Diminished RNA Primer Usage Associated with the L74V and M184V Mutations in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Provides a Possible Mechanism for Diminished Viral Replication Capacity

Abstract

The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA 3Lys and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.Although considerable progress has been made in the treatment of human immunodeficiency virus type 1 (HIV-1)-associated disease, the emergence of mutated variants of HIV-1 that are resistant to antiviral drugs represents a major problem. The prolonged clinical use of nucleoside reverse transcriptase (RT) analogue chain terminators, e.g., abacavir (ABC), 2Ј,3Ј-dideoxyinosine (ddI or didanosine), and (Ϫ)-2Ј,3Ј-dideoxy-3Ј-thiacytidine (3TC or lamivudine), gives rise to resistant viruses that contain mutations in the RT enzyme (5,8,11,48,52,54,56). These nucleoside RT inhibitors (NRTIs) compete with natural deoxynucleoside triphosphate (dNTP) pools after being phosphorylated by cellular kinases for incorporation into viral DNA. DNA synthesis is blocked once the chain terminator is incorporated, since these nucleoside analogues lack a 3Ј-OH group, which is required to continue the polymerization process (24,44,45).A single amino acid substitution in RT, i.e., M184V, is sufficient to confer high-level resistance to 3TC (5,11,47,54). The M184V mutation is rapidly selected both in tissue culture and in vivo. Experiments with recombinant mutant enzymes have show...

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

“…EFFECT OF RT MUTATIONS ON VIRAL DNA SYNTHESIS 819 step in reverse transcription, and resistance mutations, including M184V, can also impair the initiation of DNA synthesis (18,31,52). In this regard, the synthesis of U5 DNA by WT virus was reduced in hydroxyurea-pretreated cells compared to that observed in unpretreated cells, and U5 synthesis by the mutant viruses was reduced to an even greater extent in hydroxyurea-pretreated cells.…”

“…Other mutations in RT known to inhibit the incorporation of nucleoside triphosphate analogs have also been studied and have been found to impair to a variable extent RT processivity and viral replicative capacity. This is the case for mutation L74V, selected by didanosine when used as a single nucleoside analogue, and K65R, associated with resistance to zalcitabine and tenofovir (17,18,44,(46)(47)(48)53). Consistent with the possibility that the defects in RT activity produced by these mutations are accentuated by low dNTP concentrations, we found that the replication of viruses carrying an RT with these mutations, as measured by the single-cycle assay, was more sensitive to inhibition in hydroxyurea-pretreated cells than was that of WT virus.…”

“…The M184V mutation has also been shown to decrease the processivity of the RT in vitro and to impair viral replicative capacity in tissue culture systems (3,8,32,40,47). Similarly, the L74V mutation, which reduces the incorporation of dideoxyadenosine triphos-phate, the active metabolite of didanosine, has also been reported to impair the processivity of RT in most studies and to impair viral replicative capacity (18,(46)(47)(48).…”

Detection of Human Immunodeficiency Virus (HIV) Type 1 M184V and K103N Minority Variants in Patients with Primary HIV Infection

“…Our group showed that mutations associated with protease inhibitors (PIs), thymidine analogues (thymidine-associated mutations), or nonnucleoside reverse transcriptase inhibitors (NNRTIs) were present in approximately 10 to 20% of newly infected patients but that the M184V mutation in reverse transcriptase (RT), associated with resistance to lamivudine and emtricitabine, was identified in only about 4% of such subjects (12). Since M184V is known to adversely affect viral replicative capacity as well as efficiency of RT initiation and function (3,14), we speculated that this mutation might also affect human immunodeficiency virus (HIV) transmissibility.…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…It has been shown that L74V in the context of the NL4-3 laboratory strain has an 11% loss of fitness compared to that of the wild type or K70R (154). Biochemical studies have shown that this replication decrease is associated with a reduction in the incorporation of nucleotides compared to the wild type and a decrease in the initiation of minus-strand and plus-strand DNA synthesis from the tRNA 3 Lys and polypurine tract primers (47,49). A reduced ability of L74V viruses to synthesize proviral DNA was demonstrated in cell culture (61).…”