In vitro fidelity of the prototype primate foamy virus (PFV) RT compared to HIV-1 RT

Abstract

We compared the in vitro fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and the prototype foamy virus (PFV) RT. Both enzymes had similar error rates for single nucleotide substitutions; however, PFV RT did not appear to make errors at specific hotspots, like HIV-1 RT. In addition, PFV RT made more deletions and insertions than HIV-1 RT. Although the majority of the missense errors made by HIV-1 RT and PFV RT are different, relatively few of the mutations caused by either enzyme can be explained by a misalignment/slippage mechanism. We suggest that the higher polymerase activity of PFV RT could contribute to the ability of the enzyme to jump to the same or a different template.

Fig. 1

The locations of the single nucleotide substitutions generated by both HIV-1 RT and PFV RT are shown. As described in the Materials and methods, the lacZα peptide generated in our system is a fusion peptide. In our analysis, the N terminus of this peptide was not included as part of the mutational target. This leader sequence is highlighted in bold. The sequence of the plasmid that is 3′ of the termination codons, and therefore not translated, is also highlighted in bold. The first codon of the target lacZα sequence is underlined (GGA). The numbering system starts from the first nucleotide of the unique EcoRI site present in the Litmus-29 (Not). This is also the first base added by either HIV-1 RT or PFV RT when olymerizing across the template. The nucleotide substitutions detected for each RT are shown above the normal lacZα sequence. The amino acid sequence is shown below the lacZα sequence. Positions where there is more than one type of nucleotide substitution are highlighted.

Fig. 1

The locations of the single nucleotide substitutions generated by both HIV-1 RT and PFV RT are shown. As described in the Materials and methods, the lacZα peptide generated in our system is a fusion peptide. In our analysis, the N terminus of this peptide was not included as part of the mutational target. This leader sequence is highlighted in bold. The sequence of the plasmid that is 3′ of the termination codons, and therefore not translated, is also highlighted in bold. The first codon of the target lacZα sequence is underlined (GGA). The numbering system starts from the first nucleotide of the unique EcoRI site present in the Litmus-29 (Not). This is also the first base added by either HIV-1 RT or PFV RT when olymerizing across the template. The nucleotide substitutions detected for each RT are shown above the normal lacZα sequence. The amino acid sequence is shown below the lacZα sequence. Positions where there is more than one type of nucleotide substitution are highlighted.

Fig. 2

The locations of the frameshifts and short deletions/additions generated by HIV-1 RT and PFV RT. Frameshifts are defined as the gain or loss of one nucleotide. Short deletions and additions are the gain or loss of 2 or 3 nucleotides. Deletions are indicated by a Greek delta (Δ) above the lacZα sequence indicating the deleted nucleotide(s). If more than one nucleotide was deleted, the delta symbols are bracketed [ΔΔ]. Insertion of a nucleotide is indicated by showing the inserted nucleotide (highlighted) embedded within the normal flanking nucleotides. Each individual frameshift is on a separate line above the lacZα sequence. If more than one identical frameshift was found, the total number of the identical frameshift is shown as a multiplier (2X, etc).

Fig. 2

The locations of the frameshifts and short deletions/additions generated by HIV-1 RT and PFV RT. Frameshifts are defined as the gain or loss of one nucleotide. Short deletions and additions are the gain or loss of 2 or 3 nucleotides. Deletions are indicated by a Greek delta (Δ) above the lacZα sequence indicating the deleted nucleotide(s). If more than one nucleotide was deleted, the delta symbols are bracketed [ΔΔ]. Insertion of a nucleotide is indicated by showing the inserted nucleotide (highlighted) embedded within the normal flanking nucleotides. Each individual frameshift is on a separate line above the lacZα sequence. If more than one identical frameshift was found, the total number of the identical frameshift is shown as a multiplier (2X, etc).

Fig. 2

The locations of the frameshifts and short deletions/additions generated by HIV-1 RT and PFV RT. Frameshifts are defined as the gain or loss of one nucleotide. Short deletions and additions are the gain or loss of 2 or 3 nucleotides. Deletions are indicated by a Greek delta (Δ) above the lacZα sequence indicating the deleted nucleotide(s). If more than one nucleotide was deleted, the delta symbols are bracketed [ΔΔ]. Insertion of a nucleotide is indicated by showing the inserted nucleotide (highlighted) embedded within the normal flanking nucleotides. Each individual frameshift is on a separate line above the lacZα sequence. If more than one identical frameshift was found, the total number of the identical frameshift is shown as a multiplier (2X, etc).

Fig. 3

Repeats and deletions generated by HIV-1 RT. As described in Fig. 1, Bold indicates the lacZα leader sequence. GAATTC is the EcoRI site where polymerization by RT begins in the leader sequence. The vertical line (|) indicates the endpoint of the normal sequence, the beginning of a repeated sequence or the beginning of the sequence after the deletion is shown after this symbol. The locations of the endpoints of the deletion are shown by the numbers underneath the sequence.