Structure-based moloney murine leukemia virus reverse transcriptase mutants with altered intracellular direct-repeat deletion frequencies

Abstract

Template switching rates of Moloney murine leukemia virus reverse transcriptase mutants were tested using a retroviral vector-based direct-repeat deletion assay. The reverse transcriptase mutants contained alterations in residues that modeling of substrates into the catalytic core had suggested might affect interactions with primer and/or template strands. As assessed by the frequency of functional lacZ gene generation from vectors in which lacZ was disrupted by insertion of a sequence duplication, the frequency of template switching varied more than threefold among fully replication-competent mutants. Some mutants displayed deletion rates that were lower and others displayed rates that were higher than that of wild-type virus. Replication for the mutants with the most significant alterations in template switching frequencies was similar to that of the wild type. These data suggest that reverse transcriptase template switching rates can be altered significantly without destroying normal replication functions.

FIG. 1

RT structure and mutant design. (a) Ribbon diagram (23, 24) of the fingers and palm domains of MLV RT. Positions of mutagenized residues and of active site residues D150, D224, and D225 (magenta) are shown as ball and stick models. Mutant viability is represented by color coding, with green indicating viable, blue indicating delayed but viable, and red indicating not viable. (b) Positions of specific residues (black labels) relative to a primer-template model on an electrostatic potential surface rendering (26) of MLV RT fingers and palm domains. The primer, template, and incoming nucleotide are shown as stick models in yellow, green, and magenta, respectively. The view is similar to that in panel a. The position of the DNA, shown in a stick model, results from superpositioning the fingers and palm domains of MLV with HIV-1 RT from the HIV-1 RT:DNA:TTP structure (Protein Data Bank accession code 1rtd).

FIG. 2

Mutant virus replication. (A) Replication time course. Results are averages from at least two independent transfection experiments for each mutant. Black bars represent the day postinfection (indicated at the top) when spread was first detected, and a lack of detectable spread is represented by clear bars. The average first day of detection and the standard deviation (in days) are at right. Averages without standard deviations indicate cultures that tested positive in one experiment (possibly due to compensatory mutations or reversion) but not in experimental repetitions. (B) Sample viability (RT activity) assay. Culture medium was harvested from confluent cells infected with the virus stocks indicated at the left on the days shown at the top and was assayed for RT activity as described in Materials and Methods. Note that this assay was used to determine the presence or absence of detectable virus: whether variations in signal intensity reflected altered enzymatic activity or other parameters, such as cell growth rate-dependent differences in virus concentration, was not determined. (C) Reversion analysis. Virus-containing media, from the first time points shown in panel A at which spread was detectable, were used to infect 3T3 cells.

FIG. 3

Template switching assay. (A) Experimental overview: pMLV Ψ⁻ was transiently transfected into stable clonal transfectants expressing each vector to produce virus used to infect 3T3 cells. Puromycin-resistant colonies were stained with X-Gal to determine deletion frequencies. (B) MLV vectors containing direct repeats within lacZ. The parental vector, pLac-wt, contains the puromycin resistance gene transcribed from the SV40 promoter and lacZ driven by the upstream long terminal repeat. pLaac-117 and pLaac-284 contain 117- and 284-base repeats within lacZ, respectively. Long terminal repeats are represented by black boxes, and direct-repeat locations are shown. PvuII and ClaI were used to digest genomic DNA, and a HincII-ClaI fragment was used as the probe (represented by a thick bar) for Southern analysis (see Fig. 5).

FIG. 4

Template switch and error rates. Error bars represent standard deviations. (A) Deletion rates (117-base) for wild-type and mutant RTs. The data shown for the mutants selected for extensive study (WT RT, K53L, D124A, D124A/E201G, K53L/D124A, and K102A/K257A) are from at least 15 plates from two or more independent transfections and at least three independent infections for each of these mutants. Other mutants (K102A, K120L, R121L, K193R, K257A, K102A/D124A, and K53L/K102A) were included in a subset of these experiments. Data presented from the latter mutants were obtained from at least six plates from at least two independent infection experiments for each virus. (B) Deletion rates (284-base). The mutants listed above were chosen for extensive analysis, and for these, results are from at least 12 plates from two or more independent transfections and at least three independent infections. (C) lacZ inactivation rates. For these experiments, WT RT, K53L, D124A, K53L/D124A, and K102A/K257A were extensively studied (results are from at least nine plates from two or more independent transfections and at least three independent infections).

FIG. 5

Southern analysis of PvuII/ClaI-digested genomic DNA from 3T3 cells infected with a 284-base repeat vector. The 1,016-nucleotide band represents undeleted laacZ, and the 732-nt band represents deleted products. Marker band locations are indicated to the left. See Fig. 3B for a map of restriction sites and the probe. Bands were quantified by PhosphorImager, and deletion rates were calculated (bottom).

FIG. 6

Endogenous reactions. (A) Denaturing polyacrylamide gel. Numbers at left indicate lengths, in bases, of size standards. Mobility of minus-strand strong-stop DNA (−sssDNA) is indicated at right. (B) Denaturing agarose gel to visualize longer endogenous reaction products. Size standards are indicated as for panel A.