Characterization of RNA Strand Displacement Synthesis by Moloney Murine Leukemia Virus Reverse Transcriptase

Abstract

The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H−mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H− RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain. The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H−mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H− RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain. Retroviral replication requires the single-stranded RNA genome of the virus to be converted into double-stranded DNA through a complex series of reactions termed reverse transcription. This process appears to be catalyzed solely by the viral reverse transcriptase (RT) 1The abbreviations used are: RT, reverse transcriptase; SSB, single-strand binding protein; HIV-1, human immunodeficiency virus type 1; M-MuLV, Moloney murine leukemia virus; NC, nucleocapsid; LTR, long terminal repeat; bp, base pair(s); nt, nucleotide(s); DTT, dithiothreitol; SSII, RNase H− M-MuLV reverse transcriptase (SuperScript II); oligo, oligonucleotide(s). that possesses the following two distinct enzymatic activities: a polymerase activity that synthesizes DNA using either RNA or DNA templates, and an RNase H activity that cleaves RNA in hybrid duplex with DNA (1Varmus H. Brown P. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, D. C.1989: 53-108Google Scholar, 2Katz R.A. Skalka A.M. Annu. Rev. Biochem. 1994; 63: 133-173Crossref PubMed Scopus (535) Google Scholar). The current model of reverse transcription proposes that the RNase H activity of RT is critical for several steps including degradation of the 5′ end of the RNA genome following minus-strong-stop DNA synthesis to facilitate the first jump, specific cleavage at the polypurine tract to create the plus strand primer, and removal of the plus and minus strand primers (3Champoux J.J. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 103-118Google Scholar). Additionally, it is presumed that the RNase H activity is required to degrade the RNA genome following minus strand synthesis to free the minus strand DNA for use as a template during plus strand synthesis (reviewed in Ref. 3Champoux J.J. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 103-118Google Scholar). In vitrostudies, however, suggest that RNA fragments that are too large to spontaneously dissociate from the minus strand remain following cleavage of the genome (4DeStefano J.J. Mallaber L.M. Fay P.J. Bambara R.A. Nucleic Acids Res. 1994; 22: 3793-3800Crossref PubMed Scopus (51) Google Scholar, 5Ben Artzi H. Zeelon E. Amit B. Wortzel A. Gorecki M. Panet A. J. Biol. Chem. 1993; 268: 16465-16471Abstract Full Text PDF PubMed Google Scholar, 6DeStefano J.J. Buiser R.G. Mallaber L.M. Bambara R.A. Fay P.J. J. Biol. Chem. 1991; 266: 24295-24301Abstract Full Text PDF PubMed Google Scholar, 7DeStefano J.J. Mallaber L.M. Fay P.J. Bambara R.A. Nucleic Acids Res. 1993; 21: 4330-4338Crossref PubMed Scopus (78) Google Scholar, 8Mizrahi V. Biochemistry. 1989; 28: 9088-9094Crossref PubMed Scopus (23) Google Scholar, 9Oyama F. Kikuchi R. Crouch R.J. Uchida T. J. Biol. Chem. 1989; 264: 18808-18817Abstract Full Text PDF PubMed Google Scholar, 10Volkmann S. Wohrl B.M. Tisdale M. Moelling K. J. Biol. Chem. 1993; 268: 2674-2683Abstract Full Text PDF PubMed Google Scholar, 11Palaniappan C. Fuentes G.M. Rodrı́guez-Rodrı́guez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1996; 271: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Furthermore, evidence that plus strand synthesis in several retroviral systems is discontinuous demonstrates that stably annealed RNA fragments persist in vivo (3Champoux J.J. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 103-118Google Scholar,12Rattray A.J. Champoux J.J. J. Virol. 1987; 61: 2843-2851Crossref PubMed Google Scholar, 13Kupiec J.J. Sonigo P. J. Gen. Virol. 1996; 77: 1987-1991Crossref PubMed Scopus (19) Google Scholar, 14Charneau P. Clavel F. J. Virol. 1991; 65: 2415-2421Crossref PubMed Google Scholar). This raises the interesting possibility that reverse transcription requires a mechanism for the displacement of genomic RNA fragments during plus strand synthesis. Most replicative polymerases require accessory proteins such as helicases and single-strand binding proteins (SSBs) to unpair the duplex region in front of the primer terminus during DNA synthesis (15Kornberg A. Baker T. DNA Replication. W. H. Freeman & Co., New York1992Google Scholar). In contrast, studies from our laboratory 2Whiting, S. H., and Champoux, J. J. (1998)J. in and G.M. Rodrı́guez-Rodrı́guez L. C. Fay P.J. Bambara R.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, M. M. J. Virol. PubMed Google Scholar, M. C. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, Champoux J.J. J. Virol. 1994; PubMed Google demonstrate that from several retroviral systems the to displacement of the DNA strand in the of accessory although the rate of synthesis appears to be slower than that during non-displacement synthesis a single-stranded human immunodeficiency virus type and Moloney murine leukemia virus (M-MuLV) to at a to displace RNA during synthesis hybrid G.M. Fay P.J. Bambara R.A. Nucleic Acids Res. 1996; PubMed Scopus Google but this process not in reverse transcription to viral DNA in in or Skalka A.M. J. Virol. PubMed Google Scholar, M. S. A. 77: PubMed Scopus Google Scholar, E. Goff S. D. Full Text PDF PubMed Scopus Google it that or accessory proteins are required for the for the of an accessory is the viral nucleocapsid is a protein that possesses either or two zinc finger in with a binding for RNA DNA with a for and A. J. Moelling K. J. Biol. 1993; PubMed Scopus Google Scholar, S. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar, R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, F. R. 1993; PubMed Scopus Google Scholar, S.P. C. E. H. D. PubMed Scopus Google Scholar, Brown J. Virol. 1994; PubMed Google Scholar, M. H. J. Biol. PubMed Scopus Google Scholar). are of with Annu. Rev. Biochem. 1994; 63: PubMed Scopus Google Scholar, G.M. J. Biol. 1991; PubMed Scopus Google Scholar, D. M. J. 1994; PubMed Scopus Google to the that may to facilitate reverse transcription. studies have that the first and template and is during the initiation of reverse transcription from the primer B. M. C. J. 1994; PubMed Scopus Google Scholar, J.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Google Scholar, J. B. J. Virol. PubMed Google Scholar, S. Biochemistry. 1994; PubMed Scopus Google Scholar, V. C. V. J. Biol. PubMed Scopus Google Scholar, H. L. J. Virol. 1996; PubMed Google have that the of synthesis during reverse transcription V. C. V. J. Biol. PubMed Scopus Google W. R.J. A. J. Virol. 1996; PubMed Google Scholar, M. M. R. Google Scholar, P. R.J. Res. PubMed Scopus Google Scholar, Biochemistry. 1996; PubMed Scopus Google Scholar, L. Fuentes G.M. Bambara R.A. Fay P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this we have the of M-MuLV RT to RNA displacement synthesis in the or of the RNase H and we have investigated the of DNA and RNA displacement synthesis. that RT the to displace but the rate is slower than that of DNA displacement synthesis and slower than non-displacement synthesis. We find that M-MuLV displacement and that this activity is the zinc finger of the from DNA polymerase DNA and from New M-MuLV RT and from from The as for the long terminal into such that minus single-stranded DNA be by with The a of the M-MuLV from to and into DNA by minus strand viral by a from into such that single-stranded DNA plus strand viral from and be to as and by polymerase and of a region of the into at the single-stranded and by R. K. in & New Scholar). the single-stranded DNA by an that the by of the the single-stranded from the DNA to in to the regions the and with of in a of for at the single-stranded with a following the DNA and an as by the DNA by RNA polymerase as in the that to the with for at to R. K. in & New Scholar). RNA by a and into for at The through a in the of and in RNA using RNA polymerase following R. K. in & New Scholar). and the RNA for at with E. to and the RNA with and in the of and in the of RNA transcription reactions and of a of the RNA to be to by RNA of the or RNA displacement used as the end of the displacement at and of RNA have Champoux J.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). DNA and to the region of the M-MuLV genome M. C. C. Nucleic Acids Res. PubMed Scopus Google by J. L. DNA a DNA and the DNA from DNA and by DNA and DNA and RNA to and of the M-MuLV R.A. PubMed Scopus Google Scholar). primer primer and primer to the and regions single-stranded DNA from and or of the RNA transcription and of the and to single-stranded of the RNA transcription and create when annealed to single-stranded or and used to polymerase the from with the of as DNA by the J. T. Cold Spring Harbor Laboratory, Cold Spring Harbor, of RNA displacement synthesis are the used to synthesis the and using single-stranded DNA as the template The template is to the RNA displacement template The to the single-stranded DNA template from the 5′ end of the a single-stranded the primer and the RNA synthesis non-displacement and by at the and the a The of the first base of RNA displacement are by the for the by the using The the of a series of single-stranded DNA to the that the the annealed to the single-stranded template DNA in two In the first the primer annealed to the single-stranded DNA template at a of in RT by to and to In the the annealed into to either DNA RNA or in a of of The the same as the The DNA displacement as the to the 5′ end of the annealed to single-stranded DNA for at a in by for at and at for to and the with at for The by the of to with and and in the of a for the of the a the DNA and the the DNA displacement the primer annealed to the DNA displacement template at a of in by for at and for at The RNA displacement and non-displacement by the primer with single-stranded DNA for and in RNA displacement or at a of and as in a The annealed with of in RT and for at synthesis by the of and in to of the of synthesis to the first in the the of at and and with an of at the a for DNA polymerase used in of degradation of the by the activity of DNA the to and the reactions by the of the to for a and by and using the of the as fragments in DTT, and the annealed to in the of DTT, and the of of SSII, for at by the of to a of the to of and by M-MuLV RT or for reactions using DNA the to at for and synthesis by the of the and as the primer as the of primer at in the from the primer in The as the at the and the as the that of the rates and by of as a of using the template to DNA to of the template The primers used as of the RNA to create a and of the RNA to a The as that of or of primer to the the an of in RT DTT, and as a of and the rates from the of M-MuLV and finger by a and finger using as H. D. C. B. Nucleic Acids Res. 1993; 21: PubMed Scopus Google and by J. L. The in and and in at The as M. C. C. Nucleic Acids Res. PubMed Scopus Google Scholar). In a of and of in and in the of or in the of of or of or for at in The reactions by the of to with an of and to The an at and by of the to at RT possesses the to displace RNA during DNA we the of primer by RT in In this we used the RNase H− of M-MuLV RT, to the of RNase H cleavage of the RNA displacement by a RNA to single-stranded DNA of a DNA primer DNA displacement and non-displacement by RNA with a DNA or the synthesis by to of by from the a end to facilitate be that the of during displacement the of the strand the and the to the non-displacement The of with the hybrid template in the of RNase H RT the to displace of the of and the of the templates, however, that RNA displacement synthesis less than DNA displacement or non-displacement synthesis with and synthesis from by RT (1Varmus H. Brown P. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, D. C.1989: 53-108Google Scholar, 2Katz R.A. Skalka A.M. Annu. Rev. Biochem. 1994; 63: 133-173Crossref PubMed Scopus (535) Google the end with the for a of the rates of of RT RNA displacement synthesis approximately 5-fold slower than DNA displacement and at slower than non-displacement synthesis. a for DNA strand displacement used to the of the DNA polymerase the primer single-stranded but not displacement not that the stably annealed in the displacement RNA displacement synthesis by and to the rate of RNA displacement to DNA and non-displacement from the M-MuLV of synthesis by the our that RNA displacement synthesis by RT slower than DNA or non-displacement synthesis. Most primers to the end of the template the first during non-displacement synthesis less the DNA and RNA displacement not the during DNA displacement or the during RNA displacement synthesis The rates of DNA displacement, and RNA displacement synthesis and we that during RNA displacement the of the to with the of primers through this region the non-displacement and DNA displacement and the of RNA displacement synthesis at the for of the to the of the the of the to this the RNA displacement synthesis rate to be less than that of DNA displacement synthesis. of the and rates from is in rates during DNA synthesis by two two in a the or synthesis we either or the to the RNA displacement reactions the to the of that the by but by not for the DNA polymerase used as a to the of the the non-displacement template DNA polymerase the primers to but to significantly the primers the DNA and RNA displacement and the of with the of RNA displacement synthesis in a of to in that the of the primer and the template from the from the a of synthesis by the first the with the templates, the primers during non-displacement and DNA displacement synthesis and by the for non-displacement or the for DNA displacement synthesis the first from the The of in the same region at the greater during RNA displacement but not persist to and The a rate for RNA displacement the template that 5-fold greater than that with the The rates of DNA displacement, and RNA displacement synthesis the and and to the rates for the the the RNA displacement template by the of the primer to the RNA strand, a RNA displacement template from the used in the studies with synthesis from an primer that annealed of the This not the at RNA displacement is and to the synthesis at a with that at a gap. RNA displacement preceded by non-displacement synthesis during the initiation of RNA displacement The in the of the first base of RNA displacement the template and the template at or the synthesis RNA the of from non-displacement synthesis through the of the the and used to at the with the two the of the RNA displacement at the in the the the for the and the in the single-stranded region of the template from the the same with the non-displacement at to to the of RNA the template that the non-displacement template and when in the same region a DNA displacement template with non-displacement not The for be of single-stranded DNA is not the RNase H activity of RT is to cleave the genomic RNA to plus strand synthesis in it of to the to RNase H activity the rate of RNA displacement synthesis by RT in our in DNA synthesis catalyzed by M-MuLV RT the with that catalyzed by the RNA displacement template of as as the when synthesis catalyzed by M-MuLV RT but not the with and M-MuLV RT, during the first of the the of from the primer to the nearly for the two and but at in reactions RNase H activity with it of the rate by M-MuLV RT the RNA displacement template an to the the end of the template The rate by M-MuLV RT of to a of the and by an template and the same a rate of the rates of synthesis by and M-MuLV RT the DNA displacement or non-displacement We investigated the of M-MuLV displacement synthesis by RT using and the activity of our we a M. C. C. Nucleic Acids Res. PubMed Scopus Google in the of the protein to DNA The M-MuLV a zinc by regions in H. D. C. B. Nucleic Acids Res. 1993; 21: PubMed Scopus Google Scholar). The a in of the zinc and the the zinc finger as as from the and and have to in activity V. F. H. B. Nucleic Acids Res. 1991; PubMed Scopus Google Scholar). In the protein a of of the at for and of the nearly duplex by in a with of the as duplex at the to a at with to have or the rate of of the and the for RNA displacement synthesis by RNase H− by of to the RNA displacement to the of and from reactions either or not to reactions in protein The from reactions in of the same as the and to the of in reactions with and The required to this approximately to that to duplex in the reactions and not it that the the first of RNA displacement synthesis in the of and the of at and with the to RNA displacement synthesis by and to the of DNA displacement and non-displacement in the of or in the of of or The of or the rate of during non-displacement synthesis and of the from the rates when not for RNA displacement, DNA displacement synthesis by SSII, not the rate of DNA displacement synthesis in the of greater than in the of and greater than when RNA displacement synthesis the rate of by and that in the of or with the rate of RNA displacement synthesis by 2-fold, in the rate of DNA or non-displacement synthesis not In the we have the of M-MuLV RT to displace RNA during synthesis hybrid In the of RNase H RT RNA displacement synthesis either or hybrid but the rate of synthesis than during either DNA displacement or non-displacement synthesis. are with of Fuentes G.M. Fay P.J. Bambara R.A. Nucleic Acids Res. 1996; PubMed Scopus Google that and M-MuLV RNase H activity displace a RNA during DNA synthesis. We the rate of synthesis by H− to be during RNA displacement, a rate 5-fold slower than that of DNA displacement synthesis and slower than non-displacement synthesis However, the rate of RNA displacement synthesis approximately slower than non-displacement synthesis to the that during the first of synthesis the it appears that synthesis during the first several of but that this the We in the during initiation the template a of RNA displacement synthesis by it that RT a to displace we the possibility that such as or the initiation of displacement synthesis at a may have to the The possibility with the of the primer and the hybrid to a in the of at with the of with the initiation the template appears to be The the rates of synthesis the two however, evidence that displacement of RNA by RT is significantly slower than displacement of DNA This is by the rates from using the is RNA displacement than DNA displacement synthesis is a process duplex to synthesis through double-stranded it be that in the of as with duplex DNA be in the rate of synthesis the two However, the of duplex DNA is to be than that of hybrid duplex sequences of base R. H. S. A. PubMed Scopus Google Scholar, S. M. A. H. T. M. M. Biochemistry. PubMed Scopus Google Scholar, S. M. K. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar). the of the hybrid from the primer terminus the template is less than that for duplex DNA of the same the RNA displacement template RT through this region with the for DNA displacement synthesis. the and the of and that a mechanism remains a RT DNA and RNA using either an or RT in strand in RT with the RNA or DNA may be for the the rates of RNA and DNA displacement synthesis. In to that the during RNA displacement the template we that the initiation of displacement synthesis a than a preceded the region to be This since we not the process of displacement to be by synthesis of the RNA 5′ for this is that of it be non-displacement or displacement is required to a in the of the polymerase that facilitate displacement of the a from a to of synthesis for the rates an or for the of displacement synthesis through a gap. with this J.J. Buiser R.G. Mallaber L.M. Bambara R.A. Fay P.J. J. Biol. Chem. 1991; 266: 24295-24301Abstract Full Text PDF PubMed Google that the of RT from a in a of two binding for the polymerase with and that a to synthesis the of during DNA displacement synthesis. of M-MuLV RT by Wohrl B.M. M.M. A. PubMed Scopus Google that RT and the template may to of the primer terminus it is that RT may be required to to of the strand with the template strand can be The that RT of the 5′ end of the strand the displacement template may for this although such not the sequences we have that the of RT RNase H it of to displacement synthesis by M-MuLV RT the same used for the rate of synthesis by M-MuLV RT the RNA displacement template significantly greater than that catalyzed by the rates of non-displacement and DNA displacement synthesis nearly for the two it that RNase cleavage of the RNA strand for the rate of synthesis by M-MuLV However, the of the the rate of synthesis by M-MuLV RT less than that a DNA displacement the cleavage that the of the not to the RT appears to have in Additionally, the rate of synthesis by M-MuLV RT the hybrid template that with SSII, the in the of during using the rates however, that hybrid the rate of synthesis by M-MuLV RT that of DNA displacement synthesis. retroviral may be minus strand synthesis and the of plus strand synthesis for the RNase H activity to the RNA genome to fragments. of fragments may be to dissociate from the however, fragments the RNA displacement activity of RT for the B. 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