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20 A Nucleotide Substitution in the Lys tRNA Primer Binding Site Dramatically Increases Replication of Recombinant SIV Containing HIV-1 Reverse Transcriptase
K. Soderberg*1, R. C. Desrosiers2, and L. Alexander1
1Yale Univ., New Haven, CT, USA and 2 Harvard Univ., Southborough, MA, USA
Background: A recombinant SIV with reverse transcriptase sequences from HIV-1 strain HXB2 was severely impaired for replication. Detectable p27Gag levels were not observed until day 65 and peak p27Gag levels were not reached until day 75 after transfection of CEMx174 cells with the recombinant DNA. Based on this pattern of replication, we hypothesized that RT/SHIV had undergone sequence changes that significantly increased its replication.
Methods: To test our hypothesis, we investigated HIV-1 RT sequences and SIV cis-acting elements for changes in passaged RT/SHIV. Selected changes were introduced into the parental sequences to determine the sequence alterations necessary for rapid RT/SHIV replication.
Results: RT/SHIV sequences from day 75 post-transfection did not contain amino acid substitutions in HIV-1 RT; however, a single nucleotide substitution (thymidine to cytosine) was found at position 8 of the SIV primer binding site (PBS). Conversely, SIVmac239 derived from replication in CEMx174 cells in culture retained a thymidine at this position. An RT/SHIV genome with the thymidine to cytosine substitution, called RT/SHIV/TC, had dramatically faster replication kinetics than that observed with the parental RT/SHIV from which this variant was derived. Furthermore, a stock of RT/SHIV/TC replicated similarly to wild-type SIVmac239. This change resulted in a sequence that exactly complemented Lys3 tRNA sequences, the primer typically utilized by primate lentiviruses for the initiation of reverse transcription. PBS sequences in SIVmac239 exactly complement Lys5 tRNA sequences, which are only contained in rare isolates of primate lentiviruses.
Conclusions: Our studies demonstrate that the presence of thymidine at position 8 of the PBS was the primary determinant for the poor replication of the parental RT/SHIV. Our data also reveal a difference in the PBS requirements of the reverse transcriptases of SIVmac239 and HIV-1 HXB2. Furthermore, RT/SHIV/TC provides an improved system for study of the mutations in HIV-1 reverse transcriptase in a relevant animal model.
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