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Viral quasispecies may possess a molecular memory of their past evolutionary history, imprinted on minority components of the mutant spectrum. Here we report experimental evidence and a theoretical model for memory in retroviral quasispecies in vivo. Apart from replicative memory associated with quasispecies dynamics, retroviruses may harbour a “cellular” or “anatomical” memory derived from their integrative cycle and the presence of viral reservoirs in body compartments. Three independent sets of data exemplify the two kinds of memory in human immunodeficiency virus type 1 (HIV-1). The data provide evidence of re-emergence of sequences that were hidden in cellular or anatomical compartments for extended periods of infection, and recovery of a quasispecies from pre-existing genomes. We develop a three-component model that incorporates the essential features of the quasispecies dynamics of retroviruses exposed to selective pressures. Significantly, a numerical study based on this model is in agreement with the experimental data, further supporting the existence of both replicative and reservoir memory in retroviral quasispecies. [Copyright &y& Elsevier]

Protease inhibitors (PIs) are an important class of drugs for the treatment of HIV infection. However, in the course of treatment, resistant viral variants with reduced sensitivity to PIs often emerge and become a major obstacle to successful control of viral load. On the basis of a compound equipotently inhibiting HIV-1 and 2 proteases (PR), we have designed a pseudopeptide inhibitor, QF34, that efficiently inhibits a wide variety of PR variants. In order to analyze the potency of the inhibitor, we constructed PR species harboring the typical (signature) mutations that confer resistance to commercially available PIs. Kinetic analyses showed that these mutated PRs were inhibited up to 1000-fold less efficiently by the clinically approved PIs. In contrast, all PR species were effectively inhibited by QF34. In a clinical study, we have monitored 30 HIV-positive patients in the Czech Republic undergoing highly active antiretroviral therapy, and have identified highly PI resistant variants. Kinetic analyses revealed that QF34 retained its subnanomolar potency against multi-drug resistant PR variants. X-ray crystallographic analysis and molecular modeling experiments explained the wide specificity of QF34: this inhibitor binds to the PR in an unusual manner, thus avoiding contact sites that are mutated upon resistance development, and the unusual binding mode and consequently the binding energy is therefore preserved in the complex with a resistant variant. These results suggest a promising route for the design of second-generation PIs that are active against a variety of resistant PR variants. [Copyright &y& Elsevier]