Sequence, Distance, and Accessibility Are Determinants of 5′-End-directed Cleavages by Retroviral RNases H

Abstract

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...

Effects of Identity Minimization on Moloney Murine Leukemia Virus Template Recognition and Frequent Tertiary Template-Directed Insertions during Nonhomologous Recombination

“…Here, with recombination forced to occur principally from a single-template position, 16 bases of identity were sufficient to target accurate and efficient recombinogenic switching. Similar trends in acceptor recognition have previously been reported using different sequences and more-limited sets of length variation, suggesting that whereas some interactions between RT and nucleic acids differ by sequence, the observations here are not specific to the target sequences used (10,43,52). …”

RNase H activity: Structure, specificity, and function in reverse transcription

“…The third determinant is the accessibility of the end. Thus, while the 5′ end at a nick is not recognized by reverse transcriptase for RNA 5′ end-directed cleavages, a gap of 2 or more bases is sufficient for such recognition and cleavage by the HIV-1 and MoMLV enzymes (Schultz et al, 2004;Schultz et al, 2006).…”

“…Similar to DNA 3′ end-directed cleavages, two of these determinants are distance from the recessed end (DeStefano et al, 1993), and the nucleotide sequence in the vicinity of the cleavage site [see section 6.1; (Furfine and Reardon, 1991b;Fu and Taylor, 1992;DeStefano et al, 1993;Schultz et al, 2006)]. The third determinant is the accessibility of the end.…”

“…Internal RNase H cleavages most likely occur initially because reverse transcriptase can bind and cleave the genome without nucleic acid end-directed positioning. Multiple internal cleavage sites situated close together would generate the 2-3 base gaps that are sufficient to allow the kinetically favored RNA 5′ end-directed cleavages to proceed (Schultz et al, 1999;Schultz et al, 2006).…”

The Effects of Alternate Polypurine Tracts (PPTs) and Mutations of Sequences Adjacent to the PPT on Viral Replication and Cleavage Specificity of the Rous Sarcoma Virus Reverse Transcriptase

“…Rausch and Le Grice also did in vitro assays with mutant PPTs with A substitutions in the G tract of the HIV-1 PPT and showed that the A1, A2, A3, and A5 mutations affected the specificity or extent of cleavage (22). Schultz et al reported, based on in vitro cleavage of non-PPT sequences, that the RNase H of HIV-1 RT cleaves poorly if the substrate has the equivalent of an A3, T5, or A5 substitution (29).…”