Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase.

Abstract

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specifiC cleavage in HIV-1 RNA hybridized to tRNALYS, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the IIV RNAtRNALYs complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant Hil RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNAtRNALYS at two distinct sites within the PBS sequence 5'-UGGCGCCCGA | ACAG I GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALS sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.The reverse transcriptase (RT) of human immunodeficiency virus (HIV), like the enzyme of other retroviruses, exhibits several activities: RNA-and DNA-directed DNA synthesis (1-3), RNase H (4, 5), and tRNALYS binding (6, 7). These well-characterized activities participate in various stages of double-stranded proviral DNA synthesis. The synthesis of the first strand, minus-strand DNA, is primed by a tRNALYS of cellular origin hybridized to the viral genomic RNA at a complementary sequence of 18 nucleotides, the primer binding site (PBS) (8). Synthesis of the plus-strand DNA is primed by an oligoribonucleotide of virus origin hybridized to the nascent minus-strand DNA, which now serves as a template (9, 10). To produce a proviral DNA with two long terminal repeats, the enzyme has to switch templates at two stages of the reverse transcription process: during synthesis of minusstrand DNA the enzyme jumps from the 5' end to the 3' end of the RNA template and, to complete synthesis of the double-stranded proviral DNA, the enzyme switches templates at the region of the...

Three‐dimensional structural resemblance between the ribonuclease H and connection domains of HIV reverse transcriptase and the ATPase fold revealed using graph theoretical techniques

“…In addition to DNA polymerase and RNase H activity, the presence of an additional RNase D activity in RT, associated with cleavage of RNA primer, has been reported [32], although this finding has been questioned by others [33]. The presence of the two connection domains with structures analogous to that of RNase H may provide candidate sites for such activity if it is confirmed.…”

A model for reverse transcription by a dimeric enzyme

“…1 e and g). [HIV-1 may also have an RNase D activity that cuts an RNA/RNA hybrid 10 and 14 bp from the polymerization site; this might also be involved in releasing tRNA (Ben-Artzi et al, 1992).] Each half of the dimer initially binds one or other of the templates at two (and probably more) places (Fig.…”

Relationship between Retroviral Replication and RNA Interference Machineries

“…It should be noted that the HIV RT/RNase H and the murine leukemia virus (MLV) RT/RNase H have also been shown to process dsRNAs, not only RNA-DNA hybrids (Ben-Artzi et al 1992;Hostomsky et al 1994;Blain and Goff 1993). This activity has also been named RNase H*.…”