Reversion of the M184V Mutation in Simian Immunodeficiency Virus Reverse Transcriptase Is Selected by Tenofovir, Even in the Presence of Lamivudine

Abstract

The methionine-to-valine mutation in codon 184 (M184V) in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) confers resistance to (؊)-2-deoxy-3-thiacytidine (3TC; lamivudine) and increased sensitivity to 9-[2-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir). We have used the SIV model to evaluate the effect of the M184V mutation on the emergence of resistance to the combination of 3TC plus PMPA. A site-directed mutant of SIVmac239 containing M184V (SIVmac239-184V) was used to select for resistance to both 3TC and PMPA by serial passage in the presence of increasing concentrations of both drugs. Under these selection conditions, the M184V mutation reverted in the majority of the selections. Variants resistant to both drugs were found to have the lysine-to-arginine mutation at codon 65 (K65R), which has previously been associated with resistance to PMPA in both SIV and HIV. Similarly, in rhesus macaques infected with SIVmac239-184V for 46 weeks and treated daily with The rapid emergence of drug-resistant mutants of human immunodeficiency virus (HIV) has proven to be a major obstacle in antiviral therapy for AIDS (7,32,36,63). Even highly active antiretroviral therapy has been limited by the emergence of multidrug-resistant HIV (32,46,61). This has led to efforts to identify drug combinations in which resistance to one drug results in a mutant virus that suppresses normal resistance to a second drug or in a mutant virus that is hypersensitive to other drugs (2,27,28,35,52).One mutation that results in phenotypic changes in HIV type 1 (HIV-1) that are useful in certain drug combinations is the methionine-to-valine mutation in codon 184 (M184V) of reverse transcriptase (RT). This mutation arises rapidly in therapy with the oxathiolane nucleosides, (Ϫ)-2Ј-deoxy-3Ј-thiacytidine (3TC; lamivudine) and (Ϫ)-2Ј-deoxy-5-fluoro-3Ј-thiacytidine [(Ϫ)-FTC], and results in high-level (Ͼ100-fold) resistance to these drugs (49,55). This is often preceded by emergence of a methionine-to-isoleucine mutation in codon 184 (M184I), which is quickly replaced by M184V (22, 50). This M184V mutation is located in the highly conserved YMDD motif of RT, which is directly involved in binding the incoming nucleotide during reverse transcription (19,20,24,53). It results in decreased processivity (1,3,21,23,51) and increased fidelity (11,41,62) of the DNA polymerase activity of RT in biochemical assays. However, the increase in fidelity was less than twofold in an M13 phage-based assay that scored the overall mutation rate after transfection of RT products into bacteria (9). M184V mutants of HIV-1 also have reduced replication rates in certain cell lines (1, 35, 51), and they have a broad array of changes in susceptibility to nucleoside analogs (15,28,35,54,55,64).A major effect of the M184V mutation is to suppress phenotypic resistance to 3Ј-azido-3Ј-deoxythymidine (AZT; zidovudine) when M184V is present along with mutations that normally confer resistance to AZT (28,55). Clinical...

CD8+-Cell-Mediated Suppression of Virulent Simian Immunodeficiency Virus during Tenofovir Treatment

“…Because many plasma samples had insufficient viral RNA to allow sequencing, proviral DNA obtained from CEMx174 infected with PBMC isolates was used. In a previous study, this method gave sequence results that were similar to those of plasma viral RNA (49). Infected CEMx174 and PBMC cocultures were harvested as soon as culture supernatants were positive by antigen-capture enzyme-linked immunosorbent assay.…”

Suppression of Virus Load by Highly Active Antiretroviral Therapy in Rhesus Macaques Infected with a Recombinant Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1

“…Infectious stocks of RT-SHIV were prepared following transfection of the two half clones into CEMx174 cells by electroporation, as previously described (22). Virus stocks were prepared from culture supernatants of infected CEMx174 cells.…”

“…In the first round of nested PCR with RT-SHIV, 0.4 M concentrations (each) of primers 239-2675 and 239-4751 (R) were used, and in the second round, 0.4 M concentrations (each) of primers 239-2786 and 239-4615 (R) were added to each reaction. All primers are listed in Table 1, and conditions for PCRs were as described previously (22,34). Amplicons were purified by use of either a PCR purification kit (QIAGEN) or Microcon YM-50 centrifugal filter devices (Millipore, Bedford, Massachusetts).…”

“…Cell-free and cell-associated infectious viruses were isolated by cocultivation of plasma or peripheral blood mononuclear cells with CEMx174 cells in 25-cm 2 flasks. SIV p27 core antigen production was measured by previously described methods (22,34). Virus-positive cultures were centrifuged twice at 600 ϫ g for 5 min to remove cells.…”

Magnetic resonance spectroscopy reveals that activated monocytes contribute to neuronal injury in SIV neuroAIDS

“…In non-CD8-depleted monkeys and humans, the development of AIDS and CNS disease occurs with immune system dysfunction as indicated by loss of CD4 and CD8 lymphocyte function and dysregulated, activated monocytes/macrophages (3,64,65). Several studies have recently used PMPA and RCV in SIV pathogenesis models (66)(67)(68)(69)(70). To date, there are no reports that treatment with either of these agents directly inhibits cellular immune responses.…”