Processivity and drug-dependence of HIV-1 protease: determinants of viral fitness in variants resistant to protease inhibitors Stefano Menzo a , Alessia Monachetti a , Claudia Balotta b , Stefano Corvasce b , Stefano Rusconi b , Stefania Paolucci c , Fausto Baldanti c , Patrizia Bagnarelli a and Massimo Clementi d Objective: To investigate the role of processivity and drug-dependence of HIV-1 protease as fitness determinants in variants resistant to protease inhibitors (PI). Design and methods: HIV-1 protease sequences from 32 infected subjects (27 patients who failed PI-treatments and five PI-naive controls) were evaluated using a recombi- nant method. The HIV-1 phenotype to seven PI was analysed together with the replication capacity of recombinants and the processivity and drug-dependence of the HIV-1 proteases. Protease mutants (positions 10, 46, 54, 82, 84, 90, and combinations thereof) were generated invitro and studied under identical experimental conditions. Results: In the absence of PI, 24 of 27 (89%) resistant proteases from treated subjects showed decreased processivity compared with the wild type. Processivity was lower in sequences bearing fewer mutations, than in more mutated ones. Twelve sequences (44%) conferred slower replication kinetics to the recombinant viruses. Seven sequences (26%) showed higher processivity levels in the presence of PI than in their absence, suggesting that drug-dependence influences PI-resistant variants. Among the mutants generated invitro, mutations 82A and 90M determined broad cross-resistance to PI in association with 10I. A drop of processivity was observed for the 82A+90M variants; 10I allowed partial recovery for 82A and 84V, and marked recovery for 90M mutants. Conclusions: A decrease in HIV-1 protease processivity parallels early selection of primary mutations, whereas its recovery is driven by compensatory mutations. Furthermore, a PI may select drug-dependent, besides resistant, HIV-1 protease variants. Changes in processivity and drug-dependence of HIV-1 proteases have implications in the replication capacity of PI-resistant viruses. & 2003 Lippincott Williams & Wilkins AIDS 2003, 17:663–671 Keywords: HIV-1 fitness, protease inhibitors, drug-dependence, enzyme processivity Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. From the a Istituto di Microbiologia, Universita ´ di Ancona, the b Istituto di Malattie Infettive e Tropicali, Universita ` di Milano, the c Servizio di Virologia, I.R.C.C.S. Policlinico S. Matteo, Pavia and the d Cattedra di Microbiologia, Universita ` Vita-Salute San Raffaele, I.R.C.C.S. Istituto Scientifico San Raffaele, Milan, Italy. Correspondence to Massimo Clementi, MD, Universita ` Vita-Salute San Raffaele, Laboratorio di Microbiologia, Ospedale San Raffaele, via Olgettina 60, I-20132, Milan, Italy. Tel: +39 022643.2649; fax: +39 022643.2640; e-mail: massimo.clementi@hsr.it Received: 6 September 2002; revised: 8 November 2002; accepted: 19 November 2002. DOI: 10.1097/01.aids.0000050852.71999.5f ISSN 0269-9370 & 2003 Lippincott Williams & Wilkins 663