Nonpolar Thymine Isosteres in the Ty3 Polypurine Tract DNA Template Modulate Processing and Provide a Model for Its Recognition by Ty3 Reverse Transcriptase

Abstract

Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (؉) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (؉)-RNA and failure to release (؉)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (؊)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T 3 F substitution and enhanced ribonuclease H (RNase H) activity ϳ12-13 bp downstream. In the most pronounced case, dual T 3 F substitution at PPT positions ؊1/؊2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (؊)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT ϳ12-13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T 3 F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5 vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper. Although reverse transcriptase (RT)1 -associated ribonuclease H (RNase H) activity degrades RNA of the RNA/DNA replication intermediate with little sequence specificity, it must precisely remove the tRNA and polypurine tract (PPT) primers of (Ϫ)-strand (1) and (ϩ)-strand DNA synthesis (2), respectively, to generate sequences at the 5Ј and 3Ј termini of the double-stranded DNA recognized by the integration machinery (3-8). Since the PPT is most likely embedded in a considerably larger RNA/DNA hybrid, precise hydrolysis at the PPT-U3 junction observed in vitro (9) suggests unique structural features may participate by correctly positioning this junction in the RNase H catalytic center. Our recent chemical footprinting of HIV-1 PPT-containing RNA/DNA hybrids (10) and a comparison with the crystal structure of HIV-1 RT bound to a related duplex (11) support this notion. Nucleic acid in the RT-RNA/DNA co-crystal is distorted 8 -14 bp upstream of the PPT-U3 junction, comprising weakly paired, unpaired, and mispaired bases (Fig. 1A). Subsequent chemical footprinting studies (10) revealed that template thymines of this region and thymine ϩ1 (i.e. immediately 3Ј to the PPT) deviate from standard Watson-Crick base pairing in the absence of the retroviral enzyme. The finding that these naturally occurring HIV-1 PPT distortions are 10 -14 bp apart was particularly intriguing, sinc...

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

“…Flexibility in PPT architecture may, therefore, play an important role in sequestering HIV-1 RT via structural motifs in the DNA polymerase and/or RNase H domain. As a complementary approach to the use of pyrimidine isosteres (44,46), we examined here the consequences of increasing the stability of this RNA/DNA hybrid via a scanning strategy employing LNA analogs in a pairwise and triplet fashion.…”

“…More recently, the fluorescent cytosine analog pyrrolo-dC was exploited to confirm that a cytosine residue of the unzipped portion of the HIV-1 PPT (Ϫ) DNA defined crystallographically was also unpaired in the absence of RT (45). These studies coupled with a parallel evaluation of the Ty3 PPT substituted with thymine isosteres (46) illustrate the value of nucleoside analogs as probes of PPT structure.…”

Examining Ty3 Polypurine Tract Structure and Function by Nucleoside Analog Interference

“…RNase H-mediated PPT Selection-Ty3 PPT selection was evaluated as described previously (10). 5Ј-End labeling of the PPT-containing RNA was performed with T4 polynucleotide kinase and [␥-…”

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

“…8B, RNase H cleavage specificity was evaluated via hydrolysis of an RNA/DNA hybrid mimicking the Ty3 PPT/U3 junction. Because the RNA strand of this hybrid contains an additional 13 nt downstream of the junction, this substrate allows simultaneous evaluation of both specific and nonspecific hydrolyses (32). As an example, although low level hydrolysis occurred between positions ϩ1 and ϩ10, wild-type RT and mutants Q290A and Y298A cleaved preferentially at the PPT/U3 junction (Fig.…”