BACTERIOLOGICAL REVIEWS, June, 1966Copyright © 1966 American Society for Microbiology

Symposium on Replication of Viral Nucleic AcidsI. Formation and Properties of a Replicative Intermediate in the

Biosynthesis of Viral Ribonucleic Acid'

R. L. ERIKSON AND R. M. FRANKLIN

Departmenit of Pathology, University of Colorado Medical School, Denver, Colorado

INTRODUCTION..267Problem of Viral Ribonucleic Acid (RNA) Replicatio i.. 267Developmenit of Current Concepts of Viral RNA Synthesis ................. 268

BIOSYNTHESIS OF VIRAL RNA ............................. 269Formation of Virus-Specific Double-Stranded RNA .................... 269Properties of Virus-Specific Double-Stranded RNA .................... 270Viral-Induced RNA Polymerases ..275CONCLUSIONS. 275LITERATURECITED..276

INTRODUCTION

Problem of Viral Ribonucleic Acid(RNA) Replication

Most deoxyribonucleic acid (DNA) virusescontain double-stranded DNA with the well-known duplex structure which replicates accord-ing to the mechanism first proposed by Watsonand Crick (81). The replication of double-stranded viral DNA thus presents no particularproblem beyond the basic problem of DNA syn-thesis, which has been studied for the most partwith bacteria.Some DNA viruses, such as 4X174, contain

single-stranded DNA (72), and during the courseof infection intracellular double-stranded DNAis formed. This double-stranded DNA replicatesextensively as a duplex, but it has not been clearwhether the single-stranded DNA in the progenyvirion is derived from the double-stranded repli-cative form (RF) by selection of one strand or

by the synthesis of further single strands on thedouble-stranded template (73). Denhardt andSinsheimer (17) recently presented a model forthe replication of 4X174 DNA, whereby thesingle-stranded parental DNA is incorporatedinto the RF duplex, which then replicates semi-conservatively. The RF containing parental DNAmay be responsible for the synthesis of all thesingle-stranded progeny DNA, whereas theother RF molecules probably do not replicatefurther but rather serve as templates for messen-ger RNA synthesis.

1 A contribution to a symposium held at the An-nual Meeting of the American Society for Micro-biology, Atlantic City, N.J., 29 April 1965, with JohnHolland as convener and Consultant Editor.

Most RNA viruses contain single-strandedRNA (64, 66). The excellent studies of Gierer(29-31) showed that tobacco mosaic virus (TMV)RNA was a linear single-stranded polymer. Mostlater studies on viral RNA have not been as de-tailed, but in many cases the hydrodynamic prop-erties of a particular RNA have been comparedwith those of TMV RNA. The data have sug-gested that the RNA of a virion exists as a singlemolecule (38). Analysis of the enzymatic inacti-vation of infectious RNA from enteroviruses alsosuggested that viral RNA is single-stranded(39). The hydrodynamic properties of bacterio-phage R17 RNA are also compatible with thoseexpected of a linear single-stranded polymer(53). Thus, there is a large amount of publisheddata-some very convincing and some onlysuggestive-which indicate that most viral RNAis in the form of a single strand (64). Only twoRNA viruses-reovirus and wound tumor virus-contain double-stranded RNA (32, 33).There are two obvious alternatives for the

chemical composition of viral RNA: it could bechemically homogeneous, or it could be a mixtureof two species of polymers having defined linearsequences of bases, with one polymer beingcomplementary to the other in base sequence.

According to current belief, a given populationof virions contains a homogeneous populationof RNA molecules. This assumption is basedchiefly on the base ratios of viral RNA, sincegenerally the contents of adenine and guaninediffer from those of uracil and cytosine, respec-

tively (Table 1). The exceptions in Table 1 are

reovirus and wound tumor virus, as expected.The adenine-uracil and guanine-cytosine ratiosvary, but usually are not both equal to one for a

267

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ERIKSON AND FRANKLIN

TABLE 1. Base composition of the RNA from some RNA virulses

BacteriophageR17 ................

Animal virusesPoliovirus (3 types).Coxsackie A9......EMC ...............

Influenza A(PR8)Influenza B(LEE)..Rous sarcoma.......Reovirus type 3.

P'lant virusesTYMV ................

Southern bean mosaic.TMV (normal)........Wound tumor.........

(iuanine C tosine Adenine

26.3*

24.027.723.720.118.320.522.3

17.226.025.318.6

24.9

22.020.523.924.023.130.422.0

38.123.018.519.1

23.1

28.527.027.823. 123.029.928 .0

22.625.829.831.1

tUl acil.efel-ol.u

25.7 53

25.424.724.532. 8-)S 613.227 .9

2. 125. 326. 331.3

655224636362

5121)46

* Results expressed as moles per cent.

particular viral RNA. One cannot rule out thepossibility of a constant fraction of complemen-tary strands in a population of viral RNA. Itwould be extremely valuable to test further thepresence or absence of complementary RNA ina viral population by attempted hybridization ofa viral RNA with itself.The data. on single-strandedness and on base

ratios suggest that there must be an asymmetricalstep either in the synthesis of viral RNA or inthe selection of viral RNA for assembly intovirions. The asymmetrical step is probably thatof synthesis, since intracellular viral RNA hasthe sa.me base ratios as the RNA of virions, ac-cording to a recent study on poliovirus-infectedHeLa cells (87). After the inhibition of host-cellRNA synthesis with actinomycin D, the RNAsynthesized in the poliovirus-infected cell wasanalyzed by sucrose gradient sedimentation. Twovirus-specific species were found: 355 and 16SRNA. The 35S RNA, corresponding to viralRNA, had base ratios close to those of viralRNA, as did the 16S RNA. Thus, an under-standing of viral RNA replication depends onelucidation of the process whereby viral RNA ofnoncomplementary nature is the major product.

Development (f Current Concepts ofViral RNA Synthesis

Early speculations (R. Dulbecco, personalcommunication) on the nature of viral RNA syn-thesis centered on two a.lternative hypotheticalschemes (Fig. 1). In the first scheme, (a) host-cell DNA or modified host-cell DNA, or (b)some viral-induced DNA intermediate, wouldserve as a. templa.te for RNA synthesis. In thesecond scheme, (c) vira.l RNA would be synthe-

(a) viral RNA-host cell DNA - viral RNAor"modified" host cell DNA

(b) viral RNA -viral DNA intermediate - viral RNA

(c) viral RNA -viral RNAFIG. 1. Early proposals (coicerinii the m1lC/haIIiSml

of viral RNA biosAytllesis.

sized directly from viral RNA. Since the structureof RNA was not known when these schemeswere first discussed, it was not possible to specifythe molecular deta.ils of these genera.l alternatives.The inhibition of DNA synthesis, either beforeor after infection or both before and after infec-tion with poliovirus, did not affect the yield ofvirus (71). Unfortunately, similar experimentswith Newcastle disease virus (NDV) in HeLacells a-re more difficult to interpret because of thevery low yield of NDV in this cell system (71).But at least for poliovirus, newly synthesizedDNA does not pla.y a. role in vira.l RNA synthe-sis. When cells were pretreated with 5-bromoura-cil to form highly abnormal DNA, and weresubsequently infected with poliovirus of well-defined genotype, no increase in the mutationfrequency of the progeny virus wa.s noted (71).Thus, even pre-existing host-cell DNA does notplay a role in RNA virus replication.Mitomycin C induces breakdown (61, 69)

and cross-linka.ge (41, 42, 76) of DNA. Whereaslow doses inhibit only DNA synthesis, highdoses inhibit both DNA synthesis and DNA-dependent RNA synthesis, the latter due to ex-tensive destruction of DNA templates. Since evenvery high doses of mitomycin C did not affect

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the yield of mengovirus, replication of this RNAvirus does not require either associated or priorsynthesis of DNA, or integrity or expression ofany particular host-cell genome (57).

Actinomycin D inhibits DNA-dependent RNAsynthesis (58, 59) by interaction with the DNAtemplate (40, 60). Under conditions of total in-hibition of cellular RNA synthesis by actinomy-cin D, there is a normal production of mengo-virus (58, 59) and other viruses. It is also possibleto demonstrate the synthesis of viral-specificRNA in these cells (26, 87). Thus, no host-cellgenome need function during replication of someRNA viruses. Difficulties arise, however, whenRNA virus synthesis is inhibited by actinomycinD (60). The basis of this inhibition is not yetclear but may be due to secondary effects of ac-tinomycin in some cases. For example, inhibitionof reovirus synthesis (34) is dependent on theconcentration of actinomycin (68) and the lengthof time the cells are pretreated with actinomycinbefore infection (R. M. Franklin, unpublisheddata). A detailed discussion of RNA virusessensitive to actinomycin is beyond the scope ofthis review.A different approach to the problem of DNA

involvement in RNA virus replication is the useof the homology test, in which denatured DNAforms a specific hybrid with homologous RNA.The failure to find such a hybrid when Escherichiacoli DNA was tested with RNA from MS2 bac-teriophage (18) indicates that DNA, either pre-existing or newly synthesized, does not serve as atemplate for viral RNA synthesis. Thus, a varietyof approaches to the problem have led to theconclusion that, for certain RNA viruses, (i)neither host-cell DNA nor any DNA synthesizedafter infection serves as template for viral RNAsynthesis, and (ii) neither host-cell DNA norany DNA synthesized after infection need expressitself (by controlling the synthesis of specificRNA species) during viral RNA synthesis.These principles have been demonstrated only

with certain RNA viruses which have served asconvenient model viruses. It is far from clearthat the statement is valid for all RNA viruses.This qualification should be kept in mindthroughout this entire review. At this time thereis no evidence on the mechanism of replicationof the double-stranded RNA of reovirus. In thecase of the avian tumor viruses, both actinomycinand inhibitors of DNA synthesis block viral mul-tiplication under certain conditions (6, 7, 77-80).The data discussed above resulted, then, in

consideration of the alternate possibility thatviral RNA serves as template for further viralRNA synthesis.

Mengovirus-infected L cells have an alteredpattern of RNA synthesis (27). There is a viral-induced inhibition of cellular (nuclear) RNAsynthesis and an appearance of viral-specific(actinomycin-resistant) RNA synthesis in thecytoplasm (26, 27). These observations led tothe search for an RNA polymerase responsiblefor synthesis of viral RNA. Evidence for such anenzyme was first demonstrated in the mengo-virus-L cell system (10, 11), and further workwith RNA phage-infected E. coli (35, 86) hasfully confirmed the existence of such an enzyme(or enzymes).An important study demonstrated the existence

of a unique species of RNA in Krebs II ascitescells infected with encephalomyocarditis virus(EMC). This molecular species had properties ofdouble-stranded RNA and it was infectious (54).By analogy with the double-stranded DNAfound in 4X174-infected bacteria, this double-stranded RNA was termed a replicative form.After this initial report, double-stranded RNAwas detected in a number of different cells in-fected with RNA viruses, as will be discussed inmore detail later. Replicative form may not bethe best descriptive term for double-strandedRNA, since our present evidence indicates amore complicated structure to be the actualreplicative form. Rather than add further confu-sion to the literature, we have decided to continuccalling the double-stranded RNA replicative formand the more complex structure replicative inter-mediate (which will be discussed below).

In conclusion, one or several enzymes catalyzethe polymerization of viral RNA from an RNAtemplate. The precise nature of the template isnot yet understood, but a double-stranded RNAor a more complex structure is involved.

BIOSYNTHESIS OF VIRAL RNAFormation of Virus-Specific

Double-Stranded RNAFor the purposes of this discussion, we will

assume that the mechanisms of RNA replicationfor all small RNA-containing viruses are similar.

Montagnier and Sanders (54) first demon-strated conclusively that a replicative form ofviral RNA appeared in infected cells. Two formsof virus-specific RNA were identified by sucrosegradient analysis of the RNA extracted fromKrebs II ascites cells infected with EMC. Onetype of RNA had a sedimentation coefficient of37S, the same as that of RNA extracted fromintact virus. This RNA was infectious and, there-fore, was the RNA eventually packaged into thevirion. The second form of RNA had a sedimen-tation coefficient of 20S and was also infectious.

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Unlike the 375 viral RNA or the host-cell RNA,this RNA was resistant to treatment with pan-creatic ribonuclease. Its buoyant density in aCs2SO4 equilibrium density gradient was 1.57g/cc, while that of single-stranded EMC RNAwas 1.63 g/cc. Upon heating, the 20S RNA dis-played a temperature versus optical densityprofile similar to that exhibited by DNA, and amelting temperature of 96 C in 0.15 M NaCl plus0.015 M sodium citrate. All of these characteristicssuggested that the 20S component was a double-stranded form of viral RNA, analogous to thereplicative form that had been described for thesingle-stranded DNA of OX174 (73). RNA withsimilar properties has also been found in polio-virus-infected HeLa cells by Baltimore, Becker,and Darnell (9). In this case, the replicative formwas found in special cytoplasmic structures whichwere isolated from infected cells and which con-tained the viral RNA polymerase and most ofthe newly synthesized viral RNA. The replicativeform of poliovirus RNA was infectious, just aswas the replicative form of EMC virus RNA(55).

Interesting observations have also been madeon RNA phage-infected E. coli. Growth of RNAphage is independent of DNA synthesis (14), andparental bacteriophage RNA is not transferredto progeny phage (15, 19). These facts must betaken into account when mechanisms of viralRNA replication are considered.

Parental RNA is converted to a ribonuclease-resistant molecule which has sedimentationproperties consistent with those expected of adouble-stranded RNA composed of a parentalstrand and its complement (21, 22, 45). Further-more, chloramphenicol inhibited the conversionof parental RNA to the ribonuclease-resistantform. Therefore, an enzyme (or enzymes) notpresent in the uninfected cell is necessary for thisconversion (21, 45).

Ribonuclease-resistant RNA can also be de-tected in bacteriophage-infected E. coli as aspecies of RNA which is labeled after a veryshort pulse of H3-uridine (25). The time of ap-pearance of this RNA, as well as the results ofpulse-chase experiments, strongly suggests that itis a precursor of single-stranded phage RNA.The loss of ribonuclease-resistant label was ap-proximately equal to the gain of 27S label duringthe chase. If the RNA were labeled for 15 min,a period long enough to label both strands of theRNA duplex, the ribonuclease-resistant labeldecreased during the subsequent chase to 25% ofthe initial quantity. These results suggest that, ofthe two strands in the ribonuclease-resistant core,one is turning over while the other may be stable.

These results supported the earlier experimentsof Weissmann et al. (84, 85) which led to theproposal that viral RNA probably replicates byan asymmetrical semiconservative mechanismwhereby newly formed, parental-like strandsdisplace a parental-like strand from the duplexas synthesis proceeds. These experiments in-volved a hybridization technique whereby thetotal amount of replicative form in the infectedcell could be accurately determined (84). Newlysynthesized replicative form was detectable by20 min after infection, although replicative formcontaining parental RNA was detectable by 5 or6 min after infection. At 45 min after infection,approximately 1.7 % of the total RNA in the cellconsisted of replicative form; 20% of the totalRNA was viral RNA.

Ribonuclease-resistant replicative forms ofRNA have also been found in cells infected withseveral other animal and plant viruses. Amongthe plant viruses are turnip yellow mosaic virus(TYMV) (50, 56) and TMV (13, 70, 82).

Properties of Virus-SpecificDouble-Stranded RNA

Table 2 lists some characteristics of the replica-tive form of animal, plant, and bacterial RNAviruses. The assignment of a double-strandedstructure to RF is justified by these and the fol-lowing properties. (i) The molecule is resistantto hydrolysis by pancreatic ribonuclease, aknown property of double-stranded RNA (28).Most of the preparations listed in Table 2, withthe exception of two (1, 44), were prepared byribonuclease treatment of an RNA preparationfrom infected cells. (ii) The double-helical struc-ture of the replicative form of MS2 RNA hasbeen directly demonstrated by X-ray diffractionanalysis (47). (iii) Hybridization tests of de-natured ribonuclease-resistant RNA with viralRNA indicate the presence of a strand of RNAcomplementary to viral RNA. Further, the nu-cleotide compositions of TMV RNA RF and ofboth components of the replicative form arecompatible with a duplex structure containing astrand of viral RNA and a strand complementaryto viral RNA (84, 82).The values in Table 2 all reflect the double-

helical structure of RF molecules. Although thereported buoyant densities vary considerablyfrom laboratory to laboratory, all are lower thanthose of single-stranded RNA compared in aparticular laboratory. The sedimentation rate ofanimal virus RF is greater than that of bacterio-phage RF, a reflection of the higher molecularweight of animal virus RNA as compared withbacteriophage RNA.

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TABLE 2. Properties of replicative forms from various sources

Virus

EMCEMCPolioPolioMS2MS2frM12R17TMV

Tm

Temp

C

9684

103

10196

97

Buffer

SSc I1/ICSSC

SSc

0.2 M NaCI10-3 M NaCi

SSC

Measurement*

ODOD

Ribonuclease

ODOD

Ribonuclease

Buoyant densityt

CS2S04

1.57

1.651.581 .609

1. 609

1.6081.601

CsCl

1.868

Sedimenta-tion co-efficient

(S)

20

1616

1314.2

13.011.6

Reference

5454955834443

12313

* Refers to type of assay for measuring Tm. OD = increase in absorbance at 260 mr; ribonuclease =conversion of replicative form from a ribonuclease-resistant to a ribonuclease-sensitive form.

t Results expressed as grams per cubic centimeter.t SSC = 0.15 M NaCl plus 0.015 M sodium citrate.

Fraction numberFIG. 2. Sucrose gradient sedimentation patterns of RNA extracted from cells infected witlh R17 containing

P32-labeled RNA. The RNA was layered on a sucrose gradient (20 to 7%) containing 0.1 M NaCI and 0.001 MMgCI2 and centrifuged at 37,500 rev/min for 4 hr at 12 C in a Spinco model L2 ultracentrifuge. Each fraction wasdiluted to 2 ml with 0.15 M NaCl, and the optical density (a) was determined at 260 m,i. A 1-ml amount was re-moved froin each for treatment with ribonuclease (0.1 g/ml, 10 min at 37 C). After precipitation with 10% tri-chloroace-.. acid, the radioactivity for the untreated (0) and ribonuclease-treated (O) fractions was determined.Time after infection: (a) zero; (b) 6 min; (c) 12 min. This figure was originally published by Erikson et al. (21).

Experiments have been performed in thislaboratory (21, 25) to characterize all forms ofviral-specific RNA in R17 bacteriophage-infectedcells. The replicating molecule as isolated byphenol extraction at room temperature appearsto be heterogeneous, as determined by sedimenta-

tion through a sucrose gradient. This is true forpulse-labeled molecules and also for moleculescontaining parental RNA. To observe this sedi-mentation heterogeneity, the assay for ribonu-clease resistance must be carried out after cen-trifugation of the RNA (Fig. 2). If the RNA is

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O- 400zD~~~~~~~~

O 2.0 I

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8.

6.0

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10 20 30 40 10 20 30 40FRACTION NUMBER

FIG. 3. Analysis of the effect ofpancreatic ribonuclease on sedimentationi of the replicative intermediate. RNAwas prepared and then centrifuged exactly as shown in Fig. 2. After fractionationi, certain fractions were collectedcorresponding to 16S to 24S RNA and IOS to 15S RNA. These were then recentrifuged either with or withoutriboniuclease treatment (conditions described in the legend to Fig. 2). For simplicity, only the radioactivity ofparental RNA is plotted.

treated prior to centrifugation, a single homoge-neous peak of resistant RNA is observed. Asexpected from this result, if RNA is first fraction-ated by centrifugation, the subsequent ribonucle-ase treatment of any fraction which containsribonuclease-resistant RNA yields 13S ribonu-clease-resistant molecules and nonsedimentablefragments (Fig. 3). These results led to the sug-gestion (25) that nascent strands of viral RNAare attached to a double-stranded form of RNAand the presence of these strands causes the com-plex to sediment more rapidly than the double-stranded core. This molecular species was desig-nated (21) the replicative intermediate (RI) to dis-tinguish it from the molecule previously describedas the replicative form (54).Examination of viral-specific RNA in other

types of cells has revealed similar sedimentationpatterns. For example, in actinomycin-treatedchick embryo fibroblasts infected with Semiikiforest virus, two viral RNA components werelabeled with H3-adenosine. These had sedimenta-tion coefficients of 36S and 24S (74). Only thelatter component was labeled during a short

pulse, as with R17. Furthermore, ribonucleasetreatment before sedimentation resulted in ashift of the S value of the labeled material to lessthan 24S, similar to the behavior of R17 replica-tive intermediate. Such properties have also beenreported for foot-and-mouth disease virus (12).Although not enough physicochemical data

are available to establish definitively the structureof the complex which generates new strands ofviral RNA, we consider that the structures shownin Fig. 4 are compatible with the data obtainedto date. Double-stranded RNA (RF) is formedwhen parental RNA acts as a template for thesynthesis of its complement. This step is probablycontrolled by a viral-specific polymerase. Newviral strands are then synthesized on this tem-plate, perhaps in a direction opposite to that inwhich the complement was synthesized. Newviral strands may be generated by a second en-zyme, possibly a host-specific enzyme. The nas-cent strand is attached to the duplex template byhydrogen bonds close to the growing point ofthe chain. The population of duplexes with nas-cent strands would represent RI, and the different

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0-1w +

pdrentRNA1-pcirental RNA

MODEL A

a

HCX

+ H

NHHo

MODEL BFiG. 4. Mechanisms for the synthesis of viral RNA. In model A, the new viral RNA molecule would be gener-

ated on a stable duplex in a manner presumably analogous to the synthesis ofRNA on a DNA duplex. In this casethe new strand (cross-hatchedbars) wouldbe released, and theparental strand (clearbars) wouldremain in the duplexhydrogen-bonded to its complementary strand (solid bars). In model B (see 25), the viral strands would be displacedfrom the duplex during the synthesis of the new viral RNA strand which would remain hydrogen-bonded to thecomplementary strand. During the first round ofsynthesis in this case, the strand displaced would then be the paren-tal strand originally in the duplex.

lengths of the nascent chain would result in sedi-mentation heterogeneity of the molecules. Therecould be one or several nascent chains per duplex,although no information is available on thispoint. Completed viral chains would have achoice of three fates: to form new duplexes, toserve as messenger RNA, or to enter virions.This scheme (Fig 4, model A) is compatible withthe fact that parental RNA is not transferred toprogeny phage, because parental RNA wouldremain in the duplex. Alternatively, the viralRNA strand may be displaced from the comple-mentary strand as a new strand is synthesized(Fig 4, model B). This scheme was suggestedpreviously (25), but is not compatible with thelack of transfer of parental RNA to progenyphage (15, 19). There is a possibility that bothmechanisms may be operative in one cell if aduplex containing the parental strand functionsdifferently from other duplexes. Such behaviorwould be analogous to that of parental DNA-containing replicative form of 4X174 (17).

The parental-labeled replicative intermediateRNA, which has a sedimentation coefficient of12 to 16S (Fig. 2c), can be separated (22) intoits component strands by thermal denaturation(Fig. 5). The 12 to 16S RNA, when resedimentedwithout further treatment, displayed the expectedsedimentation profile (Fig. 5a). However, whenthis RNA was heated for 3 min at 95 C to breakhydrogen bonds and separate the strands, a newpeak of parental-labeled RNA appeared uponsubsequent sedimentation (Fig. 5b). This RNAhad the same sedimentation rate as the R17 RNAadded to the sample to serve as a sedimentationrate marker. Therefore, some parental RNAwhich had been converted to a double-strandedform after infection was then indistinguishable,by sedimentation analysis, from viral RNA.Moreover, after denaturation the RNA was nolonger resistant to ribonuclease, an expectedresult of the separation of the strands.

In Fig. 5c and d, the ribonuclease-resistant13S RNA (RF) is shown undenatured and de-

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ERIKSON AND FRANKLIN BACTERIOL. REV.

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FiG. 5. Analysis of 12 to 16S RNA extracted from bacteria infected for 12 min with P32-labeled R17. (a) Sedi-mentation ofan untreated sample. (b) Sedimentation ofa sample denatured 3 min at 95 C in 10-3m ethylenediamine-tetraacetic acid (EDTA) plus 10-2 M tris(hydroxymethyl)aminomethane (Tris), pH 7.2, in the presence of 60 ,lgof unlabeled R17 RNA added as a sedimentation rate marker. (c) Sedimentation of a sample after ribonucleasetreatment (0.1 ,ug/ml, 10 min, 37 C in 0.15 M NaCl). (d) Sedimentation of a sample of the ribonuclease-resistantRNA (Fig. Sc) after denaturation (3 min at 95 C in 10-' M EDTA plus 10-2 M Tris, pH 7.2) in the presence of60pug of unlabeled R17 RNA. Each sample was centrifuged through a 4.4-ml sucrose gradient at 37,500 rev/min for4 hr at 12 C. A 0.1-ml amount of each fraction was used for radioactivity determination (0) and the remainderwas diluted for optical density determination (0). This figure was originally published by Erikson et al. (22).

natured. The sedimentation profile after de-naturation (Fig. 5d) indicates that the parental-labeled RNA is smaller and more heterogeneousthan the parental RNA that entered the double-

stranded form as well as that obtained from thesample untreated with ribonuclease (Fig. 5b).This suggests that ribonuclease hydrolyzes somebonds in the component strands of the double-

274

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stranded molecule. Additional experiments arerequired to clarify the effects of ribonuclease onthese molecules.

Viral-Induced RNA PolymerasesThere are numerous reports concerning the

enzymes responsible for the synthesis of theRNA which eventually appears in progeny virus,some of which will be mentioned here.

Virus-specific RNA polymerase activity wasoriginally described by Baltimore and Franklin(10). This enzymatic activity was in the micro-some fraction of mengovirus-infected L cells andcatalyzed the incorporation of ribonucleotidetriphosphates into RNA. The incorporation re-quired Mg++ and was not inhibited by actino-mycin D. Nearest neighbor analysis of the prod-uct revealed that the four triphosphates wereincorporated in a nonrandom fashion. UsingRNA polymerase from poliovirus-infected HeLacells, Baltimore analyzed the product by sucrosegradient sedimentation (8). Radioactive labelwas incorporated into both 35S and 16S RNAby this system, and the 16S RNA had the prop-erties expected of double-stranded RNA.

Induction of an analogous enzyme occurs inRNA phage-infected E. coli (86). A 50-fold puri-fication did not free this particle-bound enzymeof RNA. All four triphosphates were requiredfor maximal activity, but there was no require-ment for added RNA. A similar enzmye wasisolated from f2 bacteriophage-infected cells (4).Subsequently, the further purification of theseenzymes has been carried out. The RNA synthe-tase from MS2-infected cells was purified in theform of haloenzyme, that is, in association withprimer RNA. At least a part of the product ofthis enzyme was double-stranded RNA indis-tinguishable from the double-stranded RNAisolated from MS2-infected cells (84).The f2-induced enzyme was purified over 100-

fold. Unfortunately, this enzyme is somewhatunstable in the purified state, but is completelyfree from RNA and has a requirement for addedprimer RNA (5). Any single-stranded RNA canbe utilized as primer. The base composition ofthe product synthesized is complementary to thatof the RNA used to direct the reaction (67). TheRNA product possesses a high degree of second-ary structure as judged from the high Tm andthe partial resistance to ribonuclease.

Very highly purified and specific RNA polym-erases have been isolated from cells infected withthe RNA bacteriophages Q,B or MS2 (35, 36,75). Each polymerase is active only when homol-ogous RNA is added to the reaction mixture.Identical copies of viral RNA are made in this

system, since the amount of RNA synthesized isdirectly proportional to the increase in titer ofinfectious RNA. In addition, newly synthesizedRNA is fully capable of serving as template forthe polymerase. The presence of a replicativeintermediate form of RNA which may have afunction in the synthesis of the identical copieshas not been reported, although sucrose gradientanalysis of the RNA synthesized in this systemsuggests the presence of double-stranded mole-cules (37). Unfortunately, the ribonuclease sensi-tivity of this RNA has not been tested. The iden-tification of RI should be easier in the cell-freesystem than in the intact cell where host RNAsynthesis interferes with the analysis of virus-specific RNA (25).Some amber mutants of bacteriophage f2 have

an altered capacity to induce synthesis of RNApolymerase (48, 49). The experiments with thesemutants suggest that in normal infection onlyinput RNA strands are templates for polymerasesynthesis, and progeny strands are templates forcoat protein synthesis. However, in mutant-in-fected nonpermissive cells, some of the progenyRNA is used for synthesis of polymerase, andamounts in excess of that in wild-type infectedcells are synthesized. At the same time, excessdouble-stranded RNA which has a sedimentationcoefficient of about 7S is synthesized. Thus, theenzyme isolated from cells infected with thisclass of amber mutants probably converts paren-tal RNA to a double-stranded form (67). AnRNA-dependent RNA polymerase has also beenisolated from Chinese cabbage leaves infectedwith TYMV RNA (2). The product of this reac-tion was partially resistant to ribonuclease andcontained RNA complementary to the viralstrand (3). This is further evidence for the gener-ality of the system for synthesis of viral RNA.

CONCLUSIONS

A double-stranded or replicative form of viralRNA exists in a variety of cells infected withsmall viruses which contain one molecule ofsingle-stranded RNA in the virion. This includesbacterial, plant, and animal cells infected under awide variety of conditions. The exact role thatdouble-stranded RNA plays in viral RNA syn-thesis is far from clear. Unfortunately, the evi-dence to date that it actually does play a role isonly circumstantial. However, a normal Watson-Crick base-pairing mechanism for defining thesequence of the progeny virus RNA is highlyattractive even in the absence of definitive experi-ments on the manner in which single strands aregenerated. Since double-stranded RNA is notproduced if protein synthesis is inhibited at the

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276 ERIKSON AND FRANKLIN

time of infection (21, 45), a viral-specific enzymemust be responsible for its production. In fact,there may be two enzymes (16) responsible forviral RNA synthesis: one which converts singlestrands to double-stranded molecules (67) andanother which generates progeny RNA strandsfrom these molecules.

Experiments in our laboratories are now di-rected towards an elucidation of the structure ofthe double-stranded molecule and of the role ofthe replicative intermediate in the synthesis ofviral RNA.

ACKNOWLEDGMENTThis investigation was supported by Public Health

Service grant AI-05320-VR from the National Insti-tute of Allergy and Infectious Diseases, and by grantB-14646 from the National Science Foundation. R. L.Erikson was a Public Health Service postdoctoralfellow (1-F2 CA-20,066) throughout the course ofthis research.

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