Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase

Gene, 122 (1992) 281-288 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

281

0378-l 119/92/$05.00

06819

Lack of evidence virus polymerase

for proofreading

(Error-prone replication; 3’ + 5’ exonuclease; virion; base substitutions)

David A. Steinhauer”, Department

Esteban

nucleoside

Domingo

of Biology and Institute for Molecular

Genetics,

associated

mechanisms

triphosphate

analogues;

pyrophosphate

with an RNA

exchange;

next nucleotide;

and John J. Holland University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116.

USA.

Tel. (619)534-2520 Received

by M. Salas:

19 May 1992; Revised/Accepted:

14 August/l7

August

1992; Received

at publishers:

1 September

1992

SUMMARY

The in vitro fidelity of the virion-associated RNA polymerase of vesicular stomatitis virus was quantitated for a single conserved viral RNA site and the usual high in vitro base misincorporation error frequencies (approx. 10e3) were observed at this (guanine) site. We sought evidence for RNA 3’ 45’ exonuclease proofreading mechanisms by varying the concentrations of the next nucleoside triphosphate, by incorporation of nucleoside[ 1-thioltriphosphate analogues of the four natural RNA nucleosides, and by varying the concentrations of pyrophosphate in the in vitro polymerase reaction. None of these perturbations greatly affected viral RNA polymerase fidelity at the site studied. These results fail to show evidence for proofreading exonuclease activity associated with the virion replicase of an RNA virus. They suggest that RNA virus replication might generally be error-prone, because RNA replicase base misincorporations are proofread very inefficiently or not at all.

INTRODUCTION

In recent years, it has become clear that most RNA viruses (both riboviruses and retroviruses) are highly mutable, very adaptable and capable of rapid evolution. This is largely due to error-prone replication producing mutation

Correspondence to: Dr. E. Domingo Biologica

Molecular,

28049 Madrid *Present

Universidad

(Spain).

address:

Abbreviations: kb, kilobase thioltriphosphate) phates; merase virus.

Tel. (34-1)397-8485;

National

way, Mill Hill, London,

at his present

Autonoma

Institute

address:

de Madrid,

Centro

Canto

de

Blanco

Fax (34-1)397-4799.

for Medical

Research,

The Ridge-

NW1 lAA, UK. Tel. (44-081)959-3666.

bp, base pair(s); DI particle,

defective interfering

or 1000 bp; nt, nucleotide(s); NTPorS, analogues of the indicated ribonucleoside

particle; 5’-O-([ ltriphos-

oligo, oligodeoxyribonucleotide; PolI (III), E. coli DNA polyI (III); PPi, inorganic pyrophosphate; VSV, vesicular stomatitis

frequencies so high that even clones of RNA viruses consist of populations of related, but differing variants (quasispecies populations). For recent reviews, see Domingo and Holland (1988) Coffin (1986; 1992) Strauss and Strauss (1988), Temin (1989), Domingo et al. (1992), Holland et al. (1992), Williams and Loeb (1992) and WainHobson (1992). It is generally believed that most RNA polymerases (both viral and cellular) are error-prone mainly because RNA synthesis lacks the proofreading and mismatch repair systems that promote high fidelity replication of DNA. In the case of DNA synthesis, nt misincorporation frequencies, although variable, are generally quite high, and proofreading and mismatch repair are required to achieve overall high fidelity of DNA replication. (For reviews and references see Loeb and Kunkel, 1982; Preston et al., 1988; Bebenek and Kunkel, 1990; Radman and Wagner, 1986; Lahue et al., 1989; Moses and Summers, 1988; Fry and Loeb, 1986; Kunkel, 1988). Proofreading

282 can correct nt misincorporations by up to orders of magnitude, and mismatch repair can further increase fidelity by orders of magnitude via repeated scanning of DNA duplexes and replacement of mismatched nt. Mismatch repair processes are not possible for the genomes of most RNA viruses because they replicate and package single-strand RNA molecules. Therefore, one major mechanism for correction of misincorporated nt would be a replicaseassociated proofreading function, but RNA proofreading has not yet been proved (nor disproved) for any RNA polymerase nor RNA virus replicase, and other (nonexcision) mechanisms have been proposed for possible RNA proofreading. DNA proofreading functions are polymerase-associated 3 ’ -+ 5’ exonucleases which remove newly-misincorporated nt from the growing points of nascent DNA chain (Brutlag and Kornberg, 1972; Muzyczka et al., 1972) and they have been well-characterized for a variety of DNA polymerases and polymerase complexes (Kunkel, 1988). Proofreading studies with a number of DNA polymerases demonstrate the expected effects on fidelity when the elongation rate is altered by varying the concentration of the next (correct) nucleoside triphosphate or the PPi concentration (Clayton et al., 1979; Fersht, 1979; Kunkel et al., 1981a; 1986; Abbots and Loeb, 198.5; Loeb and Kunkel, 1982). Another hallmark of most DNA proofreading is the ability of deoxynucleoside thiotriphosphates to abolish DNA proofreading because they cannot be excised after misinsertion (Kunkel et al., 1981b; Loeb and Kunkel, 1982). However, bacteriophage T4 DNA polymerase is capable of excising thiotriphosphates at least under idling conditions (Gupta et al., 1982) so that not all proofreading functions are completely inhibited by these thio analogues. Among RNA polymerases, very little is known regarding potential proofreading mechanisms. Ishihama et al. (1986) reported a ‘proofreading’ function for influenza virus RNA polymerase. However, this involved only the 3’+5’ exonucleolytic removal of excess G residues which had been added to cannibalized, capped host-cell RNA prints in vitro, and it is not yet known whether this is involved in true proofreading during virus RNA replication. Kasavetis et al. (1986) have shown that TLC-modified RNA polymerase of Escherichia coli can catalyze pyrophosphorylytic excision of newly-incorporated ribonucleotides during transcription. Ward et al. (1988) observed that poliovirus RNA polymerase exhibited decreased fidelity in copying homopolymer RNA templates when elongation rates were increased. In contrast, the elongation rate of avian myeloblastosis virus reverse transcriptase can be greatly altered without effect on fidelity (Kunkel et al., 1981a). In the present study, we utilize the virion-associated RNA polymerase of VSV DI particles to attempt detection of RNA virus polymerase proofreading functions. This RNA polymerase becomes

activated to copy its natural viral nucleoprotein templates upon addition of nonionic detergents to highly purified virions in in vitro reaction mixtures (Baltimore et al., 1979). We employed purified VSV DI virions because they synthesize a unique 46-nt RNA transcript in vitro (Reichmann et al., 1974; Emerson et al., 1977; Semler et al., 1978; Schubert et al., 1978). The sequence of this unique RNA transcript allows accurate determination of nt misincorporation frequencies at a single internal G residue (Steinhauer and Holland,

RESULTS

1986; Steinhauer

et al., 1989).

AND DISCUSSION

(a) Rationale Our rationale was to employ the in vitro RNA polymerase activity of purified VSV DI particles to synthesize a small leader RNA, then to measure polymerase misincorporations at a single defined nt site in this leader RNA product under conditions known to affect DNA proofreading. The major classes of DI particles of VSV are subgenomic deletion mutants of infectious virus. They depend for their replication on the replication and maturation proteins encoded by their nondefective, infectious parental helper viruses since these DI particles are replicative entities which cannot transcribe any viral mRNAs (Huang and Baltimore, 1970). The virion-associated RNA polymerase of these DI particles is a replicase which copies full-size DI particle genome RNA in vivo, but in vitro it produces a 46-nt ‘minus-strand leader RNA’ which is complementary to the DI genome 3’ terminus (Reichmann et al., 1974; Emerson et al., 1977; Semler et al., 1978; Schubert et al., 1978). The sequence of this 46-nt RNA is shown in Fig. 1. The arrows indicate the sites after each G residue at which Tl RNase cleaves to form the two consensus (21-nt and 13-nt) oligos. Base substitutions replacing the single central G residue (large arrow) will prevent Tl RNase cleavage and will cause formation of an error oligo 34 nt in length (Steinhauer and Holland, 1986; Steinhauer et al., 1989). The ratio of 32P counts in error oligos to those in consensus oligos allows estimation of the frequencies of nt substitutions at this single G site. We utilized this DI particle virion replicase system to seek evidence for an associated proofreading function analogous to those characterized for DNApolymerases (Loeb and Kunkel, 1982; Kunkel, 1988). We earlier documented very high nt substitution frequencies (10 - 3 to 10 - 4, in this system (Steinhauer and Holland, 1986), and similar results have been demonstrated for poliovirus RNA polymerase (Ward and Flanegan, 1992). (b) Varying PPi levels do not affect polymerase fidelity Because PPi is a product of DNA and RNA polymerase catalysis, its presence at high concentrations might result

283

l-

13mer -

-I

2,. mer

-

1 1 1 ACGhAGACCACAAAACCAGAUAAAAAAUAAAAACCACAAGAG’GkhC I

t

34mer I

Fig. 1. Sequence

of the 46-nt leader RNA synthesized

from purified virions of VW DI particles.

by nucleoproteins

It is complementary

to the 3’

terminal 46 nt of the template DI particle genomes. The arrowheads after each G residue show the sites at which Tl RNase cleaves this RNA. The thick arrow denotes the cleavage nt substitution oligo’. Methods: hamster

site following the central G residue. Any

at this site leads to the appearance VW

and its DI particles

cells in Eagle’s minimum

essential

inactivated

bovine calf serum. DI particles

differential

centrifugation

as described

as described

(Briendl

were harvested

et al., 1974; Holland templates

in sucrose gradients were prepared

on discontinuous

and Holland,

5% heat-

and purified by

et al., 1976). DI nucleo-

for transcription

purified DI particles by sedimentation

in BHK,,

medium containing

and velocity sedimentation

(Reichmann

capsid (ribonucleoprotein)

comprised

of a large 34-nt ‘error

were replicated

from

glycerol gradients

1975) except that 75% v/v glycerol

the lower layer. Aliquots

of purified

DI nucleocapsids

were

stored as aliquots frozen at -70°C and these were used after a single freeze-thaw cycle. VSV DI particle nucleocapsids were used as ribonucleoprotein-RNA

polymerase

natural

template

complexes,

i.e.,

VSV and its DI virions contain virion polymerase molecules associated with their nucleoprotein templates (Baltimore et al., 1970). Purified DI particle

nucleocapsids

containing

template

were incubated

mixture

containing

300 mM K.acetate/3 ribonucleoside

at 30°C

50 mM

aliquots

of about

Tris.acetate

mM dithiothreitol

triphosphates

pH

in a reaction

8.2/8 mM

Mg.acetate/

and concentrations

as indicated.

were added only where indicated.

3 to 10 pg of RNA

for the times indicated

of all four

PPi and NTPa5

Reactions

were stopped

analogues

by addition

of

25 mM EDTA/O.S% sarkosyl and 1 mg proteinase K/ml. This was followed by incubation at 37” C for 15 min and then at 45’ C for 10 min prior to extraction

with phenol/chloroform

ous phase was chromatographed

(1: 1). RNA recovered through

in the void volume. After concentration nt minus-strand

replicase

it from trace product

the gels, ethanol-precipated excess of concentrated 1986; Steinhauer

mined by Cerenkov

amounts

RNAs.

of early termination

and

This 46-nt RNA was eluted from

and dried, then thoroughly

RNase Tl as described product

digested

(Steinhauer

with an

and Holland,

oligos were fractionated

gels and the relative amounts counting

the 46-

was purified on 20% polyacryla-

et al., 1989). Digestion

in 20% polyacrylamide

in the aque-

G-75 and recovered

by ethanol precipitation,

leader RNA product

mide gels to separate readthrough

Sephadex

of excised gel bands

of each were deteras described

(Stein-

hauer et al., 1989) except that one-dimensional gel fractionation was employed. The nt substitution frequencies were estimated from the ratio of counts in the 34-nt ‘error oligo’ (not cleaved by Tl RNase at the selected G site) to the total counts

in the consensus

(non-error)

21-nt plus 13-nt

oligos. By estimating the mutation frequency at this site in the absence and in the presence of agents known to affect proofreading activity, it should be possible emphasized

to demonstrate the presence of such activity. It should be that all of the 3zP nt incorporation data represent rates of

incorporation. Incorporation of nt by VSV DI nucleocapsid-polymerase complexes (and infectious virus complexes) in vitro remains linear for hours (Reichman et al., 1974; Briendl and Holland, tions were stopped during linear incorporation.

polymerase

tion (Hopfield,

1974; Ninio,

selectivity

prior to incorpora-

1975; Loeb et al., 1981; Loeb

and Kunkel, 1982; Kunkel et al., 1986), generally predict that added PPi should increase nt substitution frequencies.

t

I

which postulate

1975), and all reac-

in decreased rates of chain elongation and decreased fidelity. This has been observed for PolI on natural DNA templates, but not on copolymer DNA templates (Kunkel et al., 1986; Loeb et al., 1981). Kinetic proofreading models,

Other models may predict the opposite (Hopfield, 1980). Doubleday et al. (1983) reported increased fidelity for PolI and DNA polymerase CIwhen 1 mM PPi was added. Also, Abbotts and Loeb (1985) and Kunkel and Loeb (1981) observed increased replicative accuracy for DNA polymerase a upon PPi addition. It is conceivable that in some systems, PPi might increase the ratio of PPi exchange vs. incorporation for mismatched nt vs. correct nt. In contrast to reports of decreased or increased fidelity resulting from PPi addition, Kunkel et al. (1986) observed no effect of PPi on the fidelity of the avian myeloblastosis virus DNA polymerase (reverse transcriptase) which apparently lacks any proofreading function. We show below that fidelity of VSV RNA polymerase is likewise unaffected by PPi addition. Fig. 2A shows one example of a number of experiments in which increasing amount of PPi to VSV polymerase template complexes caused decreasing rates of RNA chain elongation, as expected. However, Fig. 3A shows that the rate of misincorporation at the selected G site did not vary detectably over a wide range of PPi concentrations despite the decreased rates of incorporation. The two lower, heavy bands in these 20% polyacrylamide gels are the two correct consensus oligos of 13 and 21 nt, and the faint top band is the 34-nt ‘error oligo’ which results from substitution of any nt in place of the correct G at this position. In a separate experiment, we excised the gel bands and carefully quantitated the amounts of 32P in each to determine the ratios of error nt (34 nt) to consensus nt (13 plus 21 nt). Comparison of these ratios in Table IA (controls) with those in Table IB (added PPi) shows no obvious effect on fidelity (in confirmation of the apparent lack of variation in relative band intensities in Fig. 3). Thus, PPi neither greatly increases, nor decreases VSV polymerase fidelity at this single site. (c) Next nt effect on VSV polymerase fidelity in vitro Because proofreading involves 3’ +5’ exonuclease excision of misincorporated nt at the growing point, competition between excision and chain elongation should significantly affect error frequencies. Unless processivity is affected, higher rates of chain elongation beyond a misincorporated nt should tend to lock-in errors, and consequently next-neighbor effects on fidelity are a hallmark of proofreading (Loeb and Kunkel, 1982). For example, Clayton et al. (1979) and Fersht (1979) observed decreased fidelity when the concentration of correct next-neighbor nucleotide triphosphate was increased using T4 DNA polymerase on homopolymer DNA templates or PolIII on $X174 DNA, respectively. Likewise, Kunkel et al. (1981a)

284

6-

1

pp cont. Fig. 2. Effect of PPi addition

concentration

of ATP ‘next nt’ (B) on the rates of synthesis

Methods: all polymerase

reactions

EDTA/O.S%

of ATP and 10 PCi of [a-32P]CTP sarcosyl

and 1 mg proteinase

was phenol/chloroform-extracted, cpm quantitated

by Cerenkov

K/ml).

ethanol-precipitated, counting.

for labeling. Following

were carried

viral polymerase mM K,acetate/3

purified on 20% polyacrylamide

COW. (PM)

of 46-nt leader RNA by nucleoproteins

from

remain linear for

associated with approx. 3 to 10 pg of DI genome RNA template) mM dithiothreitol/50 PM UTP, GTP and CTP plus the indicated reactions

were stopped

at 37°C for 15 min for proteinase

(A) Sodium PPi was added to each reaction

500

out for 20 min at 30°C (rates of RNA synthesis

After 20 min, polymerase

incubation

1

300 ATP

hours). Equal aliquots of VSV Di particle nucleoproteins (containing were incubated in 50 mM Tris.acetate pH 8.2/8 mM Mg.acetate/300 concentrations

I

100

3

(mM)

(A), or varying

purified virions of VSV DI particles.

I

I

2

gels, visualized

by addition

K digestion,

of stop solution

(25 mM

the 46-nt leader RNA product

by autoradiography,

mixture at the following concentrations:

excised from the gel and 32P 0, 0.01. 0.1, 0.25, 0.5, 0.75,

1.0 and 2.5 mM. ATP concentration was 150 PM. (B) ATP (next nt) was added to each reaction mixture at the following concentrations: 25, 50, 100, 250 and 500 PM (no PPi added). Similar inhibitory effects of increasing PPi concentrations and stimulatory effects of increasing ATP concentrations were observed

in other experiments

(not shown).

observed similar results with PolI, but they observed no such next nt effect with the avian myeloblastosis virus reverse transcriptase which lacks an editing function. Again, our data below show no next nt effect on the fidelity ofVSV RNA polymerase. Fig. 2B shows one example of a number of experiments in which addition of increasing amounts of next nt (ATP) to our DI RNA polymerase-nucleoprotein template system caused increasing rates of chain elongation as expected. The Km for ATP incorporation into VSV DI particle 46-nt minus-strand leader RNA is approx. 133 PM (Beckes et al., 1987), and 26 of the 46 nt in this leader RNA are A residues (Fig. 1). However, when after RNase Tl digestion oligos were fractionated on 20% polyacrylamide gels no obvious difference in relative band intensities were observed at increasing ATP concentrations of 50 PM, 100 PM, 250 PM or 500 PM (Fig. 3B). Thus, there was no evidence for next nt (ATP) effects on fidelity at the selected G site. This was again confirmed in separate experiments by carefully excising gel bands and quantitating the ratios of error band [ 32P]oligo counts to those of the two consensus oligos. Comparison of these ratios in Table IB shows no significant variation in fidelity at ATP concentrations of 10 PM, 50 PM, 100 PM or 500 PM. We conclude that next nt concentrations exert little or no effect on polymerase fidelity at this single G site.

(d) Effect of ribonucleotide-[l-thioltriphosphates on VSV polymerase fidelity in vitro Kunkel et al. (1981b) demonstrated clearly that deoxynucleoside-[ l-thioltriphosphates prevent proofreading during in vitro DNA synthesis by PolI and bacteriophage T4 DNA polymerase. These substrate analogues have a sulfur atom in place of an oxygen atom on the a phosphorus. Although incorporated normally, the substituted diester bond is not cleaved efficiently by editing exonucleases (Vosberg and Eckstein, 1977; Burgers and Eckstein, 1980; Kunkel et al., 1981b). In contrast, DNA polymerase p and avian myeloblastosis virus reverse transcriptase (both of which lack a proofreading function) exhibited no change in fidelity during incorporation of deoxynucleoside-[ 1-thio] triphosphates (Kunkel et al., 1981b). We have tested the effect of incorporation of ribonucleoside-[ l-thioltriphosphate analogues on the fidelity of VSV polymerase in vitro and we have observed very little, if any, effect. Table ID shows that incorporation of these analogues caused only a slight decrease in fidelity. This several-fold decrease in fidelity provides no evidence for the presence of an efficient proofreading function in VSV polymerase. In contrast, thio analogues decreased the fidelity of PolI by 20-fold and of T4 polymerase by more than 500-fold (Kunkel et al., 1981b).

285 also consistent virus replicase,

with the results observed with another RNA the reverse

transcriptase

of avian myelo-

blastosis virus. This enzyme also had no obvious effects on fidelity when elongation rate was altered by next nt concentration (Kunkel et al., 1981a) or PPi concentration (Kunkel et al., 1986), or by nucleoside-[ 1-thioltriphosphate analogues (Kunkel et al., 1981b). Poliovirus RNA polymerase exhibited high error rates similar to our results, but they increased about five-fold on homopolymeric RNA templates in vitro when the elongation rate was increased eight-fold by raising the Mg2+ concentration and the pH (Ward et al., 1988). It is not clear whether this was due to an increase in primary misincorporation rates due to

Fig. 3. Lack of detectable effects of varying concentrations (Panel A) or ATP next nt (Panel B) on the proportion oligos’ synthesized

in vitro by virion polymerase

vitro reaction

conditions

concentrations

as indicated

trations

nt concentrations

of VSV DI particles.

were as for Fig. 2 except

In

for PPi and ATP

below. (Panel A) Added sodium PPi concen-

were (left to right in lanes l-7):

1 mM. ATP concentration

of added PPi of 34-nt ‘error

0, 10, 100,250,

500,750

PM and

in all was 150 PM. (Panel B) Added ATP next

were (left to right in lanes

l-4):

50, 100, 250, and

500 PM (no PPi added). The 46-nt leader RNA product of VSV DI particle virion polymerase was gel-purified as described for Fig. 1, and the Tl RNase fractionated

digestion

products

(21-nt,

on one-dimensional

8 M urea was incorporated oligos containing

13-m and 34-nt oligos) were also

20% polyacrylamide

gels, except that

into these gels to prevent band broadening

ribonucleotide-[

1-thiolanalogues.

of

In these urea gels, the

34-nt oligos produced two closely apposed narrow bands of similar intensity, both of which yield the expected 21-nt and 13-nt bands when uncut G residues were subjected

to further Tl RNase digestion.

given for 34-nt in Table I are the sum of these two separate reason

for their slight differences

The cpm

bands.

The

in gel mobility was not investigated.

Our overall results suggest that the RNA polymerase of VSV (a ribovirus) resembles the reverse transcriptase of avian myeloblastosis virus (a retrovirus) in lacking a proofreading function to excise misincorporated nt. It must be emphasized that our results do not rule out the possibility of a separate proofreading exonuclease which is not packaged with the virion or DI particle RNA replicase. Also, it should be recognized that our results are confined to one preselected site in the VSV RNA genome, and it is known that 3’+5’ exonuclease activity (Fersht and Knill-Jones, 198 1) and pyrophosphorolytic cleavage of 3’ terminal nt (Mizrahi et al., 1986) can vary with respect to sequence. However, error rates determined at several sites of the standard VSV virus genome both in vivo and in transcription products in vitro are similar to those obtained with DI particles (compare Table I and values reported by Steinhauer and Holland, 1986, and Steinhauer et al., 1989). Our data are consistent with the studies of DNA polymerases cited above in which fidelity effects due to next nt concentrations or to nucleoside-[ l-thioltriphosphates were observed only in polymerase preparations with associated 3’ +5’ exonuclease editing activity. They are

changed reaction conditions, or to some form of editing, since they did not investigate proofreading. The absence of proofreading must be based on negative biochemical evidence. In our assay, any alteration of the misincorporation rate greater than tenfold would have been noticeable, but no such changes comparable to those described for DNA polymerase were observed. It should be noted that our misincorporation assay is capable of detecting increased or decreased error rates because in vitro error rates regularly exceed in vivo error rates at the same specific site in VSV (Steinhauer and Holland, 1986). It is likely that most, if not all, riboviruses (and retroviruses) may be found to lack editing 3’ -5’ exonuclease activities. Proofreading entails significant energy costs (Fersht et al., 1982) and there is now good evidence that RNA viruses (riboviruses and retroviruses) not only tolerate high mutation frequencies, but can achieve great biological adaptability as a result (see reviews in Domingo and Holland, 1988; Coffin, 1986; 1992; Strauss and Strauss, 1988; Temin, 1989; Domingo et al., 1992; Holland et al., 1992; Williams and Loeb, 1992; Wain-Hobson, 1992). The classic studies of Drake (1969) and Drake et al. (1969), pointed out that very small DNA genomes can tolerate high mutation rates, and that the isolation of antimutator mutants of DNA bacteriophage shows that viruses in nature are probably frequently selected to have optimally high mutation rates. It is interesting that polymerases of at least some DNA animal viruses do have associated exonuclease activities. This has been shown for herpes simplex virus (Abbotts et al., 1987) and for Epstein-Barr virus (Grossberger and Clough, 1981; Tsurumi, 1991). Nevertheless, drug selection of antimutator mutants of herpes simplex virus indicates that these animal DNA viruses might be selected in nature for higher-than-necessary mutation frequencies even though the large size of their viral DNA genome apparently requires a polymerase-associated 3’ +5’ exonuclease function (Hall et al., 1984). It will be important to examine the RNA polymerases of a number of other RNA viruses for editing functions. RNA polymerases might have generally evolved without editing

286 TABLE

I

Estimation

of base substitution

frequencies

Experimental

of VSV polymerase

a

Concentrations (PM) of nucleoside triphosphates

group

Ratios

Corrected

32P cpm in 34-m gel band

substitution

32P cpm in 13-nt + 21-nt

estimates 5.5 x 1o-3

and PPi A (controls)

B (PPi added)

C (varying

1

ATP 150

1.1 x 1o-2

2

ATP 150

2.2x 1om3

1.1 x 1om3

3

ATP 150

2.6 x 10 - 3

1.3 x 1om3

1

ATP 15OjPPi 100

2x 1o-3

1x10m3

2

ATP 15OjPPi 1000

2x 1o-3

1 x 1o-3

next 1

nucleotide)

ATP 10

2

D ([1-thio]

nt

3.3 x lo-’ 1.7 x 1om3

3

ATP 50 ATP 100

4

ATP 500

1

ATP lSO/GTP/UTPaS

50;

frequency

1.6x 10m3

1.8 x 1o-3

8.5 x 1om4 9x 1om4

1.6 x 10m3

8x10m4

1.5 x 1om2

7.5 x 1om3

1.5 x 1om2

7.5 x lo-3

CTP5 + 100 pCi [m3’P]CTP

analogues) 2

ATPctS 150/GTPaS UTPzS

SO/CTPaS

50/ 5 + 100 pCi

[ z-32P]CTP a Reaction

mixtures

50 PM and

[ c(-32P]CTP (100 or 200 @/mmol)

leader

were as described

RNA product

polyacrylamidegels

was gel-purified, containing

for Fig. 2. Except as noted below, or in the table, the concentrations excised

5 PM. Reactions

were incubated

of ATP were 150 PM? GTP and UTP were

for 2 h at 30°C (except reaction

and eluted from the gels, digested

with RNase

8 M urea. The resulting 34-nt ‘error oligo’ and the two consensus

Al which was run 20 min), the 46.nt

Tl, and the product

oligos were fractionated

on 20%

(21-nt and 13-nt) oligos were visualized by autoradiography

(as in Fig. 3) excised from gels and carefully assayed for 32P (cpm) by Cerenkov counting. Background cpm from adjacent blank areas of gels were determined and subtracted from cpm found in adjacent gel bands, and then ratios of error/consensus oligo cpm were calculated. These ratios were divided by 2 to correct corrected

for approx.

nt substitution

50% remaining

frequency

estimates

uncut G residues

at the selected site (Steinhauer

are shown in the right-hand

3’ +5’ exonucleases. The energy costs ofhigh fidelity would not often be balanced by significant biological advantage because the error frequency of protein synthesis is high, often exceeding 10 _ 4 per codon (Lotfield and Vanderjagt, 1972; Edelman and Gallant, 1977; Ellis and Gallant, 1982; Rosenberger and Hilton, 1983; Lute et al., 1985).

columns.

and Holland,

Beckes,

J.D.,

Haller,

concentration

lation of vesicular

P.M.

stomatitis

virus messenger

function for the acid polymerases.

effect of ATP

virus leader RNAs

and Eckstein,

induction

synthesis

of DNA,

of the mechanism

triphosphate.

M.F., Branscomb,

and correction

by mutant

Kinetic error discrimination

XXXVI.

3’+5’ exonuclease activity in J. Biol. Chem. 247 (1972) 241-

F.: A study

ate analogs of deoxyadenosine

and trans-

RNA. Proc. Natl. Acad.

from Escherichia coli with diastereomeric

6889-6893. Clayton, L.K., Goodman, merases.

of DNA

phosphorothion-

J. Biol. Chem. 254 (1988) E.W. and Galas, D.J.: Error

and wild-type mechanisms.

T4 DNA

poly-

J. Biol. Chem. 254

(1979) 1902-1912. Coffin, J.M.: Genetic variation in AIDS viruses. Cell 46 (1986) 1-4. Coffin, J.H.: Genetic diversity and evolution of retroviruses. Curr. Top.

REFERENCES and Loeb, L.A.: DNA polymerase CIand models for proofNucleic Acids Res. 13 (1985) 261-274. Nishiyama, Y., Yoshida, S. and Loeb, L.A.: On the fidelity replication: herpes DNA polymerase and its associated exoNucleic Acids Res. 15 (1987) 1185-1198.

Baltimore, D., Huang, A.S. and Stampfer, M.: Ribonucleic sis of vesicular stomatitis virus, II. An RNA polymerase Proc. Natl. Acad.

stomatitis

Sci. USA 72 (1975) 2345. Brutlag, D. and Kornberg, A.: Enzymatic

polymerase

Abbotts, J. reading. Abbot& J., of DNA nuclease.

J.: Differential

of vesicular

and mRNAs. J. Virol. 61 (1987) 3470-3478. Breindl, M. and Holland, J.J.: Coupled in vitro transcription

248. Burgers,

This work was supported by NIH grant AI14627 to J.J.H. and by grant BI089-066%C03-02 and Fundacibn Ramon Arces to E.D.

et al., 1989), and the final

group B as indicated.

A.A. and Perrault,

on synthesis

A proofreading deoxyribonucleic ACKNOWLEDGEMENTS

1986; Steinhauer

PPi was added only for experimental

acid synthein the virion.

Sci. USA 66 (1970) 572-576.

Bebenek, K. and Kunkel, T.A.: Frameshift errors initiated by nucleotide misincorporation. Proc. Nat% Acad. Sci. USA 87 (1990) 4946-4950.

Microbial. Immunol. 176 (1992) 143-164. Domingo, E., Holland, J.J. and Ahlquist, P. (Eds.): RNA Genetics, 1-3. CRC Press, Boca Raton, FL, 1986. Domingo, E., Escarmis, C., Martinez, M.A., Mateu,

M.G.:

Foot and mouth disease

species. Curr. Top. Microbial. Doubleday, O.P., Lecomte, P.J. nucleotide

selection

associated

tivity of DNA polymerase. 489-499.

Martinez-Salas,

virus populations

Vols. E. and

are quasi-

Immunol. 176 (1992) 33-48. and Radman, M.: A mechanism with the pyrophosphate

UCLA

exchange

for ac-

Symp. Mol. Cell. Biol. 11 (1983)

287 Drake,

J.W.: Comparative

rates of spontaneous

E.O.: Spontaneous Edelman,

Emerson,

Nature

221

Nature

Deoxyribonucleoside

in E. coli. Cell 10 (1977)

J.: Mistranslation

J.: An estimate

Mol. Gen. Genet.

S.U., Dierks,

of the global error frequency

in

P.M. and Parsons,

J.T.: In vitro synthesis

of vesicular

stomatitis

of a

virus. .I.

A.R.:

Fidelity

of replication

tion. Proc. Natl. Acad.

of phage

spontaneous

+X174

DNA

mutation

by DNA

Sci. USA 76 (1979) 4946-4950.

dine, and pyrimidine:pyrimidine Fersht,

mismatches

Polymerases.

J.W. and Tsui, W.-C.: Kinetic basis of spon-

Barr

CRC Press, Boca Raton,

D. and Clough,

virus-induced

turnover,

deoxyribonucleic

processiveness

polymerase:

and phosphonoacetic

acid sensitivity.

Bio-

S.J.: Template-primer

M., Randall,

S., Almy,

R.E., Gelep, P.T. and Schalfer, P.A.: Generation of genetic diversity in herpes simplex virus: an antimutator phenotype maps to the DNA polymerase

locus. Virology

J.J., Villarreal,

generation

and replication

M.: Factors

of rhabdovirus

involved

defective

in the

T particles.

J.

Virol. 17 (1976) 805-815.

l-20. processes

a new mechanism

requiring

high specificity.

for reducing

errors

Proc. Natl. Acad.

Hopfield, J.J.: The energy relay: a proofreading cooperativity and lacking all characteristic in DNA

replication

and protein

scheme based on dynamic symptoms of proofreading

synthesis.

Proc.

Natl.

Acad.

Sci.

and Baltimore,

poliovirus-infected Ishihama,

D.: Initiation

of polysome

formation

in

HeLa cells. J. Mol. Biol. 47 (1970) 275-291.

A., Mizumoto,

K., Kawakami,

K., Kato, A. and Honda,

A.:

Proofreading function associated with the RNA-dependent RNA polymerase from influenza virus. J. Biol. Chem. 261 (1986) 1041710421. Kassavetis,

G.A., Zentner,

P.G. and Geiduschek,

E.P.: Transcription

at

bacteriophage T4 variant late promoters: an application of a newlydevised promoter-mapping method. J. Biol. Chem. 261 (1986) 1425614265. Kunkel, T.A.: Frameshift mutagenesis by eucaryotic DNA polymerases in vitro. J. Biol. Chem. 261 (1986) 13581-13587. Kunkel,

T.A.: Proofreading.

Cell 53 (1988) 837-840.

Kunkel, T.A. and Loeb, L.A.: Fidelity of mammalian Science 213 (1981) 765-767. Kunkel,

T.A., Schaaper,

in

Annu.

Rev.

M. and Gopinathan,

Nucleoside

monophosphate

J. Biol. Chem. 256 (1981) 3978-

D.: The frequency

of errors

in protein

J. 128 (1972) 1353-1356.

P. and Benkovic,

of DNA poly-

and misincorporation

reactions.

Sci. USA 83 (1986) 5769-5773.

R.E. and Summers,

tagenesis.

S.J.: Mechanism

activity switch and DNA sequence

American

W.C.

(Eds.):

DNA

Society for Microbiology.

Replication Washington,

and MuDC, 1988,

p. 515. N.,

biochemical

and

Bessman,

M.J.:

basis of spontaneous

Poland,

R.L.

mutation,

I. A comparison

Studies

on the of the

deoxyribonucleic acid polymerases of mutator, antimutator, and wildtype strains of bacteriophage T4. J. Biol. Chem. 247 (1972) 7116Ninio, J.: Kinetic

amplification

of enzyme discrimination.

Biochimie

57

(1975) 587-595. B.D., Zakour,

selection analogs.

by DNA

R.A., Singer, B. and Loeb, L.A.: Fidelity of base polymerases.

Site-specific

In: Moses, R.E. and Summers, American

Society for Microbiology.

ton, DC, 1988, pp. 196-207. Radman, M. and Wagner, R.: Mismatch Reichmann,

incorporation

of base

W.C. (Eds.), DNA ReplicaWashing-

repair in Escherichia coli. Annu.

20 (1986) 523-538.

M.E., Villarreal,

J.J.: RNA polymerase defective T particles

L.P., Kohne, activity

of vesicular

D., Lesnaw,

and poly(A) stomatitis

J. and Holland,

synthesizing

activity

in

virus. Virology 58 (1974)

240-249.

USA 77 (1980) 5248-5252. A.S.

synthesis.

R.A., Koplitr,

of pyrophosphorolysis

Rev. Genet.

Sci. USA 71 (1974) 4135-4139.

Huang,

Biochem.

tion and Mutagenesis.

in biosynthetic

of DNA

replication.

polymerization.

V., Benkovic,

dependence

Preston,

Holland, J.J., de la Torre, J.C. and Steinhauer, D.: RNA virus populations as quasispecies. Curr. Top. Microbial. Immunol. 176 (1992) Hopfield, J.J.: Kinetic proofreading:

correction

7122.

132 (1984) 26-37.

L.P. and Breindl,

of

13610-

Lute, M.C., Tschanz, K.D., Gotto, D.A. and Bunn, C.L.: The accuracy of protein synthesis in reticulocyte and HeLa lysates. Biochim. Bio-

Muzyczka, B.L., Weisslitz,

T.A.: Fidelity

R.B. and Vanderjagt,

biosynthesis.

Moses,

dependent

7689-7692. Fisher,

during

261 (1986)

P.: DNA mismatch

merase I: Exonuclease/polymerase

nucleotide

turnover of (Sp)-dATPcrS by T4 DNA polymerase. The stereochemistry of the associated 3’.5’-exonuclease. J. Biol. Chem. 257 (1981) Hall, J.D., Coen, D.M.,

and Modrich,

Proc. Natl. Acad.

chemistry 20 (1978) 4049-4055. Gupta, A., DeBrosse, C. and Benkovic,

Holland,

generation

allows detection

J. Biol. Chem.

Science 245 (1989) 160-164.

On fidelity of DNA

Mizrahi,

of purified Epstein-

acid

misincorporation

phys. Acta 825 (1985) 280-288.

FL, 1986, pp. 157-183.

W.: Characterization

induced

mechanisms.

Loeb, L.A. and Kunkel,

Loftfield,

taneous mutation. J. Mol. Biol. 156 (1982) 37-51. Fry, M. and Loeb, L.A.: Fidelity of DNA synthesis. In: Animal Cell DNA Grossberger,

R.A. and Loeb, L.A.: On fidelity of DNA syn-

13616. Lahue, R.S., Au, K.G.

K.P.:

dur-

78 (1981b) 6734-6738.

3987.

during DNA replication.

Sci. USA 78 (1981) 4251-4255.

A.R., Knill-Jones,

on proofread-

prevent proofreading

Biochemistry

Biochem. 52 (1982) 429-457. Loeb, L.A., Dube, D.K., Beckman,

of misincorpora-

Fersht, A.R. and Knill-Jones, J.W.: DNApolymerase accuracy and spontaneous mutation rates: frequencies of purine:purine, purine:pyrimiProc. Natl. Acad.

T.A., Beckman,

thesis. Pyrophosphate

a defined system.

III holoenzyme:

[ 1-thioltriphosphates

ing in vitro DNA synthesis.

two proofreading

188 (1982) 169-172.

Virol. 23 (1977) 708-716. polymerase

Effect of the next nucleotide

F., Mildvan, A. S., Koplitz, R.M. and Loeb, L. A.:

Kunkel, T.A., Eckstein,

R.M. and Greening,

221 (1969) 1128-1132.

unique RNA species by a T particle Fersht,

delity of DNA replication.

Kunkel,

131-137. Ellis, N. and Gallant, translation.

S.A., Preparata,

mutation.

P. and Gallant,

mutation.

ing. J. Biol. Chem. 256 (1981a) 9883-9889.

(1969) 1132. Drake, J.W., Allen, E.F., Forsberg,

R.M., Beckman,

DNA polymerase.

R.A. and Loeb, L.A.: On fi-

Rosenberger,

R.F. and Hilton,

J.: The frequency

of transcriptional

and

translational errors at nonsense codons in the 1ucZ gene of Escherichiu coli. Mol. Gen. Genet. 191 (1983) 207-212. Schubert,

M., Keene,

J.D.,

Lazzarini,

R.A.

and Emerson,

complete sequence of a unique RNA species particle of VSV. Cell 15 (1978) 103-112.

S.U.:

synthesized

The

by a DI

Semler, B.L., Perrault, J., Abelson, J. and Holland, J.J.: Sequence of a RNA templated by the 3’-OH RNA terminus of defective interfering particles of vesicular (1978) 4704-4708.

stomatitis

virus. Proc. Natl. Acad.

Sci. USA 75

Steinhauer, D.A. and Holland, J.J.: Direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA. J. Virol. 57 (1986) 219-228. Steinhauer, D.A., de la Torre, J.C. and Holland, J.J.: High nucleotide substitution error frequencies in clonal pools of vesicular stomatitis virus. J. Virol. 63 (1989) 2063-2071.

288 Strauss, J.H. and Strauss, E.G.: Evolution Microbial. 42 (1988) 657-683.

of RNA viruses. Annu. Rev.

T.: Characterization

with Epstein-Barr

of 3’ to 5’ exonuclease

virus DNA polymerase.

activity’associated

Virology 182 (1991) 376-

H.P.

and

Eckstein,

F.: Incorporation

of phosphorothioate

groups into $6 and $X DNA. Biochemistry 16 (1977) 3633-3640. Wain-Hobson, S.: Human immunodeficiency virus type 1 quasispecies in viva and ex viva. Cm-r. Top. Microbial. 194.

J.B.: Determination

error frequency

66 (1992) 3784-3793. Ward, C.D., Stokes, M.A.M. the poliovirus

of the poliovirus

RNA

at eight sites in the viral genome. J. Virol. and Flanegan,

RNA polymerase

J.B.: Direct measurement

error frequency

of

in vitro. J. Viral. 62

(1988) 558-562.

381. Vosberg,

C.D. and Flanegan,

polymerase

Temin, H.M.: Is HIV unique or merely different? J. AIDS 2 (1989) l-9. Tsurumi,

Ward,

Immunol.

176 (1992) 181-

Williams,

K.J. and Loeb, L.A.: Retroviral

frequencies

and mutagenesis.

(1992) 165-180.

reverse transcriptases:

CUT. Top. Microbial.

Immunol.

error 176