Asymmetric subunit organization of heterodimeric Rous sarcoma virus reverse transcriptase alphabeta: localization of the polymerase and RNase H active sites in the alpha subunit

Abstract

The genes encoding the alpha (63-kDa) and beta (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV alphabeta and alphaPol RTs within which one subunit was selectively mutated. When alphabeta heterodimers contained the Asp 181-->Asn mutation in their beta subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their alpha subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in alphabeta heterodimers mutated in the beta subunit but was lost when the mutation was present in the alpha subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the alpha subunit of the heterodimeric RSV RT alphabeta.

FIG. 1

Mutagenesis of baculovirus transfer vectors. The open reading frame of the gene encoding RSV RT α is shaded in light gray. The start (ATG) and stop of the gene are indicated. The restriction enzymes relevant for the cloning procedure are shown. Abbreviations for other enzymes are as follows: B, BamHI; E, EcoRV; H, HindIII; P, PmlI; S, SacII; and X, XhoI. The locations of the PCR primers are indicated by black arrows. A white circle indicates the location of Asp 181 or Asp 505. A black circle indicates the mutagenized codon for Asn 181 or Asn 505. (A) Mutagenesis of the codon for Asp 181 in pBac-α. (B) Mutagenesis of the codon for Asp 505 in pBac-α.

FIG. 1

Mutagenesis of baculovirus transfer vectors. The open reading frame of the gene encoding RSV RT α is shaded in light gray. The start (ATG) and stop of the gene are indicated. The restriction enzymes relevant for the cloning procedure are shown. Abbreviations for other enzymes are as follows: B, BamHI; E, EcoRV; H, HindIII; P, PmlI; S, SacII; and X, XhoI. The locations of the PCR primers are indicated by black arrows. A white circle indicates the location of Asp 181 or Asp 505. A black circle indicates the mutagenized codon for Asn 181 or Asn 505. (A) Mutagenesis of the codon for Asp 181 in pBac-α. (B) Mutagenesis of the codon for Asp 505 in pBac-α.

FIG. 2

Analysis of RSV RT α_D181NPol after elution from a heparin-Sepharose column. (A) SDS-PAGE of the eluted fractions. Proteins were detected by Coomassie staining. Lane M, protein molecular mass markers. The bracket indicates the pooled fractions. (B) HPLC size exclusion chromatography of the pooled fractions of α_D181NPol (37). The peak with a retention time of 26.01 min corresponds to a molecular mass of ∼175 kDa. The retention times of homodimeric Pol (25.86 min) and α (28.26 min) were determined in separate runs with the corresponding enzymes and are indicated by dotted lines. The molecular mass of α_D181NPol was determined using molecular mass standard proteins from U.S. Biochemical Corp. in the same buffer.

FIG. 3

Analysis of the purified mutant RSV RT enzymes by SDS-PAGE. Proteins were detected by Coomassie staining. Lane M, protein molecular mass markers.

FIG. 4

Polymerization activities on a DNA template catalyzed by RSV RTs selectively mutated in the polymerase active site. Reactions were performed for 10 min at 37°C in RT buffer with 10 nM M13 substrate and enzyme and a 250 μM concentration of each dNTP (37). Lane M shows DNA size markers (their sizes are indicated on the left), and lane -RT shows P-T without enzyme.

FIG. 5

RNase H activities catalyzed by RSV RTs. (A) Schematic representation of the heteropolymeric DNA-RNA P-T substrate comprising a 5′-end labeled 127-mer RNA to which a 36-mer DNA primer was hybridized. The major cleavage sites at positions 71 and 72 are indicated by arrows. RNase H activities of RSV RTs mutated in the RNase H active site (B) or in the polymerase active site (C) are shown. Reactions were performed for 10 min at 37°C in RT buffer with 10 nM 36-mer–127-mer DNA-RNA P-T and 10 nM enzyme. The sizes of the 5′ RNA cleavage products are indicated on the left. Lane -RT contains P-T without enzyme.

FIG. 5

RNase H activities catalyzed by RSV RTs. (A) Schematic representation of the heteropolymeric DNA-RNA P-T substrate comprising a 5′-end labeled 127-mer RNA to which a 36-mer DNA primer was hybridized. The major cleavage sites at positions 71 and 72 are indicated by arrows. RNase H activities of RSV RTs mutated in the RNase H active site (B) or in the polymerase active site (C) are shown. Reactions were performed for 10 min at 37°C in RT buffer with 10 nM 36-mer–127-mer DNA-RNA P-T and 10 nM enzyme. The sizes of the 5′ RNA cleavage products are indicated on the left. Lane -RT contains P-T without enzyme.

FIG. 5

RNase H activities catalyzed by RSV RTs. (A) Schematic representation of the heteropolymeric DNA-RNA P-T substrate comprising a 5′-end labeled 127-mer RNA to which a 36-mer DNA primer was hybridized. The major cleavage sites at positions 71 and 72 are indicated by arrows. RNase H activities of RSV RTs mutated in the RNase H active site (B) or in the polymerase active site (C) are shown. Reactions were performed for 10 min at 37°C in RT buffer with 10 nM 36-mer–127-mer DNA-RNA P-T and 10 nM enzyme. The sizes of the 5′ RNA cleavage products are indicated on the left. Lane -RT contains P-T without enzyme.

FIG. 6

Qualitative analysis of RNase H activities during DNA polymerization. The 127-mer RNA of the 36-mer–127-mer DNA-RNA was 5′-end labeled. Reactions were started by the addition of 10 nM enzyme to RT buffer containing a 10 nM concentration of the 36-mer–127-mer DNA-RNA P-T and a 50 μM concentration of each dNTP and stopped with formamide buffer after 10 min at 37°C. The sizes of the 5′ RNA cleavage products are indicated on the left. Complete primer extension in the presence of an active RNase H leads to a terminal RNA cleavage product of 16 nucleotides in length, as indicated at the bottom of the gel. Lane -RT, no enzyme added.