Analysis of Polypurine Tract-associated DNA Plus-strand Priming in Vivo Utilizing a Plant Pararetroviral Vector Carrying Redundant Ectopic Priming Elements

Abstract

Initiation of DNA plus-strand synthesis in most reverse-transcribing elements requires primer generation by reverse transcriptase-associated RNase H at one or more template polypurine tracts (PPTs). We have exploited infectious clones of the plant pararetrovirus cauliflower mosaic virus carrying redundant ectopic plus-strand priming elements to study priming in vivo. Ectopic priming generated an additional discontinuity in progeny virion DNA during infection of plants. We found that altering the length of the 13-base pair PPT by ؎25% significantly reduced priming efficiency. A short pyrimidine tract 5 to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5 end remained 3 nucleotides from the PPT 3 end in mutant primers that were longer or shorter than the wild-type primer. Use of an ectopic redundant primer to study replication-dependent priming was validated by demonstrating that it could rescue infectivity following destruction of the wild-type priming elements. We propose a model for plant pararetroviral plus-strand priming in which pyrimidines enhance PPT recognition during polymerasedependent RNase H cleavages, and suggest that fidelity of primer maturation during polymerase-independent cleavages involves PPT length measurement and 3 end recognition by RNase H. Retro-elements (REs),1 the propagation of which involves copying genomic RNA into DNA mediated by reverse transcriptase (RT), are found in organisms representing the major groups of eukaryotes. REs include animal retroviruses, animal and plant pararetroviruses, and retrotransposable elements (1). Although REs are diverse, most share several common features. Important among these is the pol gene product specifying multiple enzyme activities associated with reverse transcriptase (RT) involved in RE DNA synthesis and processing (2, 3).REs have a different origin of replication for synthesis of each strand of the double-stranded (ds)DNA phase. First, reverse transcription of the genomic RNA into minus-strand DNA is initiated in most REs by a host tRNA (4). During minus-strand synthesis, RT-associated RNase H activity degrades the RNA template (5,6). This degradation appears to occur in at least two stages. Processive DNA minus-strand synthesis is accompanied by a polymerase-dependent, non-processive RNase H degradation of the template leaving RNA fragments. These fragments are probably further degraded by subsequent polymerase-independent RNase H cleavages (7-9). The RNA primer for DNA plus-strand synthesis is generated during these steps by RNase H cleavage at a specific site called the polypurine tract (PPT), 13-18 nucleotides long. For REs producing an integrating linear double-stranded DNA with long terminal repeats (LTRs), precision of plus-strand initiation is essential for RE viability as one LTR border is defined by this step. Therefore, understanding the role of the PPT is important, particularly in retroviruses where plus-strand i...

Selection of Optimal Polypurine Tract Region Sequences during Moloney Murine Leukemia Virus Replication

“…A model has been proposed which suggests that the T stretch located in PPT upstream sequences might function in a DNA polymerase-dependent step of the plus-strand priming process (16). Mo-MLV RT has been shown by footprinting to contact from Ϫ27 to ϩ6 of the template strand (numbered relative to the site of polymerization) and from Ϫ26 to Ϫ1 of the primer strand when bound in the configuration to polymerize DNA (34).…”

“…In a previous study, we established that sequences as far upstream as Ϫ28 (where Ϫ1 refers to the base immediately upstream of the cleavage site) are required for Moloney murine leukemia virus (Mo-MLV) plus-strand priming and that a T-rich stretch in this region is critical (24). Noad et al have similarly established that T-rich sequences upstream of the PPT are required for plus-strand priming for the pararetrovirus cauliflower mosaic virus (16). Additionally, Ilyinskii et al have demonstrated that the T stretch upstream of the simian immunodeficiency virus (SIV) PPT is required for SIV replication (11), and a T stretch upstream of the Ty1 PPT is important for plus-strand priming and transposition of that yeast retroelement (33).…”

“…Powell and Levin showed that the cleavage site-proximal half of the human immunodeficiency virus type 1 (HIV-1) PPT is required for plus-strand priming, while the cleavage site-distal half of the PPT is expendable in vitro (20). Similarly, the PPT's cleavage site-proximal G stretch is important for plus-strand priming during the replication of both cauliflower mosaic virus and Mo-MLV, although individual targeted mutations within this region are tolerated by Mo-MLV (16,24). In this study, we examined how much genetic variation within the PPT and its upstream T stretch was compatible with replication by determining which sequences persisted in replicating Mo-MLV populations when these regions initially contained randomized sequences.…”

Reverse transcription of retroviruses and LTR retrotransposons

“…the integrity of some positions in the PPT is necessary for cleavage specificity [50]. Recently, a conserved U-stretch within the upstream sequence of several retroelements (table 2) was found to be important for their replication [61][62][63][64][65]. In Ty1 it has been shown that mutations within this U-rich region inhibit plus-strand strong-stop DNA synthesis (+sssDNA) [64].…”

Reverse transcriptase and integrase of the <i>Saccharomyces cerevisiae </i>Ty1 element

“…Several reports have suggested that the precise generation of PPT primer is sequence dependent, i.e the integrity of some positions in the PPT is necessary for cleavage specificity (Champoux, 1993). Moreover a conserved U-rich region (U-box) within the upstream sequence of several retroelements (Table 1) has been found to be important for their replication (Ilyinskii and Desrosiers, 1998;Noad et al, 1998;Telesnitsky, 1999, 2000;Wilhelm et al, 1999). Mutations within the U-box of Ty1 inhibit plus-strand strong-stop DNA synthesis (+sssDNA) (Wilhelm et al, 1999).…”