Expression and Characterization of Hepadnavirus ... - Science Direct

[11]

HEPADNAVIRUS REVERSETRANSCRIPTASES

[ 1 1] E x p r e s s i o n By

195

and Characterization of Hepadnavirus Reverse Transcriptases

JIANMING

HU

and

CHRISTOPH

SEEGER

Introduction The polymerase of hepadnaviruses is an RNA- and DNA-directed DNA polymerase (reverse transcriptase, RT) that is expressed from a polycistronic viral RNA template, called pregenomic RNA. Unlike its retrovirus counterparts, the hepadnavirus polymerase is translated from an internal A U G codon and is expressed as a mature polypeptide that apparently does not require proteolytic processing to gain enzymatic activity.1'2 In addition to the DNA polymerase activity, the RT exhibits an RNase H activity3-8 and an unprecedented protein-priming activity.9-12 Comparison of the predicted amino acid sequence of the hepadnavirus polymerase with that of retroviruses as well as results from genetic experiments allowed the assignment of specific functions to the structural domains of the polypeptide. The central and C-terminal regions constitute the DNA polymerase and RNase H domains, which share significant homologies with the corresponding domains of other RTs. In contrast, the N-terminal domain does not share any significant homologies with other known polypeptides. It is separated by a tether from the polymerase domain and bears a tyrosine residue that acts as the primer for RNA-directed DNA synthesis (Fig. 1). As a consequence of this unusual protein priming reaction, the 5' end of the viral minus strand DNA (the first DNA strand synthesized by reverse 1 L. J. Chang, P. Pryciak, D. Ganem, and H. E. Varmus, Nature (London) 337, 364 (1989). 2 H. J. Schlicht, G. Radziwill, and H. Schaller, Cell (Cambridge, Mass.) 56, 85 (1989). 3 p. M. Kaplan, R. L. Greenman, J. L. Gerin, R. H. Purcell, and W. S. Robinson, J. Virol. 12, 995 (1973). 4 j. Summers and W. S. Mason, Cell (Cambridge, Mass.) 29, 403 (1982). 5 L. J. Chang, R. C. Hirsch, D. Ganem, and H. E. Varmus, J. Virol. 64, 5553 (1990). 6 y. E. Khudyakov and A. M. Makhov, FEBS Lett. 243, 115 (1989). 7 H. Toh, H. Hayashida, and T. Miyata, Nature (London) 3115, 827 (1983). 8 G. Radziwill, W. Tucker, and H. Schaller, J. Virol. 64, 613 (1990). 9 R. Lanford, L. Notvall, and B. Beames, J. Virol. 69, 4431 (1995). 10F. Zoulim and C. Seeger, J. Virol. 68, 6 (1994). u G. H. Wang and C. Seeger, Cell (Cambridge, Mass.) 71, 663 (1992). 12 M. Weber, V. Bronsema, H. Bartos, A. Bosserhoff, R. Bartenschlager, and H. Schaller, J. Virol. 68, 2994 (1994).

METHODS

IN ENZYMOLOGY, VOL. 275

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

196

EXPRESSION, PURIFICATION, AND CHARACTERIZATION

I TERMINAL PROTEIN 1

96

I

I

SPACER

395 446

5131514

601

D

DD

G

[

666

715 785

I[

I I

I

DHBV HIV

REVERSE TRANSCRIPTASE I RNASE H

aa

I

"D .~. ~D .~ I

PR

65 110

1851186

262

[ l 1]

i

443

~1

I- ~ I N T

498 561

aa

FIG. 1. Domain structure of the hepadnavirus reverse transcriptase. The DHBV polymerase (top line) consists of 785 amino acid residues and can be divided into four structural domains, i.e., the N-terminal domain (also called the terminal protein), the central reverse transcriptase domain, the C-terminal RNase H domain, and a spacer domain separating the N-terminal and the RT domains. The RT and RNase H domains share significant homologies with other RTs; the highly conserved residues between the DHBV RT and the HIV RT (the bottom line), believed to be important for the catalytic activities, are indicated by single-letter amino acid symbols. The N-terminal domain of the DHBV polymerase does not show any significant homologies to other known polypeptides except for the corresponding domain of the other hepadnavirus polymerases. Likewise, the protease (PR) and integrase (INT) domains encoded by the HIV (and other retroviruses) polymerase gene are absent from the hepadnavirus polymerase. The position of the tyrosine residue (Y96) within the N-terminal domain that serves as the primer for initiating minus strand DNA synthesis is indicated. The spacer domain is not conserved among different viral isolates and appears to be dispensable for the known enzymatic functions of the RT.

transcription) becomes covalently linked to the N terminus of the polymerase and remains attached during the entire DNA synthesis process. 13-15 In addition to its role in DNA synthesis, the RT is essential for packaging of the pregenomic RNA into viral core particles.16-18 The packaging process is initiated by binding of the RT to a sequence motif near the 5' end of the pregenome called e. e, which has the potential to fold into a stem-loop structure, acts both as a packaging signal and as the template for initiation of viral minus strand DNA synthesis. 19-27 Two separate regions of the RT, 13 W. Gerlich and W. S. Robinson, Cell (Cambridge, Mass.) 21, 801 (1980). 14 K. K. Molnar, J, Summers, J. M. Taylor, and W. S. Mason, J. ViroL 45, 165 (1983). 15 R. Bartenschlager and H. Schaller, E M B O J. 7, 4185 (1988). 16 R. Bartenschlager, M. Junker-Niepmann, and H. Schaller, Z ViroL 64, 5324 (1990). 17 R. C. Hirsch, J. E. Lavine, L. J. Chang, H. E. Varmus, and D. Ganem, Nature (London) 344, 552 (1990). 18 S. Roychoudhury, A. F. Faruqi, and C. Shih, J. ViroL 65, 3617 (1991). 19 D. Fallows and S. Goff, J. ViroL 697 3067 (1995). 20 R. Bartenschlager and H. Schaller, EMBO J. 11, 3413 (1992). 21 T. Knaus and M. Nassal, Nucleic Acids Res. 21, 3967 (1993). 22 R. C. Hirsch, D. D. Loeb, J. R. Pollack, and D. Ganem, J. Virol. 657 3309 (1991). 23 G. H. Wang, F. Zoulim, E. H. Leber, J. Kitson, and C. Seeger, J. Virol. 68, 8437 (1994).

[1 1]


197

one in the polymerase domain and the other in the N-terminal domain, are required for formation of a stable ribonucleoprotein (RNP) complex between the polymerase and e R N A . 23'25 Following the formation of the RNP, the polymerase synthesizes a three to four nucleotide-long DNA strand, using as template the sequences located in the bulge region of e RNA. Concurrent with or immediately following the initiation of reverse transcription, the R N A pregenome along with the polymerase is packaged into nucleocapsids formed by the viral core proteins. To continue DNA synthesis, the RT has to switch templates and relocate to a specific location near the 3' end of the R N A pregenome, where the nascent D N A strand can anneal with a complementary sequence (Fig. 2). 24,27 Although the RT plays such a critical role in viral D N A replication, relatively little is known about its biochemical properties and its mechanism of action in carrying out the various functions that have been attributed to this unique enzyme. Although DNA polymerase activity could be detected in virus particles more than two decades ago, 3 expression and purification of soluble, enzymatically active protein has only recently been reported. Polymerase polypeptides in virions can extend the incomplete viral plus strand D N A but do not accept exogenous DNA or R N A sequences as templates for D N A synthesis, thus limiting their suitability for biochemical and genetic studies. 3,4,28Recently, however, conditions have been established for the expression of enzymatically active RT independent of other viral proteins. First, functional duck hepatitis B virus (DHBV) polymerase can be expressed in a rabbit reticulocyte lysate in vitro translation system. H The in vitro-expressed polymerase is active in priming DNA synthesis using e R N A as the template. Second, the D H B V RT is enzymatically active when expressed in yeast as a fusion protein with the capside protein of the yeast retrotransposon Ty1.29 The polymerase expressed in yeast is packaged into virus-like particles (VLPs) and initiates minus strand D N A synthesis in a reaction that also depends on e RNA. Third, the HBV reverse transcriptase has been expressed in an enzymatically active form in insect cells using the baculovirus expression system. 9 The H B V RT also exhibits protein priming and RNA-dependent DNA polymerase activities. In addition, it has been reported that the HBV RT expressed in Xenopus oocytes has a D N A polymerization activity that appears to be independent of the natural

24 G.-H. Wang and C. Seeger, Z ViroL 6'7, 6507 (1993). 25 j. R. Pollack and D. Ganem, J. ViroL 68, 5579 (1994). 26 j. R. Pollack and D. Ganem, J. Virol. 67, 3254 (1993). 27 j. E. Tavis, S. Perri, and D. Ganem, J. Virol. 68, 3536 (1994). 28 G. Radziwill, H. Zentgraf, H. Schaller, and V. Bosch, Virology 163, 123 (1988). 29 j. E. Tavis and D. Ganem, Proc. Natl. Acad. Sci. U.S.A. 90, 4107 (1993).

198

EXPRESSION, PURIFICATION, AND C H A R A C T E R I Z A T I O N

[ 11]

r.u,.~n~r.t

FIG. 2. Model for initiation of reverse transcription in hepadnaviruses. Priming of DNA synthesis occurs at the e RNA sequence located near the 5' end of pregenomic RNA, with a specific tyrosine residue within the reverse transcriptase polypeptide (shaded oval) acting as a primer. As depicted, e forms a highly conserved stem-loop structure with an internal bulge, which is used as the template to initiate minus strand DNA synthesis. After synthesis of three to four nucleotides, the nascent DNA strand, covalently linked to the polymerase via the priming tyrosine residue, is transferred to the 3' end of the RNA template, where minus strand DNA synthesis continues.

regulatory viral sequences known to be required for viral DNA synthesis in vivo.30 This chapter provides a detailed description of the cell-free system for the expression of the DHBV polymerase in the reticulocyte lysate developed in our laboratory and discusses in more general terms the properties of the HBV and DHBV RTs expressed in insect cells and yeast, respectively.

E x p r e s s i o n o f D u c k H e p a t i t i s B V i r u s R e v e r s e T r a n s c r i p t a s e in Rabbit Reticulocyte Lysate Expression of the D H B V p o l y m e r a s e gene in the rabbit reticulocyte lysate in vitro translation system yields an enzymatically active reverse transcriptase that is capable o f initiating D N A synthesis, using a tyrosine residue located n e a r the N terminus (Tyr 96, Fig. 1) of the p o l y m e r a s e as a p r i m e r (the p r o t e i n - p r i m i n g reaction), l°'u T h e protein-priming reaction requires the viral R N A s e q u e n c e ~ (nucleotides 2560-2616 on the D H B V g e n o m e , Figs. 2 and 3), 23-25'27 which serves as the t e m p l a t e for reverse transcription. T h e m a j o r p r o d u c t of the in vitro reaction is a 3- to 4-nucleotide-long D N A strand that is covalently attached to the polymerase. A small fraction ( a p p r o x i m a t e l y 10%) of nascent D N A strands are further e l o n g a t e d either in situ or following translocation and annealing with comp l e m e n t a r y sequences on R N A templates. B e c a u s e o f the simplicity of the 3oM. Seifer and D. N. Standring, J. Virol. 67, 4513 (1993).

[ 11]

199


2 5 7 6 \ ~-~ uACJ ~

SP6

(-)DNA-.4~

u

e

POL

1

AI

DR1

I 2527 2537

JL

I

2560 26163021

FIG.3. Structure of the in vitro transcript for expression of the DHBV RT in the reticulocyte lysate.1°'11,23Shown schematically are the polymerase coding region (Pol, open bar) and the critical RNA sequences for reverse transcription including the e stem-loop (nucleotides 2560-2616 on the DHBV genome), the UUAC motif at nucleotide 2576 within the bulge of e that is used as the template for the protein-priming reaction, and the other UUAC motif at nucleotide 2537 within the DR1 sequence (dotted bar), which was originally identified as the origin of viral minus DNA synthesis but has recently been shown to be the acceptor site for the nascent minus DNA strand transferred from the e motif. Also shown are the SP6 transcription initiation site and the unique restriction sites for linearizing the plasmid for in vitro transcription; A, AflII; S, SaII.

assay, the reticulocyte lysate system is amenable to genetic and biochemical analyses of the D H B V polymerase. Materials and M e t h o d s Molecular Clones. Plasmid pGal5.1 contains a complete genome of an infectious clone of D H B V fused to the SP6 promoter at the E c o R I site. 31,32 Plasmid p H P is a derivative of pGal5.1, in which a fragment spinning positions 1-172 on the D H B V genome is replaced with a 33-nucleotidelong D N A fragment that contains the leader sequence of R N A 5 of b r o m o mosaic virus (BMV) followed by a sequence encoding six histidine residues. a° In the latter construct, the first A U G codon of the polymerase open reading frame ( O R F ) is removed; translation of the polymerase ORF, beginning at nucleotide 173, is initiated by the A U G codon within the B M V leader, resulting in a fusion of the hexahistidine tag to the polymerase. It is believed that translation of the polymerase in vivo also begins with the second A U G codon of the polymerase O R F at nucleotide 170 on the D H B V genome. Introduction of the B M V leader in p H P significantly increases the translation efficiency of the polymerase ORF, and the N-terminal histidine tag can be used to purify the polymerase from the

31E. Mandart, A. Kay, and F. Galibert, J. ViroL 49, 782 (1984). 32j. C. Pugh, K. Yaginuma, K. Koike, and J. Summers, J. Virol. 62, 3513 (1988).

200


[ 11]

translation mix with nickel ions bound to a nitrilotriacetic acid (NiNTA) resin. I° In Vitro Transcription. To synthesize the R N A message used for in vitro translation of the D H B V polymerase gene, pHP is linearized with the restriction endonuclease Sail, which cuts outside of the viral sequences and leaves the e coding sequences downstream of the polymerase O R F intact, or with AfllI, which cuts right at the stop codon of the polymerase ORF (nucleotide 2527) and thus no e sequences will be present on the transcript (Fig. 3). The linearized DNAs are then extracted with phenol, precipitated with ethanol, and used as templates for R N A synthesis with the MEGAscript kit (AMBION Inc., Austin, TX). Following the transcription reaction, plasmid D N A is removed with DNase I and R N A is purified by phenol extraction and ethanol precipitation. In Vitro Translation. The in vitro translation reaction in the rabbit reticulocyte lysate system is carried out as recommended by the supplier (Promega Biotec). Reactions are incubated for 60 to 90 min at 30° and are stopped by the addition of cycloheximide to a final concentration of 10 ng//xl. Protein Priming and D N A Polymerization Reactions. The reticulocyte lysate containing the polymerase is incubated with an equal volume of a reaction mixture containing buffer (100 mM Tris-HC1, pH 7.5), salts (30 mM NaCI, 20 mM MgC12), dATP, dCTP, and dTTP (each 28/zM), and [a-32p]dGTP (2.4/~M, 400 Ci/mmol). n When other [a-32p]dNTPs are used, dNTP solutions are adjusted accordingly. Reactions are incubated at 30° for 30 min and are stopped by the addition of 9 volumes of protein-loading b u f f e r . 33 For the synthesis of viral D N A for Southern and primer extension analyses, the dNTP concentration is raised to 200/xM. The template for reverse transcription, e RNA, is present in cis within the polymerase R N A when it is transcribed from D N A templates linearized by Sail. However, when the polymerase R N A is transcribed from DNA templates linearized by AfllI, no e sequence is present; instead, a separate e R N A is transcribed from a synthetic D N A template containing the e coding sequences (nucleotides 2560 to 2616) and the SP6 promoter 23 and added in trans either during the translation or the priming reaction. Isolation of Viral DNA. For primer extension and Southern analysis the products of the completed D N A polymerization reaction are treated with RNase A (65 ng//xl) for 10 min at 37 ° and subsequently with SDS (0.6%) and proteinase K (1 mg/ml) for 1 hr at 45 °. D N A is extracted twice with phenol, once with phenol-chloroform and with a solution of 33 j. Sambrook, E. F. Fritsch, and T. Maniatis, eds., "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, 1989.

[ 111


3sS

32p

1

2

201

Pol -t~

Fro. 4. Protein-priming activity of DHBV RT expressed in the reticulocyte lysate. 11 Lane 1 shows the protein products obtained from an in vitro translation reaction in the reticulocyte lysate in the presence of [35S]methionine, with an SP6 RNA template transcribed from pHP linearized with AfllI. Lane 2 shows the products of the in vitro protein-priming reaction, whereby the in vitro-translated polymerase was incubated with the e RNA and [a-32p]dGTP as described under Materials and Methods. The reaction products were resolved by SDSP A G E and an autoradiograph is shown. The full-length 35S-or 32p-labeled polymerase polypeptide is indicated by an arrow.

butanol : isopropanol (7 : 3) followed by ethanol precipitation. Dried pellets are resuspended in TE (10 mM Tris-HC1, pH 7.5, 1 mM EDTA) containing 5 ng//xl RNase A and are incubated at 37 ° for 20 min. Primer extension reactions and isolation of D H B V D N A from core particles in transfected tissue culture cells are carried out by a standard procedure. 34 Results Translation of in vitro transcripts containing the DHBV polymerase ORF in the reticulocyte lysate produces a major protein product with an apparent molecular weight (MW) of approximately 90,000 (Fig. 4), which is in good agreement with the expected size of 90,500 calculated from the predicted amino acid sequence (785 amino acid residues of the polymerase plus the six-histidine tag). Incubation of the in vitro-expressed polymerase in a solution that includes [a-32P]dGTP plus dATP, dCTP, and TTP leads to the incorporation of a 32p label into the polymerase polypeptide, at tyrosine 96 of the N-terminal domain (Fig. 4). l°,11 The result of this proteinpriming reaction is a polymerase linked to a 3- to 4-nucleotide-long DNA strand with the sequence 5'-GTAA-3', the template for which resides in 34 C. Seeger and J. Maragos, J. Virol. 64, 16 (1990).

202


[11]

the bulge region of e RNA. As described earlier, e R N A can be present in cis on the polymerase mRNA template or added in trans as a separate RNA, either during or after completion of the in vitro translation reaction.

The protein-priming activity of the polymerase increases as a function of the concentration of e and is optimal at a final concentration of approximately l/./~M.23 The apparent Kd for the polymerase-e R N A interaction has been estimated to a range of 8-26 n M . 23,25 A small fraction of the nascent DNA strands are further elongated and range in length from 100 to 500 nucleotides as determined by Southern blot analysis. H The primer extension assay maps the 5' ends of the in vitrosynthesized D N A to position 2537 on the D H B V genome, which coincides with the natural initiation site for viral minus strand D N A synthesis. In addition, the in vitro reaction yields D N A strands with 5' ends mapping to position 2576, located in the bulge region of e RNA. The latter are created by in situ elongation of the short D N A oligomers synthesized from e RNA, whereas the former are products from the translocation reaction that leads to the transfer of nascent D N A strands from e to DR1 (Fig. 2). 11'24 The efficiency by which this transfer occurs in the reticulocyte lysate is approximately 50% and appears to depend only on sequence homology between the donor and the acceptor sites. Like other reverse transcriptases and the polymerase activity detected in the virus particles, the D H B V polymerase requires Mg 2+ and is resistant to actinomycin D for minus strand D N A synthesis. 4'N However, as already discussed, the D H B V polymerase is able to initiate D N A synthesis de novo, using a tyrosine residue within the polymerase polypeptide as a primer, and requires a specific viral R N A sequence (e) as the template. The in vitro-synthesized polymerase forms a stable RNP complex with the e RNA. 23'25 This RNA-protein interaction is highly sensitive to salt and divalent cation concentrations; concentrations of NaCI above 0.5 M or MgC12 above 10 mM dramatically inhibit the polymerase-e interaction and thus the protein priming activityY '35 In the absence of e R N A or when the tyrosine residue required for the protein-priming reaction is mutated, the enzyme is still able to synthesize DNA from RNA templates by initiating D N A synthesis apparently randomly, most likely using as primers R N A or D N A oligomers present in the reaction mixture. 23 It appears that the priming reaction catalyzed by the polymerase may be different mechanistically from the regular polymerization (elongation) reaction. For example, agents such as foscarnet and certain dideoxy nucleotide triphosphates, which are known to inhibit elongation of viral D N A strands, have little

32 j. M. Hu and C. Seeger, Proc. Natl. Acad. Sci. U.S.A. 93, 1060 (1996).

[ 1 1]


203

effect on the priming reaction, u'36 This phenomenon could be explained by a conformational change of the polymerase when it switches from a "priming" to an "elongation" mode. The observation that D N A synthesis commonly pauses after polymerization of 3-4 nucleotides in vitro is also consistent with a requirement for a conformational change in the enzyme following the priming reaction. Biochemical analyses revealed that the in vitro-synthesized polymerase is present in a high molecular weight complex with cellular proteins in the reticulocyte lysate. 35 Partial purification of the polymerase from the lysate can be accomplished through velocity sedimentation in sucrose gradients. Although the partially purified RT remains enzymatically active, it has lost the competence to form an RNP with e R N A for reasons that are not yet completely understood. Evidence is accumulating for a role of the cellular protein chaperones hsp90 and hsp70 in maintaining the polymerase in a conformation that facilitates the interaction with e RNA. 35 These results may explain the failure to purify an enzymatically active polymerase from a variety of expression systems.

Expression of HBV Reverse Transcllptase in Insect Cells Expression of the enzymatically active polymerase of human hepatitis B virus (HBV) has been accomplished by Lanford and colleagues. 9 These investigators employed baculovirus as a vector for the expression of the reverse transcriptase in insect cells. Critical to the success of these experiments was the inclusion of sequences coding for the e and DR1 motifs on the pregenome, which had been shown previously to be important for activation of the D H B V polymerase. In addition, a FLAG epitope was fused to the N terminus of the polymerase to facilitate its purification following expression in insect cells by a rapid immunoaffinity purification procedure. Like the D H B V RT, the partially purified HBV polymerase displayed protein priming and D N A polymerization activities.

Expression of Polymerase HBV sequences (subtype ayw) spanning nucleotides 2309-1988 that contain the polymerase gene and the D R I - e R N A region are cloned into the baculovirus transfer vector pBacPAC9 (Clontech). A FLAG epitope (IBI) is fused to the N terminus of the polymerase to facilitate purification of the polypeptide from cell extracts. A recombinant baculovirus termed FPL-pol, expressing the HBV polymerase under the control of the polyhe36 K. Staschke and J. Colacino, J. ViroL 68, 8265 (1994).

204


[111

drin promoter, is prepared and used to infect Sf9 cells. Infected cells are harvested 48 hr postinfection by lysis in a phosphate buffer with 0.5% Nonidet P-40. The soluble fraction of the polymerase, which constitutes less than 10% of the total amount of expressed polypeptide, is purified from the cell lysate with the help of an immunoaffinity resin coated with a monoclonal antibody (mAB) directed against the FLAG epitope (mAB M2, IBI). The immobilized polymerase is eluted with 0.1 M glycine, pH 3.0, and 10% glycerol and is neutralized with a neutralization buffer (0.8 M Tris HC1, pH 8.4, 3% Triton X-100, 80 mM dithiothreitol, 50 U RNasin per ml; 67/zl neutralization buffer/ml elute). In addition to the polymerase, the eluate contains at least four additional polypeptides as determined by Coomassie blue staining of S D S - P A G E gels. One of these other proteins, with an estimated molecular weight of 110,000, cross-reacts with the mAB M2 and is thus copurified whereas the other three polypeptides were either contaminating proteins from Sf9 cells or represented cellular proteins that specifically associate with the HBV polymerase. This latter possibility is intriguing in view of observations made with the D H B V RT, which invoke a role of cellular proteins for the protein-priming activityY In Vitro Priming Reaction The partially purified H B V polymerase displays a protein priming activity that is similar to the one observed with the D H B V polymerase expressed in the reticulocyte lysate. The reaction is carried out directly in the elutionneutralization buffer described earlier supplemented with 10 mM MgCI2, 100/zM dNTPs (dATP, dGTP, and dCTP), and 5/xCi of [ot-32p]TTP(NEN, 3000 Ci/mmol) at 30 ° for 30 rain. The major reaction product is a polymerase polypeptide covalently linked to a few nucleotides that, based on results obtained with the D H B V RT, most likely represent the 3 or 4 nucleotides synthesized on the ~ R N A template. Minor reaction products represent polymerase polypeptides attached to more elongated minus strand D N A species that range between 125 and 500 nucleotides in length. The 5' ends of these D N A species have been mapped to a U U C A motif near the 3' end of pregenomic RNA, which corresponds to the U U A C motif in D H B V and represents the natural initiation site for viral minus strands. It is worthy of note that the protein-priming reaction does not require the addition of e or other R N A templates, indicating that the RT is purified as an RNP from the cell lysate. This observation further suggests that the interaction between the polymerase and e R N A occurred in cells prior to or during the preparation of cell extracts. In this context, it is important to recall that previous attempts to express an enzymatically active HBV polymerase in insect cells with a similar construct but devoid of sequences

[ 11]


205

encoding e and DR1 failed, 37 which suggests that the polymerase cannot bind e R N A once isolated from insect cells. Surprisingly, only a very small fraction, constituting less than 1% of the partially purified H B V polymerase, is active in the in vitro protein-priming reaction. 9'38 In comparison, approximately 10-30% of the D H B V polymerase synthesized in the reticulocyte lysate is competent for the initiation of D N A synthesis. 39Although the nature of this discrepancy is not understood, it could indicate that the HBV RT, like D H B V RT, might require cellular factors for enzymatic activity, some or all of which could have been lost during the purification step.

Expression of DHBV Reverse Transcriptase in Yeast Tavis and Ganem 29 employed the yeast transposon Tyl as a vector to express an enzymatically active D H B V RT. The Tyl element replicates in yeast through a reverse transcription pathway similar to that employed by retroviruses. T y l carries two overlapping genes, TYA and TYB, which are expressed from a single genomic m R N A (Fig. 5). 40 While T Y A encodes the structural protein that assembles into VLPs, T Y B encodes a multifunctional protein which has protease, integrase, and reverse transcriptase activities. TYB is initially expressed as a T Y A - T Y B fusion protein by ribosomal frameshifling at the TYA/TYB overlap. This fusion protein, along with the genomic RNA, is incorporated into VLPs and is processed by the protease activity encoded at the N terminus of TYB to liberate the TYB protein. The TYB-coding region can be replaced with foreign genes, which can then be expressed as fusion proteins with TYA. Because the fusion polypeptides are sequestered into VLPs, they can be rapidly purified from yeast extracts and assayed for their enzymatic activities. Expression o f PoIymerase

To express the D H B V RT, the polymerase gene is cloned into the expression vector pJEF724 (Fig. 5A), which contains a complete genome of Tyl. 41 The polymerase gene is inserted downstream of the protease domain encoded by TYB. 29 Expression of the transposable element is con37B. Ayola, P. Kanda, and R. Lanford, Virology 194, 370 (1993). 38R. E. Lanford, personal communication. 39C. Seeger, unpublished results. 40j. Boeke and S. Sandmeyer,in "The Molecularand CellularBiologyof the Yeast Saccharomyces" (J. Broach, J. Pringle, and E. Jones, eds,), p. 193. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, 1991. 41A. Gabriel and J. Boeke, Proc. Natl. Acad. Sci. U.S.A. 88, 9794 (1991).

206

I1 1]

EXPRESSION, PURIFICATION, AND CHARACTERIZATION A Gall

LTR

Tyl

pJEF724 I

I

I

H i n d III

Sal I

TYA

TYB

ORFs

Fusion Protein

N-

,,:

' 7

,

~-C

B

pTYBDP

Gall I

TYA ORFs

Fusion Protein

LTR

DHBV Pol

i HA

"

DR1

TYB - DHBV Pol I

N-

-C

FIG. 5. Structure of Ty-DHBV recombinant plasmids. 29 (A) Plasmid pJEF724 (top line) contains a Tyl element cloned downstream of the galactose-inducible Gall promoter; downstream of the Ty coding region is the long terminal repeat (LTR) of Tyl. Below this are schematics of the TYA and TYB ORFs and the TYA-TYB fusion protein resulting from ribosomal frameshifting. (B) Plasmid pTYDBP used for expression of an epitope-tagged DHBV polymerase. The DHBV sequences (open bar) coding for the polymerase ORF and the DR1 and e motifs and location of the inserted HA epitope are shown. Below it are shown the TYA and T Y B - D H B V polymerase ORFs and the predicted primary frameshift translational product, the T Y - D H B V polymerase fusion protein (modified from Tavis and Ganem29).

trolled by the inducible Gall promoter. The resulting plasmid pTYBDP (Fig. 5B) also contains D H B V sequences encoding DR1 and e RNA downstream of the polymerase ORF and a 33-nucleotide insertion in the tether region of the polymerase that encodes an epitope derived from influenza virus hemagglutinin (HA). In addition, pTYBDP bears a yeast replication origin for the maintenance of the plasmid D N A as an episome and an URA3 gene as a selectable marker. Saccharomyces cerevisiae strain YH51 (MATaGAL+ura3-52 spt3-202 his4-539 lys2-801) 41 is used for transformation and expression of the T Y - D H B V polymerase fusion protein. Expression of endogenous Tyl elements is diminished in this strain by a mutation in the SPT3 gene to facilitate detection of the exogenously transfected Ty expression vector.

[ 11]


207

Isolation of VLPs and Analyses of Viral DNA Synthesized in Vivo Ty VLPs are isolated from YH51 cells transformed with pTYBDP and induced by galactose. Yeast cell lysates are fractionated by sucrose density gradient centrifugation. VLPs are then pelleted from VLP-containing fractions by high-speed centrifugation. Western blot analysis of the pelleted material reveals two proteins with an estimated molecular mass of 131 and 138 kDa that reacts with a mAB directed against the H A epitope. Because the expected size of the polymerase is approximately 90 kDa, it is most likely that the primary TYA-polymerase translation product (MW 155,000) is processed within TYA. To detect DHBV D N A within the Ty VLPs, nucleic acids are isolated by phenol and chloroform extraction and detected by Southern blot analysis with DHBV-specific probes or by primer extension analysis to map the initiation site(s) for D N A synthesis. The results show that DHBV D N A is synthesized in yeast cells by the viral polymerase, using viral RNA sequences as the template for reverse transcription. Only minus strand DNAs are made, which range from 300 to 2500 nucleotides long and are covalently linked to protein. Primer extension analysis demonstrated that the 5' ends of the minus strand D N A map to the U U A C motif within the e and DR1 sequences, identical to what has been observed with the DHBV polymerase expressed in vitro.

Polymerase Activity in Vitro Purified VLPs containing the TY-polymerase fusion protein are active in vitro for incorporation [c~-32p]dNTPs in a reaction similar to the endogenous polymerase reaction in virions, leading to the synthesis of D N A products that range from 50 to >1000 nucleotides long and are protein linked. D N A synthesis in this endogenous polymerase reaction in VLPs is sensitive to pretreatment with RNase or micrococcal nuclease but is resistant to actinomycin D, indicating that R N A is the template for D N A synthesis. S u m m a r y and Comments The successful expression of the hepadnavirus reverse transcriptase in several different expression systems as described earlier has greatly facilitated biochemical and genetical analysis of this most unusual enzyme. The demonstration that the polymerase is the only viral protein needed to initiate viral D N A synthesis de novo, using a novel protein-priming mechanism, has hastened efforts to identify key players in the priming reaction and has yielded important information regarding the requirement of both

208


[12]

the polymerase and the R N A template. A tyrosine residue at the N terminus of the polymerase polypeptide has been identified as the primer for reverse transcription, and two separate domains of the enzyme have been shown to participate in binding to the e R N A template. Detailed analyses using the in vitro-expressed enzyme have led to the surprising conclusion that the viral R N A template for initiation of D N A synthesis is the same sequence motif e, previously identified as the R N A packaging signal, and that a strand transfer is required for the continuation of viral D N A synthesis. However, it has not yet been possible to obtain highly purified enzyme in vitro, which is a prerequisite for further detailed biochemical and structural studies. Expression levels in the reticulocyte lysate are too low to be feasible for this purpose and the yeast system is not useful for large-scale purification due to contamination by Ty proteins and the uncertainty of proteolytic processing of the Ty-polymerase fusion. The recent success in expressing high levels of H B V polymerase in insect cells may facilitate the production of large amounts of the polymerase enzyme for further biochemical and structural studies.

[1 2] E x p r e s s i o n , P u r i f i c a t i o n , a n d C h a r a c t e r i z a t i o n o f V a c c i n i a V i r u s - E n c o d e d R N A a n d Poly(A) P o l y m e r a s e s

By

PAUL GERSHON and BERNARD MOSS

Vaccinia Virus Gene Expression Strategy The poxviruses, of which vaccinia virus is the prototype, replicate in the cytoplasm of the infected cell. 1 Their independence from host cell nuclear functions is aided by a distinctive replication and transcription apparatus encoded within the viral double-stranded D N A genome. This apparatus produces mature viral mRNAs with eukaryotic features, including a 5' cap and a 3' poly(A) tail. Thus, vaccinia provides a unique model system for combined genetic and biochemical studies of mRNA synthesis and modification. The ~200 vaccinia genes are expressed in a programmed fashion. 2 Three stages of gene expression have been characterized: an early stage, which 1 B. Moss, in "Fields Virology" (B. N. Fields, D. M. Knipe, and P. M. Howley, eds.), p. 2637. Lippincott-Raven Publishers, Philadelphia, 1996. 2 B. Moss, Annu. Rev. Biochern. 59, 661 (1990).

METHODS IN ENZYMOLOGY,VOL. 275

Copyright © 1996by AcademicPress, Inc. All rights of reproduction in any form reserved.