We cultured lamivudine-resistant human immunodeficiency virus type 1 (HIV-1) variants over an extended period of time in the presence of zidovudine and observed a premature reversion of the resistance-conferring M184V mutation. HIV-1. This mutation confers high-level resistance to lamivudine (3TC) and is also associated with resensitization of viruses that contain zidovudine (ZDV) resistance-conferring mutations (7) despite the fact that both drugs are nucleoside analogue RT inhibitors. The 184V mutation alone UK-427857 was shown to cause a two- to threefold increase in susceptibility to ZDV (12 15 Drug hypersusceptibility and the resensitization of resistant viruses are phenomena of potential clinical importance. However these phenotypes may occur only transiently as previously shown in the context of ZDV-3TC combination therapy (8). Cell culture experiments revealed that the presence of only one drug i.e. ZDV and the absence of 3TC drug pressure resulted in the loss of 3TC resistance in formerly dually resistant isolates (10). The new phenotype coincided with the reversion of the M184V mutation to wild type. Whether the loss of this mutation is solely attributable to a replication disadvantage of the mutant variant or whether improved susceptibility to ZDV exerts additional pressure that facilitates reversion to wild type is usually unknown. To address this issue we cultured M184V-made up of viruses either in the absence of drugs or in the presence of increasing concentrations of ZDV. We used two clinical HIV-1 isolates designated 3350-184V and 4246-184V and for comparison we also employed another 184V-made up of construct that was generated by site-directed mutagenesis (HXB-2D-184V). The presence of M184V was confirmed by automated sequencing of the RT region comprising residues 39 to 244 by using the protocol and CCND2 software provided by the supplier (Visible Genetics Inc. Toronto Ontario Canada). Other known resistance-associated UK-427857 mutations were not identified. The mutant variants are associated with a two- to threefold increase in susceptibility to ZDV based on both RT and p24 measurements (data not shown). These differences have been consistently measured not only in MT-4 cells (the present study) but also in CD4+ HeLa cells (12) and peripheral blood mononuclear cells (15). (This work was performed by K.D. in partial fulfillment of the requirements for a Ph.D. degree Faculty of Graduate Studies and Research McGill University Montreal Quebec Canada.) We initially used the clinical isolate 3350-184V and cultured this virus over a period of 20 weeks in MT-4 cells under different conditions as specified in Table ?Table1.1. Samples were analyzed after 5 10 15 and 20 weeks with regard to phenotypic susceptibility to 3TC (Table ?(Table1).1). The 50% infectious dose (IC50) values were determined on the basis of RT activity measurements as previously defined (15). Ahead of these measurements lifestyle supernatants had been equilibrated and iced cell pellets had been used to remove proviral DNA for genotypic evaluation. We noticed a reduction in IC50 beliefs for 3TC at week 20 when the pathogen was expanded in the lack of medication. UK-427857 The current presence of ZDV accelerated this UK-427857 reduce. Elevated susceptibilities to lamivudine have emerged in dose-dependent style after ca. 10 weeks in lifestyle. TABLE 1. Overview of IC50 beliefs for 3TC for stress 3350-184V expanded in the existence or lack of several medications As expected the current presence of 0.1 μM 3TC alone led to maintenance of 3TC resistance at the most recent time stage tested i.e. week 20. Nevertheless this focus of 3TC was inadequate to avoid the premature lack of 3TC level of resistance in the excess existence of 0.1 μM ZDV (Table ?(Table1).1). Maintenance of resistance to 3TC in the presence of 0.1 μM ZDV required concentrations of 3TC higher than 0.1 μM. We further exhibited that the premature loss of resistance to 3TC is usually a specific effect one that is usually attributable to the specific conversation between ZDV and the M184V mutation. As additional controls neither the presence of nevirapine nor the presence of stavudine caused an early reduction in 3TC IC50 values. Moreover we obtained nearly identical results with three unique 184V-made up of viruses that were generated by different methods i.e. site-directed mutagenesis (HXB-2D-184V) in vivo selection (3350-184V) and in vitro selection (4246-184V) (data not shown). The fact that an accelerated increase in susceptibility to 3TC was seen with both clinical isolates and the cloned.