The M184V mutation in reverse transcriptase can delay reversion of attenuated variants of simian immunodeficiency virus

Abstract

We previously constructed a series of simian immunodeficiency virus (SIV) mutants containing deletions within a 97-nucleotide region of the SIVmac239 untranslated region or leader sequence. However, as is common with live attenuated viruses, several of the mutants exhibited a moderate propensity for reversion. Since the M184V mutation in human immunodeficiency virus type 1 reverse transcriptase is associated with diminished fitness as well as lamivudine resistance, we introduced this substitution into several of our deletion mutants to determine its effects on viral replication and compensatory reversion. Our results indicate that M184V impaired viral fitness in pair-wise comparisons of mutants that contained or lacked this substitution. We also observed that M184V significantly impaired the potential for both compensatory mutagenesis and reversion in these mutants both in cell lines and in peripheral blood mononuclear cells.

FIG. 1.

Delay of phenotypic reversion of the SD2-M184V mutant after long-term culture in CEMx174 cells. Shown are growth curves of viruses over extended culture. Note that the scale of the ordinate is logarithmic. Equivalent amounts of virus from transfected COS-7 cells were used to infect CEMx174 cells based on levels of p27 antigen (10 ng per 10⁶ cells). Infected cells were grown over protracted periods, and culture fluids were monitored by RT assay. Mock infection denotes exposure of cells to heat-inactivated WT virus as a negative control. A representative example of the SD2-M184V and SD6-M184V variants at the fourth passage is shown (the experiment was performed three times with similar results).

FIG. 2.

Replication capacity of wild-type and mutated viruses in monkey PBMCs. Equivalent amounts of virus were used to infect rhesus macaque PBMCs based on levels of p27 antigen, typically 10 ng of WT or mutant virus per 4 × 10⁶ PBMCs. Viral replication was monitored by determining levels of SIV p27 antigen by enzyme-linked immunosorbent assay of culture fluids. Mock infection denotes exposure of cells to heat-inactivated wild-type virus as a negative control. The dotted line representing 0.01 ng of p27/ml indicates the threshold sensitivity of the assay. (A) Growth curves indicating antigen production in PBMCs from donor monkey A. (B) Growth curves for PBMCs from donor monkey B. p27 antigen results are the averages of duplicates. (C) Second passage of mutated viruses in 10⁶ CEMx174 cells by using an inoculum of 10 pg of p27 antigen derived from the infected PBMCs of monkey B. Viral replication was monitored by RT assay of culture fluids. Shown is a representative replication curve of experiments conducted in duplicate. Mock infection denotes exposure of cells to heat-inactivated WT virus as a negative control. Note that the scales of the ordinates are logarithmic.

FIG. 2.

Replication capacity of wild-type and mutated viruses in monkey PBMCs. Equivalent amounts of virus were used to infect rhesus macaque PBMCs based on levels of p27 antigen, typically 10 ng of WT or mutant virus per 4 × 10⁶ PBMCs. Viral replication was monitored by determining levels of SIV p27 antigen by enzyme-linked immunosorbent assay of culture fluids. Mock infection denotes exposure of cells to heat-inactivated wild-type virus as a negative control. The dotted line representing 0.01 ng of p27/ml indicates the threshold sensitivity of the assay. (A) Growth curves indicating antigen production in PBMCs from donor monkey A. (B) Growth curves for PBMCs from donor monkey B. p27 antigen results are the averages of duplicates. (C) Second passage of mutated viruses in 10⁶ CEMx174 cells by using an inoculum of 10 pg of p27 antigen derived from the infected PBMCs of monkey B. Viral replication was monitored by RT assay of culture fluids. Shown is a representative replication curve of experiments conducted in duplicate. Mock infection denotes exposure of cells to heat-inactivated WT virus as a negative control. Note that the scales of the ordinates are logarithmic.

FIG. 3.

(A) Reversion of the SD2-M184V variant after replication in CEMx174 cells. Growth curves of reverted viruses in CEMx174 cells. Equivalent amounts of virus from transfected COS-7 cells were used to infect CEMx174cells based on levels of p27 antigen (10 ng per 10⁶ cells). Viral replication was monitored by RT assay of culture fluids. Shown is a representative growth curve of experiments conducted in triplicate. Mock infection denotes exposure of cells to heat-inactivated WT virus as a negative control. (B) The M184V mutation restricts compensatory mutagenesis in the case of the SD2 variant. Viruses derived from COS-7 cells were standardized on the basis of p27 CA antigen and used to infect 10⁶ CEMx174 cells. RT activity of culture fluids was used to monitor replication. Shown is a representative growth curve of experiments performed in triplicate. Mock infection denotes exposure of cells to heat-inactivated WT virus as a negative control. Note that the scales of the ordinates are logarithmic in both panels.