Quantitative analysis of RNA cleavage during RNA-directed DNA synthesis by human immunodeficiency and avian myeloblastosis virus reverse transcriptases.
We have determined the extent of RNA cleavage carried out during DNA synthesis by either human immunodeficiency virus (HIV) or avian myeloblastosis virus (AMV) reverse transcriptases (RTs). Conditions were chosen that allowed the analysis of the cleavage and synthesis performed by the RT during one binding event on a given template-primer. The maximum quantity of ribonuclease H (RNase H) sensitive template RNA left after synthesis by the RTs was determined by treatment with Escherichia coli RNase H. RNA cleavage products that were expected to be too short to remain hybridized, less than 13 nucleotides in length, were quantitated. Results showed that HIV- and AMV-RT degraded about 80% and less than 20%, respectively, of the potentially degradable RNA to these short products. Survival of longer, hybridized RNA was not a result of synthesis by a population of RTs that had selectively lost RNase H activity. Using an assay that evaluated the proportion of primers extended versus RNA templates cleaved during primer-extension by the RTs, we determined that essentially each molecule of HIV- and AMV-RT with polymerase also has RNase H activity. The results indicate that although both HIV- and AMV-RTs cleave the RNA template during synthesis, the number of cleavages per nucleotide addition with HIV-RT is much greater. They also suggest that some hybridized RNA segments remain right after the passage of the RT making the first DNA strand. In vivo, these segments would have to be cleaved or displaced in later reactions before second strand DNA synthesis could be completed.
DeStefano
J J
JJ
Department of Microbiology, University of Maryland, College Park 20742.
Mallaber
L M
LM
Fay
P J
PJ
Bambara
R A
RA
D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology; D006678:HIV / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013698:Templates, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H