-infected individuals, who undergo antiretroviral therapy (Youree and D’Aquila, 2002). Resistance is one of the key factors limiting drug efficacy. Both genotyping and phenotyping methods are useful for assessing the susceptibility of HIV-1 to antiretroviral drugs 1386-6532/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2006.05.001