Escherichia coli RNase HI inhibits murine leukaemia virus reverse transcription in vitro and yeast retrotransposon Ty1 transposition in vivo.
BACKGROUND
Reverse transcription, which converts an RNA genome into double-stranded DNA, requires both the polymerase and RNase H activities of reverse transcriptase (RT). In vitro, poorly processive RT dissociates from partially copied RNA-DNA hybrids, that are usually extended by a second RT molecule. Despite similar structures, RNase HI of Escherichia coli can degrade RNA-DNA hybrids that are resistant to RNase H of RT. E. coli RNase HI is used to determine the accessibility to and requirement for RNA-DNA hybrids in reverse transcription in vivo and in vitro.


RESULTS
In the presence of E. coli RNase HI, reverse transcription yields incomplete cDNA molecules due to degradation of RNA-DNA hybrids. Delivery of E. coli RNase HI to Ty1 particles via fusion to the capsid protein can reduce retrotransposition by more than 99%, also indicating inhibition of DNA synthesis in vivo.


CONCLUSIONS
Inhibition of both reverse transcription in vitro and retrotransposition in vivo by E. coli RNase HI indicates that the poor processivity of RT exposes RNA-DNA hybrids critical for reverse transcription to degradation. Targeting a cellular RNase H to HIV may help define the site(s) of RNA-DNA hybrids that are susceptible to nonretroviral RNase H and may be useful for gene therapy to inhibit retroviral replication.
Ma
W P
WP
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Crouch
R J
RJ
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D066298:In Vitro Techniques; D009052:Leukemia Virus, Murine / Q000187:drug effects* / Q000235:genetics*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D018626:Retroelements / Q000187:drug effects* / Q000235:genetics; D016914:Ribonuclease H / Q000235:genetics / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D018626:Retroelements; D016914:Ribonuclease H; C078856:ribonuclease HI