Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 441064. Received 5 ... On the basis of its location in the RNase H domain, we propose that ... hybrids (3). In light of these findings, assigning a new name (RNase D).
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NOTES Redesignation of the RNase D Activity Associated with Retroviral Reverse Transcriptase as RNase H* ZDENEK HOSTOMSKY,' STEPHEN H. HUGHES,2 STEPHEN P. GOFF,3 AND STUART F. J. LE GRICE4 Agouron Pharmaceuticals Inc., San Diego, California 921211; ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-12012; Department of Biochemistry, Columbia University, New York, New York 100323; and Division of Infectious Diseases, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 441064 Received 5 October 1993/Accepted 12 November 1993
In the presence of Mn2+, reverse transcriptase of both human immunodeficiency virus and murine leukemia virus hydrolyzes duplex RNA. However, designating this novel activity RNase D conflicts with Escherichia coli RNase D, which participates in tRNA processing. On the basis of its location in the RNase H domain, we propose that this novel retroviral activity be redesignated RNase H*.
It has been recently reported that retroviral reverse transcriptase (RT) can cleave double-stranded RNA (1-3). Original studies of Ben-Artzi et al. (1), with substrate representing a tRNALys::primer binding site complex, suggested that this activity, which the authors designated RNase D, might be required at certain stages during replication. However, the similarity in cleavage specificities between the RT-associated RNase D and Escherichia coli RNase III raised the possibility that RNase D activity might reflect low-level contamination with the bacterial enzyme. These contentions were strengthened by the findings of Hostomsky et al. (6) that preparations of p51 and several RNase H-deficient forms of human immunodeficiency type 1 (HIV-1) RT were also capable of cleaving double-stranded RNA, including a bacteriophage RNA substrate conventionally used for analysis of RNase III. The RNase employed by Ben-Artzi et al. (1) and Hostomsky et al. (6) involved cleavage of a radiolabeled substrate and then fractionation of the products by high-resolution gel electrophoresis. Alternatively, DNA polymerase and RNase H activities of RT can be assessed by in situ methods with polyacrylamide gels in which a radioactive substrate is embedded. While the latter approach is more qualitative, it has the advantage that activity is assigned by molecular mass, i.e., for HIV-1 and HIV-2 RT, the polymerase and RNase H activities were associated with the 66-kDa polypeptide (5, 8). Subsequent data from Ben-Artzi et al. (2) has indeed confirmed that their original HIV-1 RT preparation (1) contained contaminating E. coli RNase III. However, work by the same authors with HIV RT polypeptides purified by metal chelate affinity chromatography (p51, p66, and p66/pSi) (7) revealed that an RNase activity capable of digesting double-stranded RNA was in fact associated with the p66 subunit of both homo- and heterodimer. Loss of this function with p66 HIV RT containing an inactivating mutation in the RNase H domain (Glu-478->Gln478) verified RNase D as a bona fide activity and showed that
*
it was tightly coupled with previously identified RNase H function. The ability of murine leukemia virus RT to hydrolyze duplex RNA supports observations with the HIV enzyme (2, 3). Blain and Goff present strong evidence that the RNase H domain is required for this activity, but there are distinct requirements for hydrolysis of RNA/DNA and RNA/RNA hybrids (3). In light of these findings, assigning a new name (RNase D) to this RNase H-associated activity seems misleading. Such an assignment is more appropriately reserved for the E. coli enzyme involved in tRNA processing (4). By analogy with the convention for certain restriction enzymes, we propose RNase H* as a more suitable designation. Although RNase H* activity has been clearly demonstrated in RT, its relevance in vivo remains obscure. In this context, it is worth noting that the HIV enzyme requires Mn2+ to cleave double-stranded RNA (2). In contrast, both the DNA polymerase and RNase H activities of this enzyme show a strong preference for Mg2+. S. H. Hughes is sponsored in part by the National Cancer Institute, DHHS, under contract NO1-CO-74101 with ABL. S. P. Goff is supported by Public Service Health grant CA 30488, and S. F. J. Le Grice is supported by NIH grant GM 46623.
REFERENCES 1. Ben-Artzi, H., E. Zeelon, M. Gorecki, and A. Panet. 1992. Doublestranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. Proc. Natl. Acad. Sci. USA 89:927-931. 2. Ben-Artzi, H., E. Zeelon, S. F. J. Le Grice, M. Gorecki, and A. Panet. 1992. Characterization of the double stranded RNA dependent RNase activity associated with recombinant reverse transcriptase. Nucleic Acids Res. 20:5115-5118. 3. Blain, S. W., and S. P. Goff. 1993. Nuclease activities of Moloney murine leukemia virus reverse transcriptase. J. Biol. Chem. 268: 23585-23592. 4. Deutscher, M. P. 1993. Ribonuclease multiplicity, diversity and complexity. J. Biol. Chem. 268:13011-13014. 5. Hizi, A., R. Tal, and S. H. Hughes. 1991. Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Esche-
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richia coli. Virology 180:339-346. 6. Hostomsky, Z., G. 0. Hudson, S. Rahmati, and Z. Hostomska. 1992. RNase D, a reported new activity associated with HIV-1 reverse transcriptase, displays the same cleavage specificity as Escherichia coli RNase III. Nucleic Acids Res. 20:5819-5824. 7. Le Grice, S. F. J., and F. Gruninger-Leitch. 1990. Rapid purification
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of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur. J. Biochem. 187:307-314. 8. Prasad, V., and S. P. Goff. 1989. Linker insertion mutagenesis of human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain. Proc. Natl. Acad. Sci. USA 86:3104-3108.
