No abstract

The ability of reverse transcriptase to generate, extend, and remove the primer derived from the polypurine tract (PPT) is vital for reverse transcription, since this process determines one of the ends required for integration of the viral DNA. Based on the ability of the RNase H activity of Moloney murine leukemia virus reverse transcriptase to cleave a long RNA/DNA hybrid containing the PPT, it appears that cleavages that could generate the plus-strand primer can occur by an internal cleavage mechanism without any positioning by an RNA 5-end, and such cleavages may serve to minimize cleavage events within the PPT itself. If the PPT were to be cleaved inappropriately just upstream of the normal plus-strand origin site, the resulting 3-ends would not be extended by reverse transcriptase. Extension of the PPT primer by at least 2 nucleotides is sufficient for recognition and correct cleavage by RNase H at the RNA-DNA junction to remove the primer. Specific removal of the PPT primer after polymerase extension deviates from the general observation that primer removal occurs by cleavage one nucleotide away from the RNA-DNA junction and suggests that the same PPT specificity determinants responsible for generation of the PPT primer also direct PPT primer removal. Once the PPT primer has been extended and removed from the nascent plus-strand DNA, reinitiation at the resulting plus-strand primer terminus does not occur, providing a mechanism to prevent the repeated initiation of plus strands.Reverse transcriptase converts the single-stranded retroviral RNA genome into the linear double-stranded DNA that integrates into the chromosome of a host cell (1, 2). The reverse transcriptase of Moloney murine leukemia virus (MMLV) 1 is a 75-kDa protein that contains an NH 2 -terminal DNA-and RNAdependent DNA polymerase activity and a COOH-terminal RNase H activity. Although the polymerase and RNase H activities are functionally separable (3-6), the polymerase and RNase H domains function in an interdependent manner (3, 7-10). The polymerase activity extends both RNA and DNA primers, although efficient extension from RNA primers appears limited to the host cell-derived tRNA primer used for minus-strand DNA synthesis and the primer used for plussense DNA synthesis that is derived from the polypurine tract (PPT) sequence in the viral genome (reviewed in Refs. 2 and 11) (12-20). The RNase H activity acts primarily as an endonuclease, hydrolyzing the RNA in an RNA/DNA hybrid to produce 3Ј-hydroxyl and 5Ј-phosphate ends (11,21,22). Cleavage by the RNase H activity of reverse transcriptase specifically generates the PPT primer during the process of reverse transcription (15,(23)(24)(25)(26). RNase H is also responsible for removing the tRNA and PPT primers from the nascent DNA strands after they have been extended and for general degradation of the viral genome after minus-strand DNA synthesis (reviewed in Ref. 11) (12,15,24,[27][28][29][30].Previous studies have indicated that two different modes of RNase H activity can be distingui...

The RNase H activity of reverse transcriptase is essential for retroviral replication. RNA 5-end-directed cleavages represent a form of RNase H activity that is carried out on RNA/DNA hybrids that contain a recessed RNA 5-end. Previously, the distance from the RNA 5-end has been considered the primary determinant for the location of these cleavages. Employing model hybrid substrates and the HIV-1 and Moloney murine leukemia virus reverse transcriptases, we demonstrate that cleavage sites correlate with specific sequences and that the distance from the RNA 5-end determines the extent of cleavage. An alignment of sequences flanking multiple RNA 5-end-directed cleavage sites reveals that both enzymes strongly prefer A or U at the ؉1 position and C or G at the ؊2 position, and additionally for HIV-1, A is disfavored at the ؊4 position. For both enzymes, 5-end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5-end, a distance termed the cleavage window. In examining the importance of accessibility to the RNA 5-end, it was found that the extent of 5-end-directed cleavages observed in substrates containing a free recessed RNA 5-end was most comparable to substrates with a gap of two or three bases between the upstream and downstream RNAs. Together these finding demonstrate that the selection of 5-end-directed cleavage sites by retroviral RNases H results from a combination of nucleotide sequence, permissible distance, and accessibility to the RNA 5-end.In reverse transcription, the single-stranded RNA genome of a retrovirus is transformed into double-stranded DNA by the viral-encoded reverse transcriptase (reviewed in Refs. 1 and 2). This multifunctional enzyme carries out DNA synthesis, strand displacement synthesis, and strand transfer and degrades the RNA portion of RNA/DNA hybrids. The polymerase domain of reverse transcriptase represents the aminoterminal two-thirds of the protein, whereas the RNase H domain comprises the remaining carboxyl-terminal portion. Although the polymerase and RNase H activities are functionally separable, both activities are essential for retroviral replication. The reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) 2 functions as an asymmetric heterodimer composed of p66 and p51 subunits (3), whereas that of Moloney murine leukemia virus (M-MuLV) is a 76-kDa protein that may function as a homodimer or as a monomer (4 -6).RNase H contributes to reverse transcription in three distinct ways (reviewed in Refs. 1, 7, and 8). First, RNase H carries out degradation of the RNA genome both during and after minus-strand DNA synthesis to facilitate plus-strand DNA synthesis and strand transfers. Second, RNase H creates the polypurine tract (PPT) primer from the viral genome. And third, RNase H removes the PPT and tRNA primers used to prime plus-strand and minus-stand DNA synthesis, respectively. Given the vital roles of RNase H in retroviral replication, it is important to understand how RNase H recognizes the RNA/DNA hybri...

Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3 terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3 terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3 terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3 end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3 terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3 terminus.Shortly after entrance of the viral cores into the cytoplasm of a cell, the single-stranded plus-sense RNA genome of a retrovirus is converted into a double-stranded DNA molecule that subsequently integrates into the host cell genome (4). This process, termed reverse transcription, requires two distinct RNA primers to synthesize the double-stranded DNA. The first primer is a host cell-derived tRNA that is used for the initiation of minus-strand DNA synthesis. The second is a short RNA derived by RNase H cleavages within a purine-rich sequence in the viral genome called the polypurine tract (PPT). This primer is used to begin plus-strand synthesis and is referred to as the plus-strand primer or the PPT primer (reference 2 and references therein).These RNA primers are extended by the viral-encoded reverse transcriptase, a multifunctional enzyme that carries out DNA polymerization, strand displacement synthesis, and the strand transfer reaction and that possesses an RNase H activity required at several steps during genome replication (4). The DNA polymerase activity of reverse transcriptase resides in the N-terminal two-thirds of the protein and utilizes either RNA or DNA as a template. Although displacement synthesis is not as efficient as nondisplacement synthesis (13,15,25,43), reverse transcriptase can simultaneously extend the 3Ј terminus of a DNA primer and displace a downstream nontemplate RNA or DNA strand, a...

During reverse transcription, the RNase H activity of reverse transcriptase specifically cleaves the viral genome within the polypurine tract (PPT) to create the primer used for the initiation of plus-strand DNA synthesis and nonspecifically cleaves the viral genome to facilitate synthesis of plus-strand DNA. To understand how primer length and sequence affect generation and utilization of the PPT, we employed short hybrid substrates containing or lacking the PPT to evaluate cleavage, extension, and binding by reverse transcriptase. Substrates containing RNAs with the correct 3 end for initiation of plus-strand synthesis were extended equally well by reverse transcriptase, but primer length affected susceptibility to RNase H cleavage. RNA substrates with 3 ends extending beyond the plus-strand initiation site were extended poorly but were specifically cleaved to generate the correct 3 end for initiation of plus-strand synthesis. Substrates containing RNAs lacking the PPT were cleaved nonspecifically and extended inefficiently. Specific cleavages to generate the plus-strand primer and 5-end-directed cleavages were kinetically favored over cleavages that destroyed the PPT primer or degraded other short RNA fragments. The PPT was not intrinsically resistant to cleavage by the isolated RNase H domain, and the isolated polymerase domain extended RNA primers containing the PPT sequence irrespective of the primer 3 end. These results provide insights into how reverse transcriptase generates and selectively utilizes the PPT primer for initiation of plus-strand DNA synthesis. Moloney murine leukemia virus (M-MuLV)1 converts its single-stranded plus-sense RNA genome into a double-stranded DNA molecule through the replicative process termed reverse transcription (reviewed in Ref. 1). Minus-strand DNA synthesis initiates from a host cell-derived tRNA primer and extends through a unique sequence (U5) and a terminal repeat sequence (R) at the 5Ј end of the genome before carrying out the first template jump to a second R sequence found at the 3Ј end of the viral genome. As minus-sense DNA synthesis progresses, a purine-rich sequence in the RNA genome termed the polypurine tract (PPT) that lies immediately adjacent to a downstream unique sequence (U3) is copied and subsequently cleaved to generate the PPT primer. This primer is used to initiate plus-strand DNA synthesis, which extends through U3-R-U5 and continues after a second jump to the 5Ј end of the DNA template. Displacement synthesis is required to complete duplex DNA synthesis and formation of the redundant DNA ends called long terminal repeats (LTRs) (2).The multifunctional enzyme that carries out this replicative process is the virally encoded reverse transcriptase (1). For M-MuLV, reverse transcriptase is a 75-kDa polypeptide that contains an amino-terminal RNA-and DNA-dependent DNA polymerase activity and a carboxyl-terminal RNase H activity that cleaves the RNA portion of an RNA/DNA hybrid. The RNase H and polymerase activities of reverse transcriptase are separabl...