Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 927-931, February 1992 Biochemistry
Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase (retrovirus ribonudease)
HANA BEN-ARTZI, ELISHA ZEELON, MARIAN GoRECKI, AND AMOS PANET BioTechnology General (Israel) Ltd., Kiryat Weizmann, Rehovot, 76326, Israel
Communicated by Ephraim Katchalski-Katzir, October 24, 1991
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.
of the RNA template and, to complete synthesis of the double-stranded proviral DNA, the enzyme switches templates at the region of the PBS. While the general scheme of reverse transcription is known, details of specific steps, such as template switching, are still missing (5, 8). In an endogenous reaction mixture containing detergent-disrupted virions, synthesis of a full-length proviral DNA with two long terminal repeats can be detected. It is not clear, however, whether auxiliary viral proteins other than RT are involved in the various steps of the reverse transcription reaction. To study the initiation and synthesis of the proviral DNA, we have constructed a system with recombinant RT and HIV-1 RNA-tRNALYs template-primer complex, which resembles the initiation complex utilized by the RT. During these studies, we noticed that HIV-1 RT cleaves specifically at the viral RNA PBS sequence. We propose that this newly described activity may be important in the reverse transcription process.
MATERIALS AND METHODS Construction of Plasmids. The plasmid pBENN7 (derived from HIV-1 lymphadenopathy-associated virus DNA) (11) was used for the isolation of a HindIII/Cla I restriction fragment of 2% base pairs (nucleotides 76-371; ref. 11). This DNA fragment includes part of R, U5, PBS, and part of gag. To restore the entire R region, this fragment was ligated with a synthetic DNA into the plasmid pSP64 (pHIV). The transcription of the HIV-1 gene starts with the first base of the R. The plasmid pGEM-1 (Promega) was modified and a new BspMI site was introduced adjacent to the T7 promoter. Four synthetic oligodeoxynucleotides spanning the sequence of tRNALYS and including an additional BspMI site at the 3' end of the tRNALYS gene were cloned into BspMI/HindIIIcleaved modified pGEM-I. The ptRNALYs cleaved with BspMI endonuclease was used as a template to yield complete tRNALYS molecules 76 bases long. Preparation of [32PJRNA and Synthetic tRNALYS Complexes. pHIV DNA was linearized with BssHII endonuclease at nucleotide 256 (12) and transcribed with SP6 RNA polymerase (13). [a-32P]CTP (105 cpm/pmol) was used for labeling the HIV RNA. The reaction mixture was treated with DNase I and phenol/chloroform extracted, and RNA was ethanol precipitated and purified by electrophoresis on 7 M urea/6% polyacrylamide denaturing gel (14). ptRNALYS was transcribed with T7 RNA polymerase (15). The reaction mixture was treated with DNase I, and RNA was extracted with phenol/chloroform followed by ethanol precipitation and subjected to electrophoresis on a denaturing gel (14). For complex preparation, purified HIV [32P]RNA and tRNALYS were hybridized at a 1:10 molar ratio in a solution
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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: RT, reverse transcriptase; HIV, human immunodeficiency virus; PBS, primer binding site; AMV, avian myeloblastosis virus.
927
Biochemistry: Ben-Artzi et al.
928
Proc. Nati. Acad Sci. USA 89 (1992)
containing 30%o formamide, 40 mM Tris-HCI (pH 7.4), 1 mM EDTA, and 0.3 M NaCi. Hybridization was for 10 min without salt at 650C and proceeded with salt at 420C for 2 hr. The RNA complex was purified on a nondenaturing 8% polyacrylamide gel. Elution of the RNA complex from the gel was done as described (14). A hybrid of 18-mer synthetic oligodeoxynucleotide, complementary to the PBS region, and HIV [32P]RNA was prepared under similar conditions for the RNase H reaction. RT Activities. Reaction mixtures (10 pl) contained 50 mM Tris-HCl (pH 8.0), 50 mM KCI, 2 mM dithiothreitol, 8 mM MgCl2, and the appropriate substrate. For standard DNA polymerase reactions, poly(A)/oligo(dT) and [a-32PJdTTP were used (16). One unit of enzyme activity is defined as nmol ofdTMP incorporated per 10 min under the standard reaction conditions (16). The substrate for RNase H reactions, HIV-1 [32P]RNA-DNA hybrid (2500 cpm), and the substrate for RNase D reactions, HIV-1 [32P]RNA-tRNALYs complex (2500 cpm), were incubated at 37°C. Samples (4 ,ul) were withdrawn, quenched with sample buffer (80%o formamide/50 mM EDTA), and heated at 95°C for 2 min. RNA was subjected to electrophoresis on a 6% polyacrylamide/7 M urea denaturing gel. Recombinant HIV RTs. Details of the expression and purification of HIV RT are described elsewhere (unpublished data). Briefly, Escherichia coli containing plasmid pHIV RT that expresses BH10 HIV RT (12) was the source of enzyme. RT was purified to apparent homogeneity by ammonium sulfate fractionation followed by chromatographies on four resins-phenyl-Sepharose, Q-Sepharose, phosphocellulose, and Superose gel. Specific activity of the purified RT (5000 units/mg) was determined by a standard RT assay using poly(A)/oligo(dT) as template/primer (16). The sequence of RNA fragments after RNase D reaction was determined by DNA primer extension (14) with a 17-mer 32P-labeled 5'-GCCGAGTCCTGCGTCGA-3', complementary to the 3' end of the HIV-1 RNA (Fig. 1) and avian myeloblastosis virus (AMV) RT (IBI). DNA products were analyzed by electrophoresis on a 6%o polyacrylamide sequencing gel (14). A DNA sequence ladder of HIV was generated with the same 32P 5' primer and dideoxynucleotides (14) on DNA plasmid (pHIV).
of the HIV-1 genome (R-U5-PBS) hybridized to a synthetic primer tRNALYS (Fig. 1). It should be noted that the synthetic tRNALYS is unmodified; however, it does not differ from the authentic tRNALYS in its ability to bind HIV RT and to initiate minus-strand DNA synthesis (18). The recombinant HIV-1 RT was produced in E. coli and purified by four consecutive column steps to apparent homogeneity. The HIV RT consists of two subunits, 66 and 51 kDa, as reported before (16, 19). The DNA polymerasespecific activity of this enzyme is similar to that of other purified HIV RT preparations (16, 19). The RT preparation is free of nonspecific RNase activities as incubation with HIV [32P]RNA single strand does not result in detectable degradation (Fig. 2, lanes 1 and 2). Surprisingly, we noticed that incubation of HIV RT with the template RNA annealed to the primer tRNALYS resulted in a unique cleavage (lanes 3-5). The size of the two RNA products, -200 and -60 nucleotides, suggests that cleavage occurs at the PBS sequence hybridized to tRNALYS and that the degradation products of 200 and 60 bases are derived from the 5' end and the 3' end of HIV RNA, respectively (see Fig. 1A). To investigate the origin of this unique RNase activity, we have obtained purified HIV RT preparations from two groups (16, 19). These two recombinant RTs were expressed and purified to apparent homogeneity by different procedures. The enzyme expressed by Mizrahi et al. (19) was processed in the bacterium by the HIV-1 coded protease. Thus, the purified enzyme is a heterodimer p66/pSl identical to RT purified from virions (3). The purified RT produced by Hizi et al. (16) is a dimer of 66 kDa. The three enzyme preparations have similar DNA polymerase specific activity, and they exhibit identical cleavage activity on the HIV RNA-tRNALYs complex (Fig. 2, lanes 6-9). It may be argued that the HIV RNA-tRNALYs substrate contains contaminating DNA and that the observed cleavage simply represents the RNase H activity of HIV RT. Several measures were taken to ensure removal of all DNA contaminations from the substrate HIV RNA-tRNALYs. After syn)
RT(I1)
RT0111
0 10' 0 5 15
1 010
) 10
RT(
RT(
RESULTS For the studies of HIV reverse transcription we have produced in vitro a template RNA that corresponds to the 5' end
-
1~~~~~~~~~~~~~~~~~~~~~~~~~~~
~
256 200
Go 0 -j
z n:
B
A
70 bases (see Fig. 1A). Nonetheless, the production of identical RNA molecules 60 and 64 bases long in the presence and absence of DNA synthesis indicates that cleavage occurs closer to the 3' end of the PBS site, as expected of the RNase D activity.
DISCUSSION We describe in this work an enzymatic activity associated with the HIV-1 RT. Our objective following this unexpected finding has been to analyze whether this RNase activity is indeed part of the HIV RT molecule. Several independent lines of evidence support the notion that RNase D is yet
A
A
B
(-dNTP's)
(--dNTP'so
0
T~
J ,*. ~~~~~~~~~~~~~~~~~~~~~~
~
~
r .jd
C~
~
~
10 20
0
10 20
_
A.
!i
S.
~~~~~~~~~~~~~~~~~~~~~~~~ 40 .:
.a.
*
110
-
90
-
76 -67 --a
FIG. 5. Determination of the cleavage site in HIV [32P]RNAtRNALYS complex. Lane 1, after the RNase D reaction, a 17-mer [32P]oligodeoxynucleotide was hybridized to the HIV RNA fragments for the primer-extension reaction (14). The DNA products were resolved on a 6% polyacrylamide sequencing gel. Lanes A, C, G, and T, products of dideoxynucleotide reaction with the same 5'-end-phosphorylated 17-mer primer and a plasmid harboring the
corresponding HIV sequences.
FIG. 6. Double-stranded RNA-dependent RNase activity during DNA synthesis. RT (300 ng per 3 units) was incubated with [32P]RNA-tRNALYS complex (2000 cpm) in the absence (A) or in the presence (B) of four deoxyribonucleotide triphosphates (1 mM each). Samples were subjected to electrophoresis in a 6% polyacrylamide/7 M urea denaturing gel. Numbers on left are bases.
Biochemistry: Ben-Artzi et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
another enzymatic activity of HIV RT. (i) The activity is present in several preparations of highly purified HIV RT prepared in three laboratories by different expression and purification procedures. (ii) The RNase D activity cochromatographed with RT on two different resins-phosphocellulose and Superose 6 gel. The molecular masses, under native conditions, of the heterodimer RT and of the RNase D are similar ('100 kDa). In contrast, the known E. coli nucleases, including RNase III, a double-stranded RNAdependent enzyme, have molecular masses in the range of 10-50 kDa (20). (iii) The bacterial mock protein fraction purified by the same procedure as RT did not cleave the substrate HIV RNA-tRNALYs. Our experiments indicate that RNase D activity is distinct from the RNase H of RT in terms of substrate specificity and the sequence at the cleavage site. The two small RNA products (60 and 64 bases) appear to represent two unique endonucleolytic cuts by the RNase D activity as, on a high-resolution sequencing gel, two large RNA products (200 and 196 bases) could also be detected. RNase H activity, on the other hand, functions as both endo- and exonuclease
(21-23).
The specificity of RNase D activity is indicated by analyzing different substrates. tRNALYS, which represents in the cloverleaf structure a double-stranded RNA region (12 base pairs of the 18 nucleotides complementary to the PBS), is resistant to RNase D cleavage. However, a duplex of tRNALYS hybridized to antisense [32P]tRNALYS, to produce a 76-base double-stranded region, is cleaved by RNase D (data not shown). The substrate for RNase D described here consists of a synthetic tRNALYS, which differs from the authentic tRNALYS (8) in 6 modified bases. Similar cleavage activity was, however, also observed when HIV-1 RNA was complexed to tRNALYS derived from total bovine tRNA (data not shown). The RNase D activity appears to be specific, as AMV RT does not cleave the substrate HIV-1 RNAtRNALYS. Specific retrovirus substrates, AMV RNAtRNA~rP and murine leukemia virus RNA-tRNAPro, are being prepared now to further analyze the substrate specificity of the RNase D activity. It is interesting to note in this respect that binding of tRNALYS and tRNATmr with their respective HIV RT and AMV RT is species specific (6, 18). What is the possible function of RNase D activity in the virus replication cycle? During reverse transcription, after the first template switch, the DNA minus-strand is extended by RT on the genomic RNA template to the 3' end of the PBS sequence (Fig. 7). The point at which synthesis stalls and the second template switch occurs has not been precisely determined (8). We propose that cleavage at the PBS sequence introduced by RNase D determines cessation of minus-strand DNA elongation and perhaps facilitates the template switch. Simultaneously with minus-strand DNA elongation, the plusstrand DNA is initiated. This strand is extended by copying the minus-strand DNA and the first 18 nucleotides of the tRNALYS primer (8). Utilization of tRNALYS as a template for RT should be dependent on its displacement from the PBS sequence of viral RNA. The dissociation of tRNALYS from the tRNALys
+)Dl\A 5'
(-)DNA Geneomic
5
5
5
b
b
A C C GC G
3'
+RNA 5' R
11 U5
C
t
(C
C G C CCG
p
p
p
|
TT G T C CC T G/
AtA
R'\ase[)
C A GtGG A C
(-)DNA 3'
cleavaue site
FIG. 7. Proposed model for RNase D activity and second template switch.
931
RNA structure at the PBS may be facilitated by the cleavages introduced at this sequence by RNase D (Fig. 7). Thus, RNase D activity may be involved in positioning the two complementary nascent DNA strands, plus and minus, for the second template switch (Fig. 7). While the second template switch at the PBS site has not been defined yet, it is of interest to note that nicks in the template RNAs induce template switch and recombination events during proviral DNA synthesis (17). We thank N. Sarver for valuable discussions and for useful comments on the manuscript, R. Lifshitz and B. Amit for help in the work, A. Hizi and C. Debouck for the gift of HIV RT, and L. Nir for typing the manuscript. Part of this work was supported by U.S. Public Health Service Contract NO1-A182696 of the National Institutes of Health (National Institute of Allergy and Infectious Diseases). pBENN7 was obtained through the AIDS Research and Reference Reagent Program, AIDS Program. 1. Baltimore, D. (1970) Nature (London) 226, 1209-1211. 2. Temin, H. M. & Mizutani, S. (1970) Nature (London) 226, 1211-1213. 3. Di Marzo-Veronese, F., Copeland, T. D., DeVico, A. L., Rahman, R., Oroszlan, S., Gallo, R. C. & Sarngadharan, M. G.
(1986) Science 231, 1289-1291. 4. Molling, K., Bolognesi, D. P., Bauer, H., Busen, W., Plass-
mann, H. W. & Hausen, P. (1971) Nature (London) 234, 241-243. 5. Goff, S. P. (1990) J. AIDS 3, 817-831.
6. Panet, A., Haseltine, W. A., Baltimore, B., Peters, G., Harada, F. & Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. USA 72,
2535-2539. 7. Barat, C., Lullien, V., Schatz, O., Keith, G., Nugeyre, M. T., Gnuninger-Leitch, F., Barre-Sinoussi, F., LeGrice, S. F. J. &
Darlix, J. L. (1989) EMBO J. 8, 3279-3285.
8. Weiss, R., Teich, N., Varmus, H. & Coffin, J., eds. (1985) RNA Tumor Viruses (Cold Spring Harbor Lab., Cold Spring Harbor,
NY), pp. 369-513.
9. Luo, G., Sharmeem, L. & Taylor, J. (1990) J. Virol. 64,
592-597. 10. Wohre, B. M. & Molling, K. (1990) Biochemistry 29, 1014110147. 11. Gendelman, H. E., Phelps, W., Feigenbaum, L., Ostrove, J. M., Adachi, A., Howley, P. M., Khoury, G., Ginsberg, H. S. & Martin, M. A. (1986) Proc. Natl. Acad. Sci. USA 83, 9759-9763. 12. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F., Doran, E. R., Rafalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., Pearson, M. L., Lautenberger, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Nature (London) 313, 277-284. 13. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K. & Green, M. R. (1984) Nucleic Acids Res. 12, 70357056. 14. Sambrook, J., Fritsch, E. F. & Maniatis, T., eds. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 15. Davanloo, P., Rosenberg, A. H., Dunn, J. J. & Studier, W. F. (1984) Proc. Natl. Acad. Sci. USA 81, 2035-2039. 16. Hizi, A., McGill, C. & Hughes, S. (1988) Proc. Natl. Acad. Sci. USA 85, 1218-1222. 17. Luo, G. & Taylor, J. (1990) J. Virol. 64, 4321-4328. 18. Barat, C., Le Grice, S. F. J. & Darlix, J. (1991) Nucleic Acids Res. 19, 751-757. 19. Mizrahi, V., Lazarus, G. M., Miles, L. M., Meyers, C. A. & Debouck, C. (1989) Arch. Biochem. Biophys. 273, 347-358. 20. March, P. E. & Gonzalez, M. A. (1990) Nucleic Acids Res. 18, 3293-3298. 21. Krug, M. S. & Berger, S. L. (1989) Proc. NatI. Acad. Sci. USA
86,3539-3543. 22. Oyama, F., Kikucki, R., Crouch, R. J. & Uchida, T. (1989) J. Biol. Chem. 264, 18808-18817. 23. Schatz, O., Mous, J. & Le Grice, S. J. F. (1990) EMBO J. 9, 1171-1176.
Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase (retrovirus ribonudease)
HANA BEN-ARTZI, ELISHA ZEELON, MARIAN GoRECKI, AND AMOS PANET BioTechnology General (Israel) Ltd., Kiryat Weizmann, Rehovot, 76326, Israel
Communicated by Ephraim Katchalski-Katzir, October 24, 1991
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.
of the RNA template and, to complete synthesis of the double-stranded proviral DNA, the enzyme switches templates at the region of the PBS. While the general scheme of reverse transcription is known, details of specific steps, such as template switching, are still missing (5, 8). In an endogenous reaction mixture containing detergent-disrupted virions, synthesis of a full-length proviral DNA with two long terminal repeats can be detected. It is not clear, however, whether auxiliary viral proteins other than RT are involved in the various steps of the reverse transcription reaction. To study the initiation and synthesis of the proviral DNA, we have constructed a system with recombinant RT and HIV-1 RNA-tRNALYs template-primer complex, which resembles the initiation complex utilized by the RT. During these studies, we noticed that HIV-1 RT cleaves specifically at the viral RNA PBS sequence. We propose that this newly described activity may be important in the reverse transcription process.
MATERIALS AND METHODS Construction of Plasmids. The plasmid pBENN7 (derived from HIV-1 lymphadenopathy-associated virus DNA) (11) was used for the isolation of a HindIII/Cla I restriction fragment of 2% base pairs (nucleotides 76-371; ref. 11). This DNA fragment includes part of R, U5, PBS, and part of gag. To restore the entire R region, this fragment was ligated with a synthetic DNA into the plasmid pSP64 (pHIV). The transcription of the HIV-1 gene starts with the first base of the R. The plasmid pGEM-1 (Promega) was modified and a new BspMI site was introduced adjacent to the T7 promoter. Four synthetic oligodeoxynucleotides spanning the sequence of tRNALYS and including an additional BspMI site at the 3' end of the tRNALYS gene were cloned into BspMI/HindIIIcleaved modified pGEM-I. The ptRNALYs cleaved with BspMI endonuclease was used as a template to yield complete tRNALYS molecules 76 bases long. Preparation of [32PJRNA and Synthetic tRNALYS Complexes. pHIV DNA was linearized with BssHII endonuclease at nucleotide 256 (12) and transcribed with SP6 RNA polymerase (13). [a-32P]CTP (105 cpm/pmol) was used for labeling the HIV RNA. The reaction mixture was treated with DNase I and phenol/chloroform extracted, and RNA was ethanol precipitated and purified by electrophoresis on 7 M urea/6% polyacrylamide denaturing gel (14). ptRNALYS was transcribed with T7 RNA polymerase (15). The reaction mixture was treated with DNase I, and RNA was extracted with phenol/chloroform followed by ethanol precipitation and subjected to electrophoresis on a denaturing gel (14). For complex preparation, purified HIV [32P]RNA and tRNALYS were hybridized at a 1:10 molar ratio in a solution
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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: RT, reverse transcriptase; HIV, human immunodeficiency virus; PBS, primer binding site; AMV, avian myeloblastosis virus.
927
Biochemistry: Ben-Artzi et al.
928
Proc. Nati. Acad Sci. USA 89 (1992)
containing 30%o formamide, 40 mM Tris-HCI (pH 7.4), 1 mM EDTA, and 0.3 M NaCi. Hybridization was for 10 min without salt at 650C and proceeded with salt at 420C for 2 hr. The RNA complex was purified on a nondenaturing 8% polyacrylamide gel. Elution of the RNA complex from the gel was done as described (14). A hybrid of 18-mer synthetic oligodeoxynucleotide, complementary to the PBS region, and HIV [32P]RNA was prepared under similar conditions for the RNase H reaction. RT Activities. Reaction mixtures (10 pl) contained 50 mM Tris-HCl (pH 8.0), 50 mM KCI, 2 mM dithiothreitol, 8 mM MgCl2, and the appropriate substrate. For standard DNA polymerase reactions, poly(A)/oligo(dT) and [a-32PJdTTP were used (16). One unit of enzyme activity is defined as nmol ofdTMP incorporated per 10 min under the standard reaction conditions (16). The substrate for RNase H reactions, HIV-1 [32P]RNA-DNA hybrid (2500 cpm), and the substrate for RNase D reactions, HIV-1 [32P]RNA-tRNALYs complex (2500 cpm), were incubated at 37°C. Samples (4 ,ul) were withdrawn, quenched with sample buffer (80%o formamide/50 mM EDTA), and heated at 95°C for 2 min. RNA was subjected to electrophoresis on a 6% polyacrylamide/7 M urea denaturing gel. Recombinant HIV RTs. Details of the expression and purification of HIV RT are described elsewhere (unpublished data). Briefly, Escherichia coli containing plasmid pHIV RT that expresses BH10 HIV RT (12) was the source of enzyme. RT was purified to apparent homogeneity by ammonium sulfate fractionation followed by chromatographies on four resins-phenyl-Sepharose, Q-Sepharose, phosphocellulose, and Superose gel. Specific activity of the purified RT (5000 units/mg) was determined by a standard RT assay using poly(A)/oligo(dT) as template/primer (16). The sequence of RNA fragments after RNase D reaction was determined by DNA primer extension (14) with a 17-mer 32P-labeled 5'-GCCGAGTCCTGCGTCGA-3', complementary to the 3' end of the HIV-1 RNA (Fig. 1) and avian myeloblastosis virus (AMV) RT (IBI). DNA products were analyzed by electrophoresis on a 6%o polyacrylamide sequencing gel (14). A DNA sequence ladder of HIV was generated with the same 32P 5' primer and dideoxynucleotides (14) on DNA plasmid (pHIV).
of the HIV-1 genome (R-U5-PBS) hybridized to a synthetic primer tRNALYS (Fig. 1). It should be noted that the synthetic tRNALYS is unmodified; however, it does not differ from the authentic tRNALYS in its ability to bind HIV RT and to initiate minus-strand DNA synthesis (18). The recombinant HIV-1 RT was produced in E. coli and purified by four consecutive column steps to apparent homogeneity. The HIV RT consists of two subunits, 66 and 51 kDa, as reported before (16, 19). The DNA polymerasespecific activity of this enzyme is similar to that of other purified HIV RT preparations (16, 19). The RT preparation is free of nonspecific RNase activities as incubation with HIV [32P]RNA single strand does not result in detectable degradation (Fig. 2, lanes 1 and 2). Surprisingly, we noticed that incubation of HIV RT with the template RNA annealed to the primer tRNALYS resulted in a unique cleavage (lanes 3-5). The size of the two RNA products, -200 and -60 nucleotides, suggests that cleavage occurs at the PBS sequence hybridized to tRNALYS and that the degradation products of 200 and 60 bases are derived from the 5' end and the 3' end of HIV RNA, respectively (see Fig. 1A). To investigate the origin of this unique RNase activity, we have obtained purified HIV RT preparations from two groups (16, 19). These two recombinant RTs were expressed and purified to apparent homogeneity by different procedures. The enzyme expressed by Mizrahi et al. (19) was processed in the bacterium by the HIV-1 coded protease. Thus, the purified enzyme is a heterodimer p66/pSl identical to RT purified from virions (3). The purified RT produced by Hizi et al. (16) is a dimer of 66 kDa. The three enzyme preparations have similar DNA polymerase specific activity, and they exhibit identical cleavage activity on the HIV RNA-tRNALYs complex (Fig. 2, lanes 6-9). It may be argued that the HIV RNA-tRNALYs substrate contains contaminating DNA and that the observed cleavage simply represents the RNase H activity of HIV RT. Several measures were taken to ensure removal of all DNA contaminations from the substrate HIV RNA-tRNALYs. After syn)
RT(I1)
RT0111
0 10' 0 5 15
1 010
) 10
RT(
RT(
RESULTS For the studies of HIV reverse transcription we have produced in vitro a template RNA that corresponds to the 5' end
-
1~~~~~~~~~~~~~~~~~~~~~~~~~~~
~
256 200
Go 0 -j
z n:
B
A
70 bases (see Fig. 1A). Nonetheless, the production of identical RNA molecules 60 and 64 bases long in the presence and absence of DNA synthesis indicates that cleavage occurs closer to the 3' end of the PBS site, as expected of the RNase D activity.
DISCUSSION We describe in this work an enzymatic activity associated with the HIV-1 RT. Our objective following this unexpected finding has been to analyze whether this RNase activity is indeed part of the HIV RT molecule. Several independent lines of evidence support the notion that RNase D is yet
A
A
B
(-dNTP's)
(--dNTP'so
0
T~
J ,*. ~~~~~~~~~~~~~~~~~~~~~~
~
~
r .jd
C~
~
~
10 20
0
10 20
_
A.
!i
S.
~~~~~~~~~~~~~~~~~~~~~~~~ 40 .:
.a.
*
110
-
90
-
76 -67 --a
FIG. 5. Determination of the cleavage site in HIV [32P]RNAtRNALYS complex. Lane 1, after the RNase D reaction, a 17-mer [32P]oligodeoxynucleotide was hybridized to the HIV RNA fragments for the primer-extension reaction (14). The DNA products were resolved on a 6% polyacrylamide sequencing gel. Lanes A, C, G, and T, products of dideoxynucleotide reaction with the same 5'-end-phosphorylated 17-mer primer and a plasmid harboring the
corresponding HIV sequences.
FIG. 6. Double-stranded RNA-dependent RNase activity during DNA synthesis. RT (300 ng per 3 units) was incubated with [32P]RNA-tRNALYS complex (2000 cpm) in the absence (A) or in the presence (B) of four deoxyribonucleotide triphosphates (1 mM each). Samples were subjected to electrophoresis in a 6% polyacrylamide/7 M urea denaturing gel. Numbers on left are bases.
Biochemistry: Ben-Artzi et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
another enzymatic activity of HIV RT. (i) The activity is present in several preparations of highly purified HIV RT prepared in three laboratories by different expression and purification procedures. (ii) The RNase D activity cochromatographed with RT on two different resins-phosphocellulose and Superose 6 gel. The molecular masses, under native conditions, of the heterodimer RT and of the RNase D are similar ('100 kDa). In contrast, the known E. coli nucleases, including RNase III, a double-stranded RNAdependent enzyme, have molecular masses in the range of 10-50 kDa (20). (iii) The bacterial mock protein fraction purified by the same procedure as RT did not cleave the substrate HIV RNA-tRNALYs. Our experiments indicate that RNase D activity is distinct from the RNase H of RT in terms of substrate specificity and the sequence at the cleavage site. The two small RNA products (60 and 64 bases) appear to represent two unique endonucleolytic cuts by the RNase D activity as, on a high-resolution sequencing gel, two large RNA products (200 and 196 bases) could also be detected. RNase H activity, on the other hand, functions as both endo- and exonuclease
(21-23).
The specificity of RNase D activity is indicated by analyzing different substrates. tRNALYS, which represents in the cloverleaf structure a double-stranded RNA region (12 base pairs of the 18 nucleotides complementary to the PBS), is resistant to RNase D cleavage. However, a duplex of tRNALYS hybridized to antisense [32P]tRNALYS, to produce a 76-base double-stranded region, is cleaved by RNase D (data not shown). The substrate for RNase D described here consists of a synthetic tRNALYS, which differs from the authentic tRNALYS (8) in 6 modified bases. Similar cleavage activity was, however, also observed when HIV-1 RNA was complexed to tRNALYS derived from total bovine tRNA (data not shown). The RNase D activity appears to be specific, as AMV RT does not cleave the substrate HIV-1 RNAtRNALYS. Specific retrovirus substrates, AMV RNAtRNA~rP and murine leukemia virus RNA-tRNAPro, are being prepared now to further analyze the substrate specificity of the RNase D activity. It is interesting to note in this respect that binding of tRNALYS and tRNATmr with their respective HIV RT and AMV RT is species specific (6, 18). What is the possible function of RNase D activity in the virus replication cycle? During reverse transcription, after the first template switch, the DNA minus-strand is extended by RT on the genomic RNA template to the 3' end of the PBS sequence (Fig. 7). The point at which synthesis stalls and the second template switch occurs has not been precisely determined (8). We propose that cleavage at the PBS sequence introduced by RNase D determines cessation of minus-strand DNA elongation and perhaps facilitates the template switch. Simultaneously with minus-strand DNA elongation, the plusstrand DNA is initiated. This strand is extended by copying the minus-strand DNA and the first 18 nucleotides of the tRNALYS primer (8). Utilization of tRNALYS as a template for RT should be dependent on its displacement from the PBS sequence of viral RNA. The dissociation of tRNALYS from the tRNALys
+)Dl\A 5'
(-)DNA Geneomic
5
5
5
b
b
A C C GC G
3'
+RNA 5' R
11 U5
C
t
(C
C G C CCG
p
p
p
|
TT G T C CC T G/
AtA
R'\ase[)
C A GtGG A C
(-)DNA 3'
cleavaue site
FIG. 7. Proposed model for RNase D activity and second template switch.
931
RNA structure at the PBS may be facilitated by the cleavages introduced at this sequence by RNase D (Fig. 7). Thus, RNase D activity may be involved in positioning the two complementary nascent DNA strands, plus and minus, for the second template switch (Fig. 7). While the second template switch at the PBS site has not been defined yet, it is of interest to note that nicks in the template RNAs induce template switch and recombination events during proviral DNA synthesis (17). We thank N. Sarver for valuable discussions and for useful comments on the manuscript, R. Lifshitz and B. Amit for help in the work, A. Hizi and C. Debouck for the gift of HIV RT, and L. Nir for typing the manuscript. Part of this work was supported by U.S. Public Health Service Contract NO1-A182696 of the National Institutes of Health (National Institute of Allergy and Infectious Diseases). pBENN7 was obtained through the AIDS Research and Reference Reagent Program, AIDS Program. 1. Baltimore, D. (1970) Nature (London) 226, 1209-1211. 2. Temin, H. M. & Mizutani, S. (1970) Nature (London) 226, 1211-1213. 3. Di Marzo-Veronese, F., Copeland, T. D., DeVico, A. L., Rahman, R., Oroszlan, S., Gallo, R. C. & Sarngadharan, M. G.
(1986) Science 231, 1289-1291. 4. Molling, K., Bolognesi, D. P., Bauer, H., Busen, W., Plass-
mann, H. W. & Hausen, P. (1971) Nature (London) 234, 241-243. 5. Goff, S. P. (1990) J. AIDS 3, 817-831.
6. Panet, A., Haseltine, W. A., Baltimore, B., Peters, G., Harada, F. & Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. USA 72,
2535-2539. 7. Barat, C., Lullien, V., Schatz, O., Keith, G., Nugeyre, M. T., Gnuninger-Leitch, F., Barre-Sinoussi, F., LeGrice, S. F. J. &
Darlix, J. L. (1989) EMBO J. 8, 3279-3285.
8. Weiss, R., Teich, N., Varmus, H. & Coffin, J., eds. (1985) RNA Tumor Viruses (Cold Spring Harbor Lab., Cold Spring Harbor,
NY), pp. 369-513.
9. Luo, G., Sharmeem, L. & Taylor, J. (1990) J. Virol. 64,
592-597. 10. Wohre, B. M. & Molling, K. (1990) Biochemistry 29, 1014110147. 11. Gendelman, H. E., Phelps, W., Feigenbaum, L., Ostrove, J. M., Adachi, A., Howley, P. M., Khoury, G., Ginsberg, H. S. & Martin, M. A. (1986) Proc. Natl. Acad. Sci. USA 83, 9759-9763. 12. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F., Doran, E. R., Rafalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff, L., Petteway, S. R., Pearson, M. L., Lautenberger, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T., Gallo, R. C. & Wong-Staal, F. (1985) Nature (London) 313, 277-284. 13. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K. & Green, M. R. (1984) Nucleic Acids Res. 12, 70357056. 14. Sambrook, J., Fritsch, E. F. & Maniatis, T., eds. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 15. Davanloo, P., Rosenberg, A. H., Dunn, J. J. & Studier, W. F. (1984) Proc. Natl. Acad. Sci. USA 81, 2035-2039. 16. Hizi, A., McGill, C. & Hughes, S. (1988) Proc. Natl. Acad. Sci. USA 85, 1218-1222. 17. Luo, G. & Taylor, J. (1990) J. Virol. 64, 4321-4328. 18. Barat, C., Le Grice, S. F. J. & Darlix, J. (1991) Nucleic Acids Res. 19, 751-757. 19. Mizrahi, V., Lazarus, G. M., Miles, L. M., Meyers, C. A. & Debouck, C. (1989) Arch. Biochem. Biophys. 273, 347-358. 20. March, P. E. & Gonzalez, M. A. (1990) Nucleic Acids Res. 18, 3293-3298. 21. Krug, M. S. & Berger, S. L. (1989) Proc. NatI. Acad. Sci. USA
86,3539-3543. 22. Oyama, F., Kikucki, R., Crouch, R. J. & Uchida, T. (1989) J. Biol. Chem. 264, 18808-18817. 23. Schatz, O., Mous, J. & Le Grice, S. J. F. (1990) EMBO J. 9, 1171-1176.
