The removal of RNA primers from DNA synthesized by the reverse transcriptase of the retrotransposon Tf1 is stimulated by Tf1 integrase

Abstract

The Tf1 retrotransposon represents a group of long terminal repeat retroelements that use an RNA self-primer for initiating reverse transcription while synthesizing the minus-sense DNA strand. Tf1 reverse transcriptase (RT) was found earlier to generate the self-primer in vitro. Here, we show that this RT can remove from the synthesized cDNA the entire self-primer as well as the complete polypurine tract (PPT) sequence (serving as a second primer for cDNA synthesis). However, these primer removals, mediated by the RNase H activity of Tf1 RT, are quite inefficient. Interestingly, the integrase of Tf1 stimulated the specific Tf1 RT-directed cleavage of both the self-primer and PPT, although there was no general enhancement of the RT's RNase H activity (and the integrase by itself is devoid of any primer cleavage). The RTs of two prototype retroviruses, murine leukemia virus and human immunodeficiency virus, showed only a partial and nonspecific cleavage of both Tf1-associated primers with no stimulation by Tf1 integrase. Mutagenesis of Tf1 integrase revealed that the complete Tf1 integrase protein (excluding its chromodomain) is required for stimulating the Tf1 RT primer removal activity. Nonetheless, a double mutant integrase that has lost its integration functions can still stimulate the RT's activity, though heat-inactivated integrase cannot enhance primer removals. These findings suggest that the enzymatic activity of Tf1 integrase is not essential for stimulating the RT-mediated primer removal, while the proper folding of this protein is obligatory for this function. These results highlight possible new functions of Tf1 integrase in the retrotransposon's reverse transcription process.

Fig 1

Schematic description of the substrates used for assaying the removal of Tf1 SP or PPT substrates. The substrates were prepared as described in detail in Materials and Methods. The 11-nt-long RNA segments with sequences of either the Tf1 SP or PPT were 5′ end labeled with ³²P. The labeled RNA was then annealed to Tf1-derived DNA segments with the appropriate 3′ ends that match either the SP sequences (the PBS-derived 40-nt-long DNA) or the PPT (61-nt-long DNA) These heteroduplexes were then elongated with the Klenow fragment of E. coli polymerase I (lacking the 3′→5′ exonuclease), thus producing full-length substrates.

Fig 2

SP removal by Tf1 RT without and with IN. (A) Primer removal was assayed for 30 min at 37°C with 200 ng of either native wild-type Tf1 RT (lane 3, marked RT), heat-inactivated RT (lane 2, HI), or RNase H-deficient mutant (lane 4, RH-RT). S, substrate only (lane 1). (B) Reactions with constant amounts of wild-type Tf1 RT in the absence (lane 2) or the presence of Tf1 IN, with increasing molar ratios of IN to RT ranging from 0.25:1 to 2:1 (lanes 3 to 6). 2(HI), a 2:1 IN/RT ratio of Tf1 RT with heat-inactivated Tf1 IN (lane 7); 2(NR), with the same concentration of Tf1 IN with no RT present (lane 8); 2(HIV), a 2:1 IN/RT molecular ratio of HIV-1 IN with Tf1 RT (lane 9).

Fig 3

PPT removal by Tf1 RT in the absence or in the presence of IN. (A) Primer removal was assayed for 30 min at 37°C with 200 ng of wild-type Tf1 RT (lane 3, marked RT), heat-inactivated RT (lane 2, HI), or RNase H-deficient mutant (lane 4, RH-RT). S, substrate only (lane 1). (B) Reactions with constant amounts of wild-type Tf1 RT in the absence (lane 2) or the presence of Tf1 IN, with increasing molar ratios of IN to RT from 0.25:1 to 2:1 (lanes 3 to 6). 2(HI), a 2:1 IN/RT ratio of Tf1 RT with heat-inactivated IN (lane 7); 2(NR), with the same concentration of Tf1 IN with no RT present (lane 8); 2(HIV), a 2:1 IN/RT molecular ratio of HIV-1 IN with Tf1 RT (lane 9).

Fig 4

The RNase H activity of Tf1 RT in the absence or presence of increasing Tf1 IN/RT molar ratios. The soluble RNase H assay that measures the release of [³H]AMP from [³H]poly(rA)-poly(dT) substrate into the TCA-soluble fraction was carried out as described in Materials and Methods. The activity of Tf1 RT by itself with no IN present has a value of 1, and all other IN/RT ratios exhibit activities relative to this value (relative activity [RA]). The numbers represent the results of three independent experiments after subtracting the nonspecific backgrounds (with the indicated SD).

Fig 5

The removal of the SP (A) or the PPT (B) by various RTs in the absence or presence of Tf1 IN. Primer removal activities of wild-type Tf1, HIV-1, and MLV RTs as well as of the Tf1 RT that lacks RNase H activity (RH-RT) were tested in the absence or presence of Tf1 IN (at IN/RT molar ratios of 2:1).

Fig 6

The removal of the nonhomologous MLV primer by Tf1 RT. All reactions were performed with a constant amount of wild-type Tf1 RT in the absence (lane 2) or the presence of Tf1 IN, with increasing molar ratios of IN to RT ranging from 0.25:1 to 2:1 (lanes 3 to 6). 2(HI), a 2:1 IN/RT ratio of Tf1 RT with heat-inactivated Tf1 IN (lane 7); 2(NR), with the same concentration of Tf1 IN with no RT present (lane 8); MLV RT, a 2:1 Tf1 IN/MLV RT molar ratio (lane 9); HIV-1 RT, a 2:1 Tf1 IN/HIV-1 RT ratio (lane 10). S, substrate only (lane 1).

Fig 7

A schematic description of the Tf1 IN variants generated and used in this study. Wild-type Tf1 IN was genetically modified either by introducing mutations in the IN active site (the DM IN) or by truncating parts of the protein in the expression vectors for generating the different variants of Tf1 IN (see Materials and Methods). Except for the first residue, all other residues that specify the boundaries between the different domains are the last residue in each domain.

Fig 8

SP and PPT removal by Tf1 RT and Tf1 IN mutants. (A) Removal of the SP. (B) Removal of the PPT. In both panels, lane 1 shows substrate only. In all other lanes, reactions were performed with a constant amount of Tf1 RT in the absence (−, lanes 2) or the presence of the different Tf1 IN variants, assayed with a 2:1 molar ratio of the IN variant over Tf1 RT (lanes 3 to 8). WT, wild-type IN (lanes 3); CD-IN, chromodomain-minus IN (lanes 4); DM IN, double mutant IN (lanes 5); NT-IN, N-terminally truncated IN (lanes 6); CT-IN, C-terminally truncated IN (lanes 7); CCD IN, catalytic core (lanes 8).