Nucleotide sequence and molecular genetic analysis of the large subunit of ribonucleotide reductase encoded by vaccinia virus

VIROLOGY

164, 121-131

(1988)

Nucleotide

Sequence and Molecular Genetic Analysis of the Large Subunit Ribonucleotide Reductase Encoded by Vaccinia Virus

LESLIE A. TENGELSEN,* Departments

MARY B. SLABAUGH,t

of

JODY K. BIBLER,t AND DENNIS E. HRUBY*,’

of *Microbiology and tBiochemistry and Biophysics, Center for Gene Research, Oregon State University, Con/al/is, Oregon 9733 J-3804 Received November

5, 1987; accepted January 11, 1988

We have mapped the vaccinia virus (W) gene encoding the large subunit of ribonucleotide reductase (W Ml) within the Hindill I restriction fragment by using an oligonucleotide probe. Nucleotide sequencing revealed a 2340-bp open reading frame (or-f), l-3, whose amino acid sequence is highly homologous to the mouse Ml protein. The l-3 gene was expressed as an immediate-early gene product, being transcribed in a leftward direction into a 2.7-kb polyadenylated transcript. Hybrid-selected translation of cycloheximide-amplified immediate-early viral RNA demonstrated that this mRNA encoded an 86-kd protein, which agrees with the expected size of the reductase large subunit. The 5’- and 3’boundaries of the l-3 transcriptional unit were determined by primer extension and Sl -nuclease analysis, respectively, and shown to contain sequence elements typical of other VV early genes. Suprisingly, the predicted amino acid sequence of the VV enzyme subunit shares 72.5% homology with the mouse large subunit, Ml. (Gois88 Academic Press.

Inc.

INTRODUCTION Vaccinia virus (VV), the prototype of the Poxvirus family, replicates its large double-stranded 185-kb DNA genome in the cytoplasmic compartment of infected cells within discrete foci known as “virus factories” or “viroplasm” (Dales and Pogo, 1981; Moss, 1985). Since viral replication requires enzymatic activities not normally found in the cytoplasm of host cells, e.g., DNA polymerase, the presence of these extranuclear replicating virus factories suggested that VV must encode or recruit many of the enzymes required to replicate and/or transcribe viral DNA. This hypothesis has been substantiated by the discovery of a number of VV enzymes including DNA polymerase (Jones and Moss, 1984; Traktman et al., 1984), thymidine kinase (Hruby and Ball, 1982), DNA-dependent RNA polymerase (Jones et al., 1987), guanylmethyl transferase (Barbosa and Moss, 1978) nucleophosphohydrolase (Munyon et al., 1968), and most recently ribonucleotide reductase (Slabaugh and Mathews, 1984; Slabaugh el a/., 1984, 1988) which are virus-encoded and/or packaged within the virion. Regardless of its location within the cell, DNA replication is dependent upon the availability of a ready supply of deoxyribonucleotides. In both eukaryotes and prokaryotes, these metabolic precursors are supplied by the enzyme ribonucleotide reductase which catalyzes the reduction of the 2’-hydroxyl group of ribo’ To whom requests for reprints should be addressed.

nucleotides to produce and maintain highly regulated pools of the four deoxyribonucleotides used in DNA synthesis (Lammers and Follmann, 1983). This enzyme has been identified in a number of organisms and studied most extensively in calf thymus, mouse, and fscherichia co/i (Thelander, 1973; Thelander et al., 1980; Caras et al., 1985). Despite the presumed availability of the host enzyme, several representatives of the Herpesvirus family have been shown to possess the coding information for both subunits of nbonucleotide reductase (Lankinen et a/., 1982, Gibson eta/., 1984). This would seem to indicate the importance of this function in the replication of nuclear DNA viruses. Similarly, a cytoplasmic DNA virus such as VV must also have access to all four DNA precursors, or be able to catalyze their production. Thus, not suprisingly, a nbonucleotide reductase activity with unique allostenc properties has been described and characterized from VV-infected cells (Slabaugh and Mathews, 1984). The experiments described in this manuscript report the genomic location of the gene encoding the large subunit of VV ribonucleotide reductase, VV M 1, and provide a molecular genetic analysis of the VV M 1 transcript and its translational product. MATERIALS Oligonucleotide

AND METHODS

probes and Southern

blots

VV DNA (WR strain) was purified from infected BSC-40 cells as previously described (Weinrich ef al.,

122

TENGELSEN

1985). Genomic VV DNA, or appropriate subcloned fragments, was digested with restriction endonucleases. The resulting fragments were separated by agarose gel electrophoresis and transferred to Zeta-probe nylon membranes (Bio-Rad). Southern blot hybridization analyses were carried out using either a 5’-end-labeled mixed octadecamer oligonucleotide probe (A/G)TA(A/G)TACAT(A/C/G/T)CC(AK/G/T)GT(C/T)TT (128-fold degenerate), designated MS4, or nick-translated cloned fragments of VV DNA (Maniatis et a/., 1982). Restriction

map and nucleotide

sequencing

A recombinant plasmid containing the HindIll I fragment of VV DNA cloned into the HindIll site of pBR322 was originally obtained from B. Moss (NIH). Plasmid DNA was amplified and purified by standard methods (Maniatis et a/., 198.2). A rudimentary restriction map of the HindIll I fragment was constructed by analyzing the results of a number of multiple restriction enzyme digestions. By reference to this map, appropriate subfragments of HindIll I were cloned into the multiple cloning site of M 13 phage vectors and sequenced by the dideoxynucleotide chain-termination method (Sanger et al., 1977). The sequencing reactions were subjected to electrophoresis on 8 M urea, 6.66% acrylamide gels and visualized by autoradiography using Kodak XAR-5 film. Where necessary, oligonucleotide primers which hybridized to regions of determined sequence were synthesized in order to confirm the nucleotide sequence between adjoining restriction fragments or to sequence regions lacking convenient cloning sites. The radioactive nucleotides used for sequencing reactions were obtained from New England Nuclear Corp. Other enzymes and cloning vectors were obtained from Bethesda Research Laboratories, Boehringer-Mannheim, and International Biotechnologies, Inc. Northern

analysis

Cytoplasmic RNA was isolated from VV-infected HeLa cells (10 PFU/cell) at 2 hr postinfection (hr p.i.) (early RNA) or 8 hr p.i. (late RNA) in the absence of drug treatment, or at 5 hr p.i. from cells treated with 100 pg/ml cycloheximide (CHX RNA). RNAs were purified by centrifugation through cesium chloride gradients, containing IV-lauryl sarkosyl (Weinrich et a/., 1985), and several subsequent cycles of ethanol precipitation. Ten-microgram aliquots of RNA were separated under denaturing conditions using electrophoresis at 75 V for 6 hr through 1% agarose gels containing formaldehyde and then transferred to nitrocellulose. The immobilized RNA was subjected to Northern blot

ET AL.

hybridization procedures (Lehrach et a/., 1977) using a nick-translated probe fragment internal to the l-3 orf. Hybrid selection/cell-free

translation

CHX RNA or late viral RNA was hybrid-selected at 37” with a cloned internal portion of the l-3 or-f which had been immobilized on a nitrocellulose filter (Mason and Williams, 1985). A filter on which pUCl9 plasmid DNA was immobilized served as a negative control. Hybrid-selected RNAs were translated in a mRNA-dependent rabbit reticulocyte lysate cell-free protein synthesizing system (Hruby and Ball, 1981) in the presence of L-[35S]methionine (1 129 Ci/mmol, New England Nuclear). Radioactive proteins were analyzed by SDS:polyacrylamide gel electrophoresis and fluorography (Studier, 1973). Sl -nuclease

mapping

and primer extension

The 3’-end of the l-3 transcript was mapped by using VV CHX RNA to protect a 3’-end-labeled DNA fragment from nuclease Sl digestion (Maniatis et a/., 1982). For primer extension analysis, 20 pg of CHX RNA was annealed for 5 min at 60” to 10 ng of a 5’-end-labeled complimentary nonadecamer oligonucleotide and allowed to cool to 42” for 1 hr. the annealing reaction contained 0.25 M KCI, 10 mM Tris-Cl (pH 8.0) and 1 mM EDTA. Primer extension reactions were carried out in 10 mM Tris-Cl (pH 8.3), 10 mM MgC12, 5 mM DlT, 0.2 mM of each of the four deoxyribonucleotides, and 8 U of reverse transcriptase (Bethesda Research Laboratories) at 37” for 1 hr. the reaction was stopped by precipitation with 0.2 M NaOAc and 100% ETOH. Primer extension products were analyzed by coelectrophoresis with dideoxy sequencing reactions primed with the same oligonucleotide. Sequence

analysis

The sequence of the l-3 or-f was assembled and analyzed using the Microgenie sequencing program (Beckman Instruments, Inc.) on an IBM personal computer. The VV Ml amino acid sequence was aligned (Mount and Conrade, 1986; Gotoh, 1987) with the mouse protein sequence with the assistance of the personnel in the Center for Gene Research Central Service Facility at Oregon State University. Protein homologies were based on the National Biomedical Research Foundation (NBRF) Protein Data Bank obtained from Beckman Instruments, Inc., and compared to sequence published by Caras et al. (1985).

VV RIBONUCLEOTIDE

REDUCTASE

RESULTS Mapping

the genomic

location

of the VV Ml gene

At the time these experiments were initiated, the nucleotide sequences (and hence, the predicted amino acid sequences) of ribonucleotide reductase large subunit proteins from E. co/i, herpes simplex virus type l(HSV-l), Epstein-Barr virus (EBV), and mouse were available (Caras et al., 1985). Although the overall amino acid sequence identity among these proteins was only 20%, a region near the carboxy-termini contained a site in which five of six consecutive amino acids were conserved in the eukaryotic and viral proteins [Lys-Thr-X-Met-Tyr-Tyr]. Assuming that this sequence might be similarly conserved within the VV M 1 gene, a 128-fold degenerate mixed oligonucleotide (MS4) was synthesized which was designed to hybridize to DNA sequences encoding the amino acid sequence Lys-Thr-Gly-Met-Tyr-Tyr. Cytoplasmic DNA isolated from cells infected with wild-type VV was digested with several different restriction enzymes (Xhol, Sall, HindIll, and Kpnl) for which complete genomrc maps were available (DeFillipes, 1982) and subjected to Southern blot hybridization procedures using the MS4 oligonucleotide as a probe (Fig. 1A). In each case MS4 hybridized to a single restriction fragment: Xhol A, SalI B, Hindlll I, and Kpnl, respectively. The specificity of these interactions was demonstrated by hybridization of the MS4 probe to cloned HSVl DNA containing Ml coding sequences but not to plasmid or other VV DNA fragments. TheXhol, Sail, Hindlll, and Kpnl restriction maps of the VV DNA molecule have been aligned and the fragments which hybridize to the MS4 probe are indicated (Fig. 1 B). Taken together, these results map the site of hybridization to the left side of the 6.5-kb HindIll I fragment. In order to improve the resolution of mapping, the restriction enzyme cleavage sites for EcoRl and Kpnl within the Hindlll I fragment were determined (data not shown). The cloned HindIll I DNA fragment was then digested with Hindlll, Kpnl, or EcoRI, and the Southern blot hybridization analysis with the MS4 probe was repeated (Fig. 2A). Positive hybridization signals were obtained with the 6.5.kb Hindlll I, 2.65-kb HindlllKpnl, and 3.0.kb Hindlll-EcoRI fragments. These data mapped the site of MS4 hybridization to within the leftmost one-third of the Hindlll I DNA fragment (Fig. 2B). As part of a larger ongoing project in the laboratory, the VV Kpnl I DNA fragment (encompassing the right side of Hindlll E, all of HindIll 0, and the left side of Hindlll I fragments, see Fig. 1A) has been subjected to detailed restriction enzyme cleavage site mapping. The data pertaining to the region to which MS4 hybrid-

LARGE SUBUNIT

123

ized have been summarized in Fig. 3. Note that a previously unrecognized small (- 150-bp) Hindlll restriction fragment was found during restriction analysis, designated here as P, lying between the 0 and I Hindlll fragments. Nucleotide sequence of the VV l-3 gene

and computer

analysis

Using the restriction map shown in Fig. 3 (expanded map), small DNA fragments were subcloned into M 13 vectors and sequenced. Synthetic primers were used to clarify regions of sequence ambiguity and to confirm the sequences spanning adjoining restriction fragments in instances where overlapping clones were not available. Using this approach the entire sequence of both strands was independently obtained. The sequence of the leftward reading strand is presented in Fig. 4, and the probable site of MS4 hybridization is indicated by brackets. Additional sequence analysis of Kpnl I revealed three additional leftward-reading or-fs partially or completely encoded within HindIll I (data not shown), orfs I-1, l-2, and l-4 in Fig. 3. Computer-assisted analysis of the coding capacity of each of the six possible reading frames indicated the presence of a single large leftward-reading or-f extending from positions 64 to 2377. The derived amino acid sequence of the protein encoded by the l-3 or-f is shown below the nucleotide sequence and yields a protein with a predicted molecular weight of 86,944 Da. The amino acid sequence contained 11 1 basic residues and 49 acidic residues, suggesting that it would be positively charged at neutral pH. When analyzed for possible secondary structure, the protein appeared to have features consistent with it being a soluble globular polypeptide. A hydrophobicity plot of the l-3 protein did not reveal any obvious domains which might Interact with membranes. An examination of the predicted l-3 amino acid sequence for the presence of potential glycosylation sites (Asn-X-Ser/Thr) revealed two sites, amino acids 280-282 and 613-615. It is not known if either site is subject to modification in viva. The nucleotide sequences flanking the l-3 orf are typical of most poxvirus immediate-early genes sequenced to date. The 50 bp immediately upstream of the putative l-3 start codon is 80% A/T rich, which is typical of VV immediate-early promoter elements (Weir and Moss, 1983; Yuen et al., 1987). The possibility that the l-3 gene is expressed as an immediate-early gene during the viral replicatrve cycle IS strengthened by the absence of the TAAATG signal which is found at the 5’.end of most VV genes expressed late in VV Infection (Rose1 et al., 1986). The sequence TATTAATGAAAAGTTA, recently suggested to be a bind-

TENGELSEN

124

ET AL.

B.

A

H,JtG

F ,L,KO

1

OqlJK,Hy t

C

B

C

I I

EPJ,R,

N I !

D

B

E

M I Xhol

A

I

L

I

F,G,l,NyTP

Sal

Hind III

FIG. 1. Genomic mapping of the gene encoding the large subunit (M 1) of VV ribonucleotide reductase. (A) Cloned HSV-1 DNA (pSG124) digested with EcoRl (lane 1) or wild-type VV DNA digested with Xhol (lane 2) SalI (lane 3). HindIll (lane 4) or Kpnl (lane 5) were electrophoresed in an agarose gel, blotted to nitrocellulose, and hybridized at 44” with a 3ZP-labeled MS4 mixed oligonucleotide probe. The left side of the figure shows the gel stained with ethidium bromide and the right side is the autoradiograph of the Southern blot hybridization. The numbers at the left refer to the fragment sizes (in kbp) of molecular weight markers which were coelectrophoresed as standards. (B) Maps of the cleavage sites of the restriction enzymes used to digest VV DNA are shown. The restriction fragments which hybridized to the MS-4 probe are highlighted in black.

ing site for an early VV transacting factor (Yuen et al., 1987) is present 12-27 nucleotides upstream of the transcriptional l-3 start site (see below), although no evidence has been obtained to indicate that the sequence has a similar binding activity in the context of the l-3 gene. The sequence TTTTTAT is found 10 bp downstream of the l-3 orf stop codon. This signal has been shown to function both in vitro and in vivo in the termination of early gene transcripts (Rohrmann et a/., 1986). Transcriptional

analysis

In order to determine whether orf l-3 was transcriptionally active, Northern and Sl analyses were per-

formed. Figure 5A is a schematic drawing of the portion of the VV genome containing the l-3 orf and identifies the probes used in this series of experiments. To detect I-3-derived transcripts, viral RNA isolated from VV-infected cells was subjected to Northern blot analysis. Hybridization procedures used probe fragment 2, which is internal to the l-3 or-f (BarnHI-Kpnl). The data in Fig. 56 indicate that prior to VV DNA synthesis a transcript with an estimated size of the 2800 nucleotides is heavily expressed from the l-3 locus. The amount of transcript is greatly enhanced by the cycloheximide (CHX) treatment confirming that the l-3 gene is expressed as a VV immediate-early gene. There is also a small amount of a larger (-4000 nucleotide) transcript present in the CHX RNA. It is not

VV RIBONUCLEOTIDE

A.

REDUCTASE

12

1234

125

LARGE SUBUNIT

34

kb

12: 4-

2-

l-

C

NMK

F

EOIGLJH

‘ .**

.’

z

t

f

D --..

I *-...*

2’

--.....*

Pi

KE

I

A

I

I

B 1

-....

-...

-.....*

-. -....

:.E

KE-

_

1 kb

FIG. 2. Frne mapprng the location of the VVMl gene within the VV HindIll I fragment. (A) Lane 1, VV DNA drgested with HindIll. Lanes 2-4, pBR322:VV HindIll I plasmid DNA digested with HindIll, Kpnl, and EcoRI, respectively. Products of the Indicated restrictron reactions were seperated by agarose gel electrophoresis, and the gel was stained with ethidrum bromide (left side), transferred to nltrocellulose, and hybridized to 3ZP-labeled MS4 probe (right side). The numbers at the left refer to the sizes (in kbp) of the molecular weight markers. (B) The HindIll map of VV DNA is shown. The VV HindIll I fragment has been expanded and the positions of Kpnl (K) and fcoRl (E) cleavage sites are indicated. Fragments which hybridized to the MS4 probe are highlighted in black.

clear if this transcript arises from transcriptional readthrough from an adjoining gene (Hruby and Ball, 1982) or perhaps activation of a cryptic VV promoter by the drug treatment (Weinrich and Hruby, 1987). The RNA from infected cells at late times contained only a trace amount of 2800-nucleotide transcript amongst a heterogeneous mixture of molecules that were both

Hind111

map ..,....

Predicted 0rfs

*""I-

0

larger and smaller. The pattern of detected transcripts was identical if the RNA was first subjected to oligo(dT)-cellulose chromatography, suggesting the presence of a 3’-terminal poly(A) tract (data not shown). To locate the ends of the 2800-base l-3 transcript, a combination of nuclease Sl and primer extension

I

,P,

,....

.

I-1

250 bp I R

I R

I-2

P I I c

I-4

1-3

s I

I R

BP I I R

/\ R

09 I I(

I R

1[ I

I c

s I I R

I R

I R

FIG. 3. Restriction map and transcriptional organization of the region of the VV genome which encodes the VV Ml gene. The top lrne represents the HindIll map of this portion of the VV DNA molecule. Below it is the predicted transcnptronal organlzatron based on nucleotide sequence and northern blot analyses (Tengelsen and Hruby, in preperation). The region containing the l-3 gene has been expanded and the location of salient restriction enzyme cleavage sites is indicated. 6, BarnHI; Bg, Bglll; C, C/al; E, EcoRI; K, Kpnl; P, Psi?; R, Rsal; X, Xhol.

TENGELSEN

126 1

CCCTACGT

9 64

ATG TTT GTC ATT AAA CGA AAT GGA TAC AAG GAA AAT GTC ATG MET Phe Val Ile Lys Arg As" Gly Tyr Lys Glu As" Val MET

a 63 105

106

TTT GAT AU ATC ACG TCT CGT ATT AGA AAA TTA TGT TAT GGC Phe Asp Lys Ila Thr Ser Arg Ile ArS Lys Leu Cys Tyr Gly

147

148

TTA AAC ACG GAT CAT ATA GAT CCT ATT AAA ATA GCT ATG AAG Leu Asn Thr Asp His Ile Asp Pro Ile Lys Ile Ala MET Lys

la9

190

GTT ATT CAA GGA ATA TAT AAT GGA GTA ACA ACG GTA GAA TTG Val Ilc Gln Gly Ile Tyr As" Gly Val Thr Thr Val Glu Leu

231

232

GAC ACT CTG GCA GCC GAA ATA Asp Thr Leu Ala Ala Glu Ile CAT CCG GAT TAT GCC ATT CTA His Pro Asp Tyr Ala Ile Leu

CAA Gin TCA Ser

273

316

AAT CTA CAC AAG GAA ACA AAA AAA CTA TTT AGT GAA GTG ATG As" Leu His Lys Glu Thr Lys Lys Leu Phe Ser Glu Val MET

357

358

GAG GAT TTA TTC AAC TAT GTT AAT CCT AAA AAT GGG AAA CAT Glu Asp Leu Phe Asn Tyr Val Asn Pro Lys Asn Gly Lys His

399

400

TCT CCG ATT ATT TCA AGT ATC ACC ATG GAT ATA GTT AAC AAA Ser Pro Ile Ile Ser Ser Ile Thr MET Asp Ile Val As" Lys

441

442

TAT AAG G&T AAA CTC AAC TCG GTT ATT ATT TAC GAA CGA GAC Tyr Lys Asp Lys Leu Asn Ser Val Ile Ile Tyr Glu Arg Asp

483

484

TTT TCA TAC MC TAT TTT GGT TTT AAA ACT TTG GAA AAA TCC Phe Ser Tyr As" Tyr Phe Gly Phe Lys Thr Leu Glu Lys Ser

525

526

TAC TTG TTG AAA ATA AAC AAC AAG ATC GTT GAA AGA CCT CAG Tyr Leu Leu Lys Ile As" As" Lys Ile Val Glu Arg Pro Gin

567

568

CAC ATG TTA ATG CGT GTC GCA GTA GGA ATT CAT CAA TGG CAT His MET Leu MET Arg Val Ala Val Gly Ile His Gln Trp Asp

609

610

ATA GAC TCA GCT ATT GAG ACG TAC AAT CTA CTT TCT GAA AAA Ile Asp Ser Ala Ile Glu Thr Tyr As" Leu Leu Ser Glu Lys

651

652

TGG TTT ACG CAC GCT TCT CCT ACC TTA TTT AAT GCG GGA ACT Trp Phe Thr His Ala Ser Pro Thr Leu Phe As" Ala Gly Thr

693

694

ACT CGT CAC CAA ATG TCT AGC TGT TTT CTA CTT AAC ATG ATC Ser Arg His Gln MET Ser Ser Cys Phe Leu Leu As" MET Ile

735

736

GAT GAT AGC ATA GAG GGT ATC TAT GAC ACG TTA &'A CGA TGC Asp Asp Ser Ile Glu Gly Ile Tyr Asp Thr Leu Lys Arg Cys

777

778

GCA TTA ATC TCT AAA ATG GCA GGG GGA ATA GGT CTA TCA ATT Ala Leu Ile Ser Lys MET Ala Gly Gly Ile Gly Leu Ser Ile

819

a20

AGT AAT ATT CGT GCC AGT GGA AGC TAT ATC TCC GGT ACC AAT Ser As" Ile Arg Ala Ser Gly Ser Tyr Ile Ser Gly Thr As"

861

862

GGT ATA TCA AAC GGT ATT ATT CCA ATG TTG AGA GTT TAT AAT Gly Ile Ser As" Gly Ile Ile Pro MET Leu Arg Vs.1 Tyr As"

903

904

AAC ACC GCT AGA TAC ATA GAT CAG GGA GGA AAC AAA CGG CCT As" Thr Ala Arg Tyr Ile Asp Gln Gly Gly As" Lys Arg Pro

945

946

GGA GTT ATG GCC ATA TAC TTG GAA CCG TGG CAT TCT GAT ATT Gly Val MET Ala Ile Tyr Leu Glu Pro Trp His Ser Asp Ile

987

988

ATG GCG TTC CTC GAT CTT AAA AAG MT ACA CGA AAC GAG GAA MET Ala Phe Leu Asp Leu Lys Lys As" Thr Gly As" Glu Glu

1029

CAT AGA ACC AGA GAT CTA TTT ATA GCT CTT TGG ATT CCT GAT His Arg Thr Arg Asp Leu Phe Ile Ala Le" Trp Ile Pro Asp

1071

1072

CTC TTT ATG MA CGA GTG AAG GAT GAC GGA GAG TGG TCG TTG Leu Phe MET Lys Arg Val Lys Asp Asp Gly Glu Trp Ser Leu

1113

1114

ATG TGT CCG GAT GAA TGT CCT GGA TTG GAC AAT GTT TGG GGA MET Cys Pro Asp Glu Cys Pro Gly Leu Asp As" Vs.1 Trp Gly

1155

1156

GAC GAG TTC GAA CGA TTG TAT ACA CTA TAC GAA AGA GAA AGG Asp Glu Phe Glu Arg Leu Tyr Thr Leu Tyr Glu Arg Glu Arg

1197

1198

AGA TAC AAA TCT ATA ATA AAG GCT CGA GTC GTC TGG AAA GCG Arg Tyr Lys Ser Ile Ile Lys Ala Arg Val Val Trp Lys Ala

1239

274

GCA GCC ACT Ala Ala Thr GCC GCC AGA Ala Ala Arg

TGT Cys ATA Ile

ACT ACA Thr Thr GCC GTA Ala Val

ET AL. 1240

ATT ATA GAA TCT CAG ATT GAA ACT GGT ACT CCA TTC ATT CTT Ile Ile Glu Ser Gln Ile Glu Thr Gly Thr Pro Phe Ile Leu

1281

1282

TAT AAG GAT GCG TGT AAC AAA AAG AGT AAT CAA CAA AAT TTA Tyr Lys Asp Ala Cys As" Lys Lys Ser As" Gln Gln As" Leu

1323

1324

GGA ACT ATC AAG TGT AGT AAT CTT TGC ACT GAG ATA ATA CAA Gly Thr Ile Lys Cys Ser As" Leu Cys Thr Glu Ile Ile Gin

1365

1366

TAT GCG GAT GCT AAT GAG GTA GCC GTT TGT AAT CTG GCA TCT Tyr Ala Asp Ala As" Glu Val Ala Val Cys As" Leu Ala Ser

1407

1408

GTT GCC TTG AAC ATG TTT GTA ATA GAT GGG CGA TTT GAT TTT Val Ala Leu As" MET Phe Val Ile Asp Gly Arg Phe Asp Phe

1449

1450

CTC AAA CTG AAG GTA GTG GTC AAA GTA ATT GTC AGA AAT CTC Leu Lys Leu Lys Vs.1 Val Val Lys Val Ile Val Arg As" Leu

1491

1492

AAT AAA ATT ATA GAT ATT AAT TAT TAT CCT ATT CCA GAA GCT As" Lys 11s Ile Asp Ile As" Tyr Tyr Pro Ile Pro Glu Ala

1533

1534

GAA ATC TCT AAT AAG AGA CAT AGA CCT ATC GGT ATT GGT GTT Glu Ile Ser As" Lys Arg His Arg Pro Ile Gly Ile Gly Val

1575

1576

CAA GGA TTA GCG GAC GCG TTT ATT CTC TTA AAT TAT CCA TTT Gln Gly Leu Ala Asp Ala Phe Ile Leu Leu As" Tyr Pro Phe

1617

1618

GAT AGC CTG GAA GCA CAA CAT ATA AAT AAG AAG ATC TTC GAA Asp Ser Leu Glu Ala Gin Asp Ile As" Lys Lys Ile Phe Glu

1659

1660

ACC ATT TAT TAC GGT GCA TTA GAG GCG AGT TGT GAA CTA GCT Thr Ile Tyr Tyr Gly Ala Leu Glu Ala Ser Cys Glu Leu Ala

1701

1702

GAG AAG GAA GGA CCA TAC GAT ACA TAT GTA GGA TCG TAC GCC Glu Lys Glu Gly Pro Tyr Asp Thr Tyr Val Gly Ser Tyr Ala

1743

1744

AGT AAC GGT ATT CTA CAA TAT GAT CTT TGG AAT GTT GTA CCG Ser As" Gly Ile Leu Gln Tyr Asp Leu Trp As" Val Val Pro

1785

1786

TCG GAT CTT TGG AAT TGG GAA CCT CTA AAA GAT AAA ATC AGA Ser Asp La" Trp As" Trp Glu Pro Leu Lys Asp Lys Ile Arg

1827

la28

ACA TAC GGT CTT AGA AAT AGT TTA TTG GTG GCA CCT CTG CCG Thr Tyr Gly Leu Arg As" Ser Leu Leu Val Ala Pro Leu Pro

1869

1870

CTG CAT CAA CAT GCT CAA ATT TTG GGA h4T AAT GAG TCG GTG Leu His Gln His Ala Gln Ile Leu Gly Asn As" Glu Ser Val

1911

1912

GAA CCG TAT ACC AGT AAT ATT TAC ACT CGG AGA GTA TTG TCT Glu Pro Tyr Thr Ser As" Ile Tyr Thr Arg Arg Val Leu Ser

1953

1954

GGA GAA TTT CAA GTA GTT AAT CCG CAT CTC CTT AGA GTT TTA Gly Glu Phe Gln Val Val As" Pro His Leu Lsu Arg Val Lsu

1995

1996

ACC GAG AGA AAA TTA TGG AAT GAT GAG ATC A4G AAT AGG ATT Thr Glu Arg Lys Leu Trp As" Asp Glu Ile Lys As" Arg 11s

2037

2038

ATG GCA GAT GGT GGA TCC ATT CAG MT ACA AAC CTT CCA GAA MET Ala Asp Gly Gly Ser Ile Gln As" Thr As" Leu Pro Glu

2079

2080

GAT ATT AAG CGA GTT TAT AU ACT ATT TGG GAA ATT CCA CAA Asp Ile Lys Arg Vs.1 Tyr Lys Thr Ile Trp Glu Ile Pro Gln

2121

2122

AAG ACG ATC Lys Thr Ile GAT CAA AGT Asp Gin Ser

ATG GCT GCA GAC AGG GGA GCC TTC ATC MET Ala Ala Asp Arg Gly Ala Phe Ile ATG AAT ATC CAT ATA GCA GAT CCG AGT MET As" Ile His Ile Ala Asp Pro Ser

2163

2206

TAT TCC AAA CTA ACG AGT ATG CAT TTT TAC GGA TGG AGT CTC Tyr Ses Lys Leu Thr Ser MET His Phe Tyr Gly Trp Ser Leu

2247

2248

ACG GGA ATC TAC T GGT CTA CTA CGT ACG AAA Gly Leu P ys Thr Gly MET Tyr $yr Leu Arg Thr Lys TCC GCT CCC ATT CAA TTC ACA TTG GAC AAG GAT AAA Ser Ala Pro Ile Gln Phe Thr Leu Asp Lys Asp Lys

CCC GCA Pro Ala

2289

ATA AAA Ile Lys

2331

2332

CCA CCC GTG GTT TGT GAT TCC GAA ATC TGT ACA TCA TGC AGT Pro Pro Val Val Cys Asp Ser Glu Ile Cys Thr Ser Cys Ser

2373

2374 2427

GGT TAA CAAAUCAGTTTTTATTCTCAAATGAGATAAAG PGAAAATATATATC 2426 Gly TER ATTATATTACAAAGTACAATTATTTAGGTTTAATCATGAGTAAGGTAATCAAGAAG 2482

2483

AGAGTTGAAACTTCACCA

315

2164

2290

ATA Ile CAA Gln

AAA Lys TCT Ser

2205

2500

FIG. 4. Nucleotide

sequence of the l-3 gene and flanking regions. The sequence of the message strand of the l-3 gene is given, being numbered from right to left with reference to the VV Hindlll map. The predicted amino acid sequence of the WM 1 protein is shown below the l-3 open reading frame. The sites of transcriptional initiation (v) and termination (A), as determined by primer extention and nuclease Sl mapping, are indicated. Nucleotide sequences previously suggested to be involved with VV immediate-early gene expression have been underlined (Yuen et a/., 1987; Rohrman et a/., 1986). The predicted binding site for the MS4 oligonucleotide probe has been bracketed.

methods were used. Probe fragment 1 (HindlllBarnHI) was 3’-end-labeled and used to map the 3’-end of the l-3 transcript approximately 365 nucleotides to the left of the BarnHI site (Fig. 5C). The 5’-end of the l-3 transcript was mapped using a 5’-labeled synthetic oligonucleotide of antimessage polarity which hybridized to the region marked as probe 3 in Fig. 5A. This probe was used in a primer extension reaction, and generated a doublet of 5’-ends (Fig. 5D) which map to posi-

tion -15 and -16 relative to the first codon of the l-3 orf. It is not obvious from this analysis if the doublet represents heterogeneity in the site used for transcriptional initiation of the l-3 RNA or is an artifact due to reverse transcriptase partially copying the cap structure (Ahlquist and Janda, 1984). The 5’- and 3’ends of the l-3 transcript which were determined in these experiments have both been indicated along with the nucleotide sequence in Fig. 4.

VV RIBONUCLEOTIDE

Translational

REDUCTASE

analysis

LARGE SUBUNIT

127

sages were specifically hybridized to plasmid DNA from VV CHX RNA (lane P) or to the internal l-3 BarnHI-Kpnl fragment from VV late RNA (lane L). In contrast, the indicated fragment selected mRNA which when translated gave rise to 86- 65-, and 30kDa proteins. The 86-kDa protein corresponds to the

The coding potential of the 2800-base l-3 transcript was assessed by the use of hybrid-selected cell-free translation procedures (Fig. 6). Other than the bands due to endogenous reticulocyte proteins, no mes-

(A) PROBE STRATEGY Hind111 IIIIIIIIIlII

’

BamHI

I

KpnI

I

I

I-2

I-l

IIIIIIII I-3

M-4

2

* 3

1

(B)

E

CC)

C

CD)

L

ACGT

1652

517 506

3234-

+365 2114-

876-

FIG. 5. Transcriptlonal analysis of the VV l-3 gene. (A) A schematlc diagram of the left portion of the VV HindIll I DNA fragment is shown together with the predicted transcriptional units. The 3’.end of probe fragment 1 (HindIll-BarnHI) was asymmetrically labeled at the BamHl site using the Klenow fragment of DNA polymerase. Probe fragment 2 was uniformly labeled by nick-translation. Probe 3 was a 19-mer oligonucleotide of antimessage polarity which was labeled at the 5’.end using polynucleotide kinase. (B) Northern blot of VV early (E), cycloheximide-amplified (C), or late RNA using probe 2. (C) VV late (L) or cycle (C) RNA was used to protect probe 1 from nuclease Sl digestion and the protected fragments were analyzed by gel electrophoresls. The numbers at the left correspond to the fragments (in bp) of a pBR322:Hinfl digest which were Included as size markers. (D) A 19.mer oligonucleotide which was complimentary to positions 292-3 10 of the sequence in Fig. 4 was used as a primer to extend from W cycle RNA and for dideoxy sequencing reactions from an Ml 3 template with an insert corresponding to the 5’.end of the l-3 gene. The products of both reactions were subjected to electrophoresis under denaturing conditions. The deduced nucleotide sequence IS shown at the right with the start sites indicated (*).

TENGELSEN

128 P

L

c

-97.4 -6Kd -68.0

-25.7

ET AL.

ther of these pieces of data prove that the l-3 gene product is the large subunit of VV-encoded ribonucleotide reductase. To address this question, the predicted amino acid sequences of the VV l-3 and mouse M 1 proteins were compared (Fig. 7). The two proteins shared similar or identical amino acid residues at 72.5% of the positions. Furthermore, cysteine residues that have been found to be conserved in all large subunit sequences reported to date were present in the same regions in the l-3 protein u, Fig. 7). In addition, nine other cysteine residues (2, Fig. 7) were exactly conserved between the mouse Ml sequence and the VV l-3 protein. DISCUSSION

FIG. 6. Hybrid-selected cell-free translation of l-3 gene product. W cycle (C) or late (L) RNA was hybridized to filters containing immobilized pUC13:BamHI-Kpnl (see Fig. 5A) plasmid DNA. As a negative control, cycle RNA was also hybridized to a filter containing pUC13 DNA (P). Bound RNA was eluted, translated in a rabbit reticulocyte lysate, and the products were analyzed by SDS:polyacrylamide gel electrophoresis. Numbers at the right indicate the positions of standard protein markers (in kDa). The arrow indicates the position of the putative l-3 gene product.

predicted molecular weight of the I&encoded VV M 1 subunit. The source of the RNA giving rise to the other two proteins is unknown. The two most probable sources are (1) premature termination products of the cell-free translation system due to structural features of the l-3 mRNA or (2) hybrid selection of other mRNAs which contain sequences which cross hybridize with l-3 sequences, The first possibility seems most likely for several reasons. First, only a single major 2800base transcript was detected by Northern blot hybridization (Fig. 5A). Second, increasing the stringency of conditions used to hybrid select RNA did not alter the ratios of the encoded polypeptides (data not shown). Third, the amino terminus of the l-3 or-f which would be capable of encoding a 30-kDa protein is extremely rich in methionine residues (13 of the 26 found in the l-3 orf) which would agree with the labeling pattern seen in Fig. 6. Similarity

of mouse and VV Ml proteins

Although the genomic location of the putative VV M 1 subunit has been mapped by hybridization with the MS4 oligonucleotide probe, and the protein which is encoded by the l-3 gene at this location was similar in size to E. co/i and mouse large subunit proteins, nei-

The experiments reported here provide data concerning the location, structure, and expression of the VV gene encoding the large subunit of ribonucleotide reductase. Using a degenerate oligoncleotide probe, the VV M 1 gene was mapped to the l-3 transcriptional unit near the left end of the VV HindIll I DNA fragment (Figs. 1-3). Sequence analysis of this region of the viral genome revealed an open reading frame encoding an 86-kDa protein running in the leftward direction (Fig. 4). The regions flanking the l-3 open reading frame contained nucleotide sequences implicated in regulation of expression of VV immediate-early genes (Yuen et al., 1987). ln viva studies confirmed that the VV l-3 gene was expressed prior to DNA synthesis and its transcription was amplified in the presence of cycloheximide (Fig. 5). Hybrid-selected cell-free translation of the l-3 transcript demonstrated that it could be translated into an 86-kDa protein (Fig. 6). Computer analysis of the predicted amino acid sequence of the VV l-3 protein indicated that it is quite similar to the mouse Ml protein and contains conserved cysteine residues, thought to be essential for enzymatic activity (Thelander, 1974) (Fig. 7). These data suggest that the VV l-3 gene encodes the regulatory M 1 subunit of the VV ribonucleotide reductase enzyme, although final proof awaits confirmation by genetic means. It was of interest to note that hybridization of VV immediate-early RNA with an internal l-3 DNA fragment selected mRNA which was translated into three proteins (86-, 65- and 30-kDa) despite efforts to carry out the hybrid selection at high stringency. Explanations for this unexpected result include premature termination by the cell-free system, cross-hybridizing RNA species encoded elsewhere in the genome, or perhaps the presence of a second “silent” cistron within the I-3-encoded mRNA. The latter possibility seems unlikely since computer analyses of all three l-3 reading frames do not reveal a second reading frame

VV RIBONUCLEOTIDE

REDUCTASE

sufficient in size to encode either the 30- or 65-kDa protein. When the internal l-3 DNA fragment used to hybrid select RNA was used as a probe in Southern blot hybridization of restriction enzyme digests of VV DNA, only the VV Hindlll I region was detected (data not shown). This indicates that it is unlikely that l-3 sequences are duplicated either entirely or in a cryptic form elsewhere in the genome. It can be noted in Fig. 55 that there is a small amount of a larger 4000-base

MLJSMl WMl MUS Ml WMl

transcript detected in the VV CHX RNA by the l-3 probe. This band was ascribed to either read-through of the l-3 transcript through the downstream gene or read-through of the upstream gene through the l-3 gene. If this were true, the RNA would have been selected by the l-3 DNA fragment used in the hybrid selection experiment and could potentially be the source of the 65- and 30-kDa bands. In view of the relative quantities of the two messages versus the rel-

60 1 MHVIKRDGRQERVMFDKITSRIQKLCYGLNMDFVDPAQITMKVIQGLYSGVTTVELDTLA * *Jr**** xx ****************** * *** *******************X*** 60 1 AETAATLTTKHPDYAILAARIAVSNLHKETKKVFSDVMEDL~INPHNG~SPMVASST 120 ** *** **************************************%* ********** * 120 61

61

MUS Ml 121 W Ml

129

LARGE SUBUNIT

121

~~~~KDRLNSAIIYDRDFSYNYFGFKTLERSYLLKINGKVAERPQHMLMRVSVGIHK 180 ****Jr* ************************** ** **************** 180 MDIVNKYKDKLNSVIIYERDFSYNYFGFKTLEKSYLLKINNKIVERPQHMLMRVAVGIHQ

EDIDAAIETYNLLSEKWFTHSPTLFNAGTNRPQLSS~FLLS~DDSIEGIYDT~Q~AL 240 ****%*********%******%*******%% ***-k-k**%%--* *********-ii**-%%-'--'240 181 WDIDSAIETYNLLSEKWFTHASPTLFNAGTSRHQMSSFFLLNMIDDSIEGIYDTLKRCAL

MUS Ml 181 W Ml

MUS Ml 241 W Ml

241

MUS Ml 301 W Ml

301

ISKSAGGIGVAVSCIRATGSYIAGTNGNSNGLVPMLRVYNPGAFAIY *** ********* ************* **********************%**** **-': ISKMAGGIGLSISNIRASGSYISGTNGISNGIIPM~~NT~YIDQGG~PGV~IY

300 300

LEPWL-DIFEFLDLKKNTGKEEQRARDLFFALWIPDLFMKRVETNQDWS~QPNE~PGLD * .'-**** ***.'-*Q.'--'< 359 **** ** *********************%%********* LEPWHSDIMAFLDLKKNTGNEEHRTRDLFIALWIPDLFMKPGLD 360

. MUS Ml 360 W Ml

I~P~I~~~~~ESYEKQGRVRKWKAqqLWYAIIESQTETGTPmLYKDS~NRKSNQQNL 419 **** * * ***** ** ****** ***************%;t*-'-*-'-* 420 361

MUS Ml 420 W Ml

421

MUS Ml 480 W Ml

480

MUS Ml 540 W Ml

540

MUS Ml 600 W Ml

600

479 GTIK~SNL~TEIVEYTSKDEVAV~NNLASLALNMYNTPEHTYDFEKLAEVTKVIVRNLNKI **************** ****;~*********** * *-** ** * *****.'-.+.+.'--'GTIKFSNLFTEIIQYADANEVAVFNLASVALNMFVIDGR-FDFLKIl
MUS Ml 660 ACNGSIQSIPEIPDDLKQLYKTVWEISQKTVLKMAAERGAFIDQSQSLNIHIAEPNYGKL 719 I 1 ************~~*****~-~~~~~~~~*~~*********~~*****~~*~~***~-~~-~~ W Ml 660 ~DG~~~;;~-TNLPEDIKTIWEIPQKTII~DRGAFIDQSQS~IHIADPSYSKL 718 779 MUS Ml 720 TSMHFYGwKQGLKTGMYYLRTRPAANPIQFTLNKEKLKDKEKALKEEEEKERNTAAMV& ***.2.> *************** ************ ******** 761 W Ml 719 TSMHFYGWSLGLKTGMWLRTKPASAPIQFTLDKDKIK----------------PPWC-

MUS Ml 780 W Ml

762

2 2 LENREECLMCGS *** *** --DSEI$TS$SG

791 771

FIG. 7. Similarity between VV and mouse Ml polypeptides. The predicted amino acid sequence of the l-3 gene product is aligned against the amino acid sequence of the mouse enzyme. Positions at which identical of equivalent amino acids occur are denoted by asteriks. Cysteine residues conserved between M 1 proteins from VV , mouse, f. co/i, and EBV are indicated (1) as are those which are conserved only between VV and mouse proterns (2).

TENGELSEN

130

ative quantities of the translation products, the 4000base message would have to be postulated to be a much more efficient message. In any case, to definitively address what relationship, if any, the three polypeptides have to one another, it will be necessary to microsequence their amino termini and/or generate M 1-specific antisera and test their immunoreactivity. The genomic location of the VV ribonucleotide reductase M2 subunit has recently been determined and its nucleotide sequence determined (Slabaugh et al., 1988). Together with the data presented here, this should provide the opportunity to use molecular genetic approaches to address a number of interesting questions, including how VV ribonucleotide reductase interacts with other VV replicative enzymes during DNA synthesis; the role of both the Ml and M2 subunits in catalysis and/or regulation of enzymatic activity; whether these proteins are essential for the VV replicative cycle in tissue culture or in the animal; and whether either of the subunit genes would be an appropriate insertion site for expressing foreign gene products (Smith and Moss, 1984). ACKNOWLEDGMENTS We thank E. M. Wilson for excellent technical assistance and C. K. Mathews for useful discussions. The oligonucleotides used for this project were synthesized by the Center for Gene Research Central Services Facility at Oregon State University. This work was supported by Public Health Service grants (Al-21335, Al-24594, and GM-37508), a Research Career Development Award (Al-0666) to D.E.H. from the National Institutes of Health, and a Patricia Harris fellowship to L.A.T. This is Oregon Agricultural Experiment Station Technical Paper No. 8413.

REFERENCES AHLQUIST, P., and JANDA, M. (1984). cDNA cloning and in vitro transcription of the complete brome mosaic virus genome. ILlol. Cell. Biol. 4, 2876-2882. BARBOSA, E., and Moss, B. (1978). mRNA (nucleoside-2’-)-methyltransferase from vaccinia virus. J. Biol. Chem. 253, 7698-7702. CARAS, I. W., LEVINSON, B. B., FABRY. M., WILLIAMS, S. R., and MARTIN, D. W. (1985). Cloned mouse ribonucleotide reductase subunit Ml cDNA reveals amino acid sequence homology with Escherichia co/i and herpesvirus ribonucleotide reductases. 1. Biol. Chem. 260, 7015-7022. CONDIT, R., MOTYCZKA, A., and SPIZZ. G. (1983). Isolation, characterization and physical mapping of temperature sensitive mutants of vaccinia virus. Virology 128, 429-443. DALES, S., and POGO, B. G. T. (1981). ln “Biology of Poxviruses” (D. Kingsbury and H. Zurhausen, Eds.). Springer-Verlag, New York. DEFILLIPES, F. M. (1982). Restriction enzyme mapping of vaccinia virus DNA. J. Viral. 43, 136-l 49. ENGSTROM, Y., ROZELL, B., HANSSON, H.-A., STEMME, S., and THELANDER, L. (1984). Localization of ribonucleotide reductase in mammalian cells. EMBO J. 3, 863-867. ESPOSITO.J., CONDIT, R., and OBIJESKI,J. (1981). The preparation of orthopoxvirus DNA. J. Viral. Methods 2, 175-l 79.

ET AL. GIBSON, T., STOCKWELL, P., GINSBERG, M., and BARRELL, B. (1984). Homology between two Esptein-Barr virus early genes and herpes simplex virus ribonucleotide reductase and 38K genes. Nucleic Acids Res. 12, 5087-5099. GOTOH, 0. (1987). Pattern matching of biological sequences with limited storage. CAB/OS 3, 17-20. GRASLUND, A., SAHLIN, M., and SJOBERG,B. M. (1985). The tyrosyl free radical in ribonucleotide reductase. Environ. Health Perspect. 64, 139. HRUEY, D. E., and BALL, L. A. (1981). Cell free synthesis of enzymatically active vaccinia virus thymidine kinase. Virology 113, 594-601. HRUBY, D. E., and BALL, L. A. (1982). Mapping and identification of the vaccinia virus thymidine kinase gene. 1. Viral. 43, 403-409. HRUBY, D. E., and GUARINO, L. A. (1984). Novel codon utilization within the vaccinia virus thymidine kinase gene. Virus Res. 1, 315-320. JONES, E. V., and Moss, B. (1984). Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA. 1. Viral. 49, 72-77. JONES,E. V., PUCKETT,C., and Moss, B. (1987). DNA dependent RNA polymerase subunits encoded within the vaccinia virus genome. /. Virol. 61, 1765-1771. LAMMERS. M., and FOLLMANN, H. (1983). The ribonucleotide reductases-A unique group of metalloenzymes essential for cell proliferation. Struct. Bonding (Berlin) 54, 27-91. LANKINEN, H., GRASLUND,A., and THELANDER,L. (1982). Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus. J. Viral. 41, 893-900. LEHRACH, G., DIAMOND, D., WOXNEY, J. M., and BOEDTKER,H. (1977), RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16, 4743-4752. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor NY. MASON, P. J., and WILLIAMS, J. G. (1985). “Nucleic Acid Hybridization: A Practical Approach” (E. D. Hames and S. 1. Higgins, Eds.). p. 129. IRL Press, England. Moss, B. (1985). Replication of Poxviruses. ln “Virology” (B. Fields, D. M. Knipe, R. M. Chanock, J. L. Melnick, B. Roizman, and R. E. Shope, Eds.), pp. 685-704. Raven Press, New York. MOUNT, D. W., and CONRADE, B. (1986). Improved programs for DNA and proteins on the IBM-PC and other standard computer systems. Nucleic Acids Res. 14, 443-454. MUNYON, W., PAOLETTI, E., OSPINA, J., and GRACE, J. T., JR. (1968). Nucleotide phosphohydrolase in purified vaccinia virus. 1. Viral. 2, 167-172. PERKUS, M. E., PANICALI, D., MERCER, S.. and PAOLE~I, E. (1986). Insertion and deletion mutants of vaccinia virus. Virology 152, 285-297. REICHARD, P. (1987). Regulation of deoxyribotide synthesis. Biochemistry 26, 3245-3248. ROHRMANN, G., YUEN, L., and Moss, B. (1986). Trranscription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell 46, 1029-l 035. ROSEL,J. L., EARL, P. L.. WEIR, J. P., and Moss, B. (1986). Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the Hindlll H genome fragment. J. Viral. 60, 436-449. SANGER, F., NICKLEN, S., and COULSON, A. E. (1977). DNA sequenc-

VV RIBONUCLEOTIDE

REDUCTASE

Ing with chain-terminating Inhlbltors. Proc. /Vat/. Acad. Sci. USA 74, 5463-5467. SHUMAN, S., BROYLES,S. S.. and Moss, B. (1987). Purification and characterization of a transcription termination factor from vaccinia virions. /. Viol. Chem. 262, 12372-l 2380. SLABAUGH,M. B.. and MATHEWS, C. K. (1984). Vaccinia virus-induced ribonucleotide reductase can be distinguished from host cell activity. 1. Viral. 52, 501-506 SLAEIAUGH,M B., JOHNSON, T. L., and MATHEWS, C. K. (1984). Vaccinia virus Induces ribonucleotide reductase in primate cells. J. Viral. 52, 507-514. SLABAUGH, M. B., ROSEMAN, N., DAVIS, R., and MATHEWS, C. K. (1988). Vaccinia virus-encoded ribonucleotide reductase: Sequence conservation of the gene for the small subunit and its amplification in hydroxyurea-resistant mutants. /. Viral. 62, 519-527. SMITH, G. L.. and Moss, B. (1984). Vaccinia virus expression vectors: Construction, propertles, and applications. BioTechniques 1, 306-312. STUDIER, F. W. (1973). Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248. THELANDER, L. (1973). Physicochemlcal characterization of ribonucleoside dlphosphate reductase from fscherichia co/i. J. Viol. Chem. 248, 4591. THELANDER, L (1974). Reaction mechanism of ribonucleoslde di-

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phosphate reductase from Escherichia co/i. Oxldation-reductionactive disulfides In the Bl subunit. J. Biol. Chem. 249, 4858. THELANDER, L., and REICHARD, P. (1979). Reduction tides. Annu. Rev. Biochem. 48, 133-l 58.

of ribonucleo-

THELANDER, L., ERICKSSON,S., and AKERMAN, M. (1980). Ribonucleotide reductase from calf thymus, separation of the enzyme into two non-identical subunits, proteins Ml and M2. /. Biol. Chem. 255, 7426-7432. TRAKTMAN, P., SRIDHAR. R., CONDIT, C., and ROBERTS,B. E. (1984). Transcnptional mapping of the DNA polymerase gene of vacclnia virus. 1. Viroi. 49, 125-l 31. WEINRICH, S. L., and HRUBY, D. E. (1987). Noncoordinate regulation of a vaccinla virus late gene cluster J. Viroi. 61, 639-645. WEINRICH, S. L., NILES, E. G., and HRUBY, D. E. (1985). Transcriptional and translational analysis of the vaccinia virus late gene L65. J. VifOl. 55, 450-457. WEIR, J., and Moss, B. (1983). Nucleotide sequence of vacclnia virus thymidine klnase gene and the nature of spontaneous frameshift mutations. /. Vfroi. 46, 530-537. YUEN, L., DAVISON, A. J., and Moss, B. (1987). Early promoter-binding factor from vacclnia vlnons. Proc. Nat/. Acad. Sci. USA 84, 6069-6073.