Nucleic Acids Research, Vol 24, Issue 9 1719-1726, Copyright © 1996 by Oxford University Press
GM Fuentes, PJ Fay and RA Bambara
During retroviral reverse transcription the genomic RNA is degraded by the
RNase H activity of reverse transcriptase (RT). Previous results suggest
that after RNA-directed DNA synthesis, fragments of RNA remain annealed to
the newly synthesized DNA [DeStefano et al.(1991) J. Biol.Chem. 266,
7423-7431]. These must be removed to allow synthesis of the second DNA
strand. We measured the ability of HIV-, AMV- and MuLV- RT to coordinate
DNA-dependent DNA synthesis and removal of downstream segments of RNA. The
substrates employed were DNA templates having upstream DNA and downstream
RNA primers. We found that none of the wild type RTs elongated the upstream
DNA without simultaneous degradation of the RNA. Consistent with these
results, HIV-, AMV- and MuLV-RT showed relatively higher affinity for RNA
than for DNA oligonucleotides bound to a DNA template. Differences were
observed in the RNA degradation and DNA extension patterns generated by the
different RTs. AMV-RT degraded the RNA to segments 11-12 nt long, and
readily elongated the upstream DNA to the end of the template. MuLV- and
HIV-RT degraded the RNA primarily to segments 15-16 nt long. At low
concentrations of the latter two RTs, the DNA primer stalled when it
encountered the 5'-end of the RNA. In sufficient excess, all of the RTs
elongated the upstream primer without stalling. Even though we were unable
to detect displacement of the downstream RNA by the wild type RTs, MuLV-
and HIV- RT lacking RNase H, were able to elongate the upstream DNA to the
end of the template without degradation of the RNA. This suggests that
degradation of downstream pieces of RNA is not absolutely required before
synthesis of the plus strand DNA. The implications of these findings for
viral replication are discussed.
ARTICLES
Relationship between plus strand DNA synthesis removal of downstream segments of RNA by human immunodeficiency virus, murine leukemia virus and avian myeloblastoma virus reverse transcriptases
Department of Microbiology, University of Rochester,School of Medicine and Dentistry, NY 14642, USA.
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