Recognition of internal cleavage sites by retroviral RNases H.
The RNase H activity of reverse transcriptase is essential to complete retroviral replication. Many studies have characterized how reverse transcriptase associates with recessed and exposed DNA 3' ends or RNA 5' ends to position the RNase H domain for cleavage, but little is known about how a nick might affect RNase H cleavages, or how RNase H carries out internal cleavages, which do not require positioning by a nucleic acid end. We have addressed these issues using model hybrid substrates and the reverse transcriptases of Moloney murine leukemia virus (M-MuLV) and human immunodeficiency virus type 1 (HIV-1). Our results show that a nick separating an upstream RNA and a downstream RNA annealed to DNA is essentially ignored by RNase H, indicating that the RNA 5' end at a nick is not sufficient to position 5' end-directed cleavages. Cleavage sites that are located close to the 5' end of the downstream RNA are not recognized in the absence of the upstream RNA, and the 5' ends of the shorter upstream RNAs enhance cleavage at these sites. The recognition of an internal cleavage site depends on local sequence features found both upstream and downstream of the cleavage site, designated as the -1/+1 position. By analyzing the nucleotide frequencies in the sequence surrounding strong internal cleavage sites, preferred nucleotides have been identified in the flanking sequences spanning positions -14 to +1 for HIV-1 and -11 to +1 for M-MuLV. These data reveal that general degradation of the retroviral genome after minus-strand synthesis can occur through sequence-specific cleavages.
Schultz
Sharon J
SJ
Department of Microbiology, Box 357242, School of Medicine, University of Washington, Seattle, WA 98195-7242, USA.
Zhang
Miaohua
M
Champoux
James J
JJ
D001483:Base Sequence; D017931:DNA Primers; D015497:HIV-1 / Q000201:enzymology* / Q000502:physiology; D006868:Hydrolysis; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000502:physiology; D010766:Phosphorylation; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity; D014779:Virus Replication
D017931:DNA Primers; D016914:Ribonuclease H