Studies on the biosynthesis and characterization of rhinovirus ribonucleic acid

WXOLOGY

48,221-229

(1972)

ies 0

the

Biosynthesis

and

Characterizatio

Ri bonucieic SARVAN Departlnent

K. SETHP

Stanford, Accepted

CARLTOP\;

AND

of Medical Microbiology,

Acidi E. SCHWERDT

Stanford University California 94305 December

School

of Medicine,

30, 19i’l

The synthesis of rhinovirus type 20 specific RNA in infected HeLa cells was examined in the presence of actinomycin D and uridine-5-3K. The virus induced in the infected cells the synthesis of two major components of RNA sedimenting at 32 S and 18 S, respectively, and a heterogeneous component with a sedimentation coefficient of 24-26 S. Treatment of virus-specific RNA components with ribonuclease resulted in the complete degradation of 32 S and 24-26 S components, while the 18 S component showed partial resistance to ribonuclease. Poliovirus type I underidentical conditions induced the synthesis of two components of RNA sedimenting at 35 S and 18 S, respectively. Rhinovirus specific RNA in infected HeLa cells was first detectable betTeen 4 and 5 hr postinfection at about the same time that infectivit:r first appearedintraeellularly. The maximal synthesis of virus specific RNA took place between 6 and 7 hr postinfection. The purification of rhinovirus in cesium chloride resulted in the loss of infectivity and appearance of phosphotungstic acid-penetrable virus particles. RNA extracted from the purified virions sedimented with a major peak at 32 S but with a heterogeneous skewed type of sedimentation along the t’railing edge. INTRODUCTION

Rhinoviruses are classified as a subgroup of picornaviruses on the basis of the size, morphology, ribonucleic acid (RNA) genome of their virions and their lack of an outer Iiprotein envelope. They are distinguishable, however, from other members of the picornavirus group by the lability of their infectivity at pII 3.0-5.0 (Ketler et al., 1962), their higher buoyant density in cesium chloride (Chappel and Harris, 1966; Dans et al., 1966)) and their optimal growth temperature of 33” (Tyrrell and Parsons, 1960)0 Replication of the RNA genome of several picornaviruses such as polio, Mengo, enceph1 This investigation was supported by a grant from the Brown-Kazen Fund of the Research Corporation and by U. S. Public Health Service Training Grant AI-00082 from the National Institute of Allergy and Infectious Diseases. 2 Present address: Division of Virology, Central Research Institute, Kasauli (Simla Hills), India 222 Copyright

@ 1972 by

hzdemic

Press,

Inc.

alomyocarditis (EMC) viruses and others has been studied in considerable detail (Franklin and Rosner, 1962; Dalgarno and Martin, 1965; Baltimore and Girard, 1966). The synthesis of RNA of picornaviruses as well asof small RNA bacteriophages appears to involve essentially similar rephcative processes. In each ease, the virus specifies in infected cells a replicative form (RF) of RNA which is h.ehcal, base-paired, doublestranded in nature (Montagnier and Sanders, 1963; Baltimore et al., 1964; Billeter et ai., 1966; Baltimore, 1966) and a repiica‘tive intermediate (RI), presumably a multistranded form of RNA through which the single-stranded viral RNA is syn-thesized (Franklin, 1966; Baltimore and Girard, 1966; Baltimore, 1968; Granbouiaa and Franklin, 1968). Little is known about the nature of rhinovirus RXA or its mode of synthesis in infected cells. By analogy to the RNA rep%cation of picornaviruses, it might

222

SETHI

AND

that steps involved in the synthesis of rhinovirus RNA are similar to those of other small RNA viruses. Nevertheless, the lower st,ability, higher buoyant density of rhinoviruses in cesium chloride solution and the suggestion of a genome larger than that of other picornaviruses (McGregor and Mayor, 1968) warrant an investigation of the properties of rhinovirus RNA and its synthesis in infected cells. In this report, we describe the synthesis of rhinovirus-specified RNA in infected HeLa cells and compare its characteristics with that isolated from purified virions.

SCHWERDT

Virus propagation and isotopic labeling. HeLa cell monolayers grown at 35” for 4872 hr after seeding in loo-mm petri dishes were infected at an input multiplicity of 10 plaque-forming units (PFU) per cell. After adsorption for 1 hr at 33”, the inoculum wa#sreplaced with 10 ml of MEM containing 5 % fetal bovine serum. Actinomycin D (1 lg/ml) was added to the growth medium of infected cell monolayers at 2 hr postinfection (pi) followed 1 hr later with 200 $Zi of uriiine-5-3H per culture. The cultures were then incubated at 33” in the presenceof both drug and label for 18-20 hr. The infected cells, showing a marked cytopathic effect at this MATERIALS AND METHODS time, were disrupted by freezing at -70” Reagents. Tris buffer (NTE) contained, and thawing three times to release virus. 0.1 &’ NaCl, 0.01 M TrissHCl (pH 7.4), Poliovirus was labeled similarly except that actinomycin D was added to infected cul0.001 M EDTA (ethylenediamine tetraacetate) ; standard solution of citrate (SSC) con- tures at 1 hr pi and the label 1 hr later. Infectivity assays. Rbinovirus and poliosisted of 0.15 M NaCl and 0.15 II1 sodium virus infectivity were measured by a modificitrate (pH 7.0). Uridine-5-3H (specific accation of the agar cell-suspension plaque tivity 25 Ci/mmole) was purchased from assay method (Cooper, 1961) described elseNew England Nuclear Inc. Actinomycin D where (Sethi and Schwerdt, 1970). Infectious was a gift from Merck, Sharp and Dohme, RNA from infected cells or purified virus Rahway, New Jersey. Pancreatic ribonuwas assayed by the plaque method described clease(RNase) was obtained from Worthington Biochemical Corp., Freehold, New by Bishop and Koch (1969). Virus purification. Clarified labeled virus Jersey, and ribonuclease-free sucrose from suspension was emulsified once with an equal Mann Laboratories, New York. Crystalline volume of trichlorotrifluoroethane, “genephenol was purchased from Mallinckrodt tron,” (Allied Chemical Corp., Morristown, Chemical Works, St. Louis, Missouri, sodium dodecyl sulfate (SDS) from Matheson, Cole- New Jersey) by vigorous shaking and the two phaseswere separated by centrifugation. man and Bell Laboratories, East Rutherford, New Jersey, and polyvinyl sulfate (potas- Virus was sedimented from the aqueousphase sium salt) from Eastman Organic Chemicals, by centrifugation at 28,000 rpm in the 30 rotor of a Spine0 Model L ultracentrifuge for New York. Viruses and cells. Rhinovirus serotype 20 4 hr at 4”. The pellet containing virus was and HeLa cells were obtained from Dr. V. V. held overnight in a small volume of 0.01 $2 Hamparian, and an attenuated strain Tris . HCI (pH 7.8) at 4“ and suspended thoroughly by pipetting. Cesium chloride (Sabin) of poliovirus type 1 was used from the stock maintained in this laboratory. The was then mixed with the virus suspensionto viruses were pIaque purified three times, give an average density of 1.40 g/cm3 and cells were frequently checked for the pres- centrifuged in a SW 39 rotor for 20 hr at ence of Mycoplasma. HeLa. cells were grown 30,000 rpm. Fractions were collected from the bottom of the tube and an aliquot of in 12-0~ prescription bottles or loo-mm plastic petri dishes (Falcon Plastics, Los each fraction used for the determination of Angeles) in Minimum Essential Medium radioactivity and buoyant density. A single (MEM) containing Earle’s balanced salt cycle of virus centrifugation through cesium solution, 10% fetal bovine serum, and anti- chloride usually resulted in a heterogeneous biotics (penicillin 200 units/ml, streptomycin radioactive profile with a sharp peak at 100 pug/ml). buoyant density 1.40 g/cm3. The fractions

RHINOVIRUS

RIBONUCLEIC

with maximum radioactivity around density 1.40 g/cm3 were pooled and recentrifuged to equilibrium in fresh cesium chloride solution of the samedensity. Fractions were collected and assayedfor infectivity and radioactivity and examined by eiectron microscopy. The refractive index of each fraction was measured in an Abbe refract’ometer (Bausch and , New York) and density calculated as bed by Vinograd afnd Hearst’ (1962). In someexperimenk, virus was purified by a single cycle of centrifugation in cesium chloride followed by rate zonal sedimentation in a sucrose gradi& of 5 7%to 30 7c in KTE. RNA extraction jrorn purijied virus. RXA from purified poliovirus and rhinovirus was extracted at 4’ or at 60” in the presence of polyvinyl sulfate (IO pg/ml). Purified virus ?;l-a,s diluted in 0.01 ii/r sodium acetat’e (pH 5.1) to which sodium dodecyl sulfate was added to a final concentration of P%. An equal volume of KTE-saturated phenol was then added to t,he suspensionand vigorously shaken for 3 min at 4” or in a water bath at 60”. The mixture was chilled in ice bath and centrifuged at 2000 rpm for 5 min to separate the phenolic and aqueousphases.The phenol extra&ion of the aqueous soUion was repeated twice. The us RNA solution was there adjusted t M NaCl and precipitated at -20” two volumes of ethanol in the presence of uninfected HeLa ccl1 RNA as carrier. The RKA precipitate was collected after 18 hr by centrifugat’ion, dissolved in NTE and reprecipitatcd with alcohol. RNA extraction from infected cells. HeLa cell monolayers in 100 mm petri dishes lvere infected a,t an input multiplicit,y of lo-20 PFU/cell and after adsorpt’ion for 1 hr \?Tere covered with 5 ml of growt,h medium. Aetinomyein D at a concentration of 10 fig/ml was added to the infected cells at given times for I hr followed by P hr of labeling with uridine-S-3 (200 pCi/culture). The cell sheets were then washed three times with cold phosphate-bufi”ered saline and solubilizcd by the addition of 10 ml of 0.01 M sodium acetate (pH 3.1) containing SDS to a fine1 eoncentratiorr of 0.5 %. An equal volume of NTE-saturat’ed phenol was then

223

ACID

added and RNA extracted at 60” 2s dcscribed above and precipitated twice ~+
Virus-SpeciJied RLVA

in

Infected Cells

Rate of accuvmlation. Rhinovirus-specified RNA synthesized in infected cells during each hour throughout one cyle of virus growth at 33” was measured by its incorporation of uridine-&31B. &La cell vultures in. 60-mm petri dishesinfected at, an inp;ic multiplicity of SO PFU/celL were exposed Lo actinomycin II (10 gg/ml) for 1 hr followed by I hr of labeling (200 &i uridine-5-3H) in the presenceof the drug. RNA was th.eE cx-

224

SETH1

AND

tracted by phenol-SDS at 60”, and TCAprecipitable radioactivity was determined. In uninfected cells exposed to actinomycin D under the same conditions the incorporation of uridine-5-3H was less than 1% of that occurring in untreated cells. The incorporation of label into the virus-specified RNA of infected cells, corrected for labeled RNA found in uninfected controls, was first detected at 4 hr pi and accumulated exponentially thereafter for 3 hr, after which the rate became approximately linear (Fig. 1). Sedimentation analysis. Alcohol-precipitated rhinovirus and poliovirus-specified RNAs obtained from infected cells at the time of maximal RNA synthesis were collected by centrifugation and dissolved in small volumes of NTE. One-tenth milliliter

SCHWERDT

volumes were then layered over 5 ml of 5 % to 20 % sucrose gradient in NTE and centrifuged. As shown in Fig. 2, uridine-5-3H labeled rhinovirus-specific RNA has two major components sedimenting at 32 S and 18 S. A small amount of radioactivity is also incorporated into the 24-26 S region and was regularly present in all experiments. In the uninfected cells, no incorporation of radioactivity occurred in the 28 S or 18 S ribosomal RNA; some is found in the 4-5 S RNA (Fig. 3a), the synthesis of which appears to he less sensitive to actinomycin D (Perry and Kelley, 1968). Poliovirus-specified RNA also exhibited two major components sedimenting at 35 S and 18 S, which is in agreement with previous studies (Zimmerman et al., 1963; Baltimore et al., 1966). When sedimentation velocity rates of poliovirus and rhinovirus RNAs were determined in parallel runs, the major (35 S) component of poliovirus-specific RNA always sedimented more rapidly than that of rhinovirus. The minor compo-

FRACTION

IO31

’

I

’

2

’

3

HOURS

’

4

’

5 AFTER

’

6

’

7

’

8

’

9

’

IO

1

INFECTION

1. Rate of accumulation of rhinovirus specified RNA. A measure of virus specified RNA synthesized during hourly intervals throughout a single cycle of virus growth in HeLa cells was made by estimating the amount of uridine-5-3H incorporated as described in the text. These estimates corrected for label incorporation by uninfected cells under the same conditions, were accumulated and plotted on a logarithmic scale for each hour beginning at 4 hr pi when virus-specified RNA was first detectable. FIG.

NUMBER

2. Comparison of RNAs synthesized in HeLa cells infected with rhinovirus type 20 and poliovirus type 1. Cell monolayers were infected at an input multiplicity of 10 PFU/cell. After 1.5 hr of infection in the case of poliovirus and 5 hr for rhinovirus, actinomycin D (10 pg/ml) was added to the infected cells for 1 hr followed by labeling with uridineZ9H for 1 hr in presence of the drug. RNA was then extracted by phenol-SDS method and analyzed on a 5% to 20y0 sucrose gradient. Fractions were collected and TCAinsoluble radioactivity was determined. O---O, Cpm rhinovirus-specific RNA; X--X, cpm poliovirus-specific RNA; O--O, UV absorbance at 280 nm of HeLa cell ribosomal RNA. FIG.

RHINOVIRUS

C.

4c

+2es

+1ss

RIBONUCLEIC

d.

/4s

R

ACID

czss

4185

“14s

-I- 1.0

i

t

3

A,

6 4

0.5

2 0

0 IO

20

30

IO

FRACTION

NUMBER

20

30

FIG. 3. Velocity sedimentation analysis of RNA synthesized at different time intervals in HeLa ceils infected with rhinovirus type 20. HeLa cell monolayers in 60 mm petri dishes were infected at an input multiplicity or 10 PFU/cell and labeled for 1 hr with uridine-5-3H (200 ;uCi). Cells were pretreated with actinomycin D (10 pg/ml) for 1 hr prior to the addition of radioactive label. Uninfected control cells were also similarly treated with actinomycin and labeled with uridine-5-3H. RXA from the infected or uninfected cells was extracted with phenol-SDS at 60” and analyzed on the sucrose gradient 5Lj0 to 20% in SW 39 rotor at 38,000 rpm for 4 hr. Fractions were collected and an aliquot of each treated with ribonuelease. TCA-precipitable radioactivity was determined and expressed a,s cpm. The labeling periods were: (a) uninfected cells, (b) 1-2 hr postinfection (pi), (c) 2-3 hr pi, (d) 34 hr pi, (e) 4-5 hr pi; (f) 5-6 hr pi, (g) 6-7 hr pi, (h) 7-8 hr pi. @- - 0, TCA insoluble radioacti-vit,y (cpm) ; X - - X, cpm after RNase treatment,; 0- - 0, UV absorbance at 260 nm.

nents (18 S) in both cases, however, sedimented at the samerate (Fig. 2). Sequential appearance of virus-specified RNA co?nponents.The appearnce of rhinovirus-specific RNA components in HeLa cells was followed by exposing infected cells for 1 hr to actinomycin D (10 pg/ml) at different intervals followed by 1 hr of labeling with uridine-5-3H. RNA from the infected cells was extracted and analyzed on sucrose graients. The radioactivity profiles of virus specified RNA components synthesized at

hourly imervals after infection are shown in Fig. 3. Virus specified R&A in the infected cells was first readily detected between 4 and 5 hr of infection, although low levels of radioactivity were incorporated between 3 and 4 hr of infection (Fig. 3d). Sedimentation velocity profiles of RNA synthesized to 3 hr of infection (Fig. 3b, c) did not differ sign%i~~ cantiy from that of uninfected ceils treated with actinomycin D (Fig. 3a). The RNA components detectable between 4 and 5 hr

226

SETH1

AND

was predominately 32 S (Fig. 3e). As shown in Figs. 3f, g, h the synthesis of both 32 S and 18 S components of virus-specified RNA progressed rapidly after 5 hr of infection. The pattern of labeling in the two components of RNA remained essentially similar at 6-7 and 7-8 hr postinfection. At later intervals, however, the incorporation of radioactivity became more heterogeneous and the 32 S peak of virus specified RNA became broader. Treatment of rhinovirus specified RNA with ribonuclease resulted in complete degradation of the 32 S component indicating its single strandedness. The R1VA sedimenting at 18 S was partially resistant to ribonuclease (Fig. 3f, g, h) and thus resembled the mixture of virus-specified double-stranded RNA and replicative intermediate (RI) RNA reported for poliovirus-infected cells (Baltimore and Girard, 1966) and in vitro poliovirus RPU’A synthesis (Girard, 1969). RATA from pur@ed virus. Uridine-5-3H labeled rhinovirus preparations extracted with genetron were purified by two cycles of isopycnic centrifugation in cesium chloride or by one cycle in cesium chloride followed by rate zonal centrifugation in a sucrose gradient. Purification by either procedure yielded a single componen.t with coincident peaks of infectivity and radioactivit,y which, in the cesium chloride gradient, occurred at the 1.40 g/cm3 density level. Prolonged contact of rhinovirus with cesium chloride resulted in 50-90% loss of infectivity during purification. Electron microscopic observation of negatively stained, purified virus revealed a large fraction (30-50%) of phosphotungstic acid (PTA)-infiltrat,ed virus particles among the intact virions despite the fact that the stained specimens were prepared from a single, apparently homogeneous isopycnic or rate zonal centrifugation component. Poliovirus similarly purified by either of the two methods noted above did not suffer significant loss in infectivity or reveal an appreciable number of PTA-filled virus particles. Labeled RNA from purified rhinovirus and poliovirus virions was extracted and analyzed on sucrose gradients. The major peak

SCHWERDT

FRACTION

NUMBER

4. Velocity sediment,ation of uridine-5-3H labeled RNA from purified rhinovirus type 20 and poliovirus type 1. Purification of virus was done by equilibrium centrifugation in cesium chloride followed by rate zonal centrifugation in sucrose gradient. RNA was extracted by phenol-SDS at 60” and velocity sedimentation analysis done by centrifugation on sucrose gradient 5% to 200% in SW 39 rotor at 38,000 rpm for 4 hr. Fractions were collected from the bottom of the tubes and radioactivity measured. O--O, Cpm rhinovirusRNA; X--X, cpm poliovirus RNA; e--O, cpm rhinovirus RNA treated with RNase; O---O, UV absorbance of HeLa cell ribosomal marker RNA at 260 nm. FIG.

of RNA extracted from rhinovirus corresponded to 32 S relative to the 28 S ribosomal RNA marker. RNA extracted from poliovirus sedimented at 35 S in a single, sharp peak (Fig. 4). The radioactive profile of RNA from rhinovirus was heterogeneous with a broad shoulder to the right which persisted whether extraction with phenolSDS was carried out at 60” or 4”. This RNA is degraded by treatment with RNase and is, therefore, single stranded (Fig. 4). These results suggest that the 32 S component of RNA synthesized in the infected cells is incorporated in the virion. Infectivity of rhinovirus RNA. The ribonucleic acid from purified virions or infected cells was infectious. The ratio of infectivity of purified rhinovirus to that of RNA isolated from an equivalent amount of purified virions was 104. The yield of infectious RNA from infected cells was about 1O-3 PFU/cell. The infectivity of RNA from both sources was lost on treatment with RNase but was not neutralized by type-specific antiserum (Table 1). These observations confirm ear-

RHINOVIRUS

TABLE 1

RIBONUCLEIC

ACID

9rr; --

about 3 hr, after which it becomeslinear and then declines (Baltimore et al., 1966). The: longer eclipse period and lower rate of a~ cumulation of rhinovirus-specific RNA may well be an intrinsic property rhino I viruses. Contributing extrinsic factors, how Average number Preparation of plaques per ever, may be the lower temperature of in 60-mm petri dish cubaCon (33”) and the fact that only 50%. of the cells appear to be infected at an input. RNA from infected cells (10-l) 75 multiplicity of IO PFU/cell as determined After treatment with 68 by infective center assay (unpublished obrhinovirus type specific servations) . antiserum (1: 150) The intracellular appearance of infectivit> After treatment with 0 a,s determined by a one-step growth curve ribonuclease 10 fig/ml RNA from purified virions 64 (unpublished observat ) coincided with (10-l) that of virus specified A in the infected After keatment with 56 dis. These findings suggest that thtb rhinovirus type specific encapsidation of virus specified R antiserum (1: 150) rapid as is the case with poliovirus After treatment with 0 more et al., 1966). ribonuclease 10 fig/ml Velocity sedimenta’tion analyses in sucrow Rhinovirus type 20 (10-j) 82 gradients of rhinovirus type 20 spe After treatment with type 0 labeled for I plr with uridine-5 specific antisercm (1: 150) After treatment with 74 experimental growth revealed t ribonuclease 10 pg/ml components with sedimentation veloeit:., rates of 32 S and I8 S and a minor shoulder a Heka cell monolayers in several 100 mm at about the 24-26 S position. The F petri dishes were infected at an input multiplicity and significance of this last, minor of 10 PFU/cell and RNA extracted 7 hr later by component is not known. Its persistent apphenol-SDS method, precipitated with alcohol pearance and reproducible sedimentatior~ and dissolved in 2 ml of buffer. b Rhinovirus was purified by one cycle of velocity in replicate experiments, however, equilibrium centrifugation in CsCl followed by suggest that it is not merely randomly derate zonal centrifugation in a sucrose gradient. graded viral RNA. The 32 S RNA compo. nent is single st.randed since it is completeI;. degraded by ribonuclease and appears to bc lier reports of infectious R.NA from other identical to the RNA isolated from purified rhinovirus serotypes (Dimmock, 1966) ; virions. The 18 S component, the rats; ot Fiala and Saltzman, 1969; Nair and synt,hesis of which roughly paraileled thar of the 32 S component, is partially resistant, to degradation by ribonuclease and, t,hereDISCUSSION fore, resembles the mixture of the double.To the extent that rhinovirus serotype stranded replicative form and replicativc 20 RNA biosynthesis has been characterized intermediate RKA components described in this study, it, resembles tha,t of other for poliovirus (Pons, 1964; Bishop et al., picornaviruses. When HeLa cell cult’ures 1965; Watanabe, 1965; Baitimore aud were infected with 10 PFU per cell of rhinoGirard, 1966). virus in the presence of actinomycin D, The purification of rhinovirus type 20 and virus-specified RNA was first detected at poliovirus type B under identical conditions 4 hr, rose exponentially to about 7 hr and revealed several differences. Rhinovirus ap then linearly for an additional 3 hr. Poliopeared to be unstable when centrifuged irl virus viral RNA (35 S) synthesis in HeLa cesium el;loride solution. This was manicells is detected as early as 75 min postinfection and accumulates exponentially until fested by a significant, loss in infcctivit:; a?~(: TNFECTIOUSRIYONUCLEICACID FROMHEL~CELLS INFECTED WITIZ RHINOVIRUS TYPE 200 AND FROM THE PURIFIED VIRIOXSb

228

SETH1

AND

by the appearance of a large number of PTAinfiltrated particles in the purified preparations negatively stained and examined by electron microscopy. Poliovirus infectivity and integrity of virions did not change appreciably under similar conditions. The buoyant density of rhinovirus type 20 in cesium chloride was 1.40 g/cm3 which is in agreement with the values obtained for other rhinovirus serotypes (Chappel and Harris, 1966; Dans et al., 1966). Why it is higher than that of poliovirus and other enteroviruses is not known but probably is not due to a larger rhinovirus genome as previously suggested (McGregor and Mayor, 1968). Rhinovirus RNA extracted from purified virions revealed a sedimentation profile with a peak at 32 S and a heterogeneous trailing edge of radioactivity. The profile’s skewed appearance could be due to the degradation of RNA either during its extraction or during the purification of virus or to the existence of the viral genome in several components. The latter explanation seems unlikely since the nucleic acid isolated from the purified virus is infectious and the distribution of the radioactivity in the trailing edge is random. The most probable explanation is the degradation of viral RNA in situ during the purification procedure. The appearance of a large number of PTA-infiltrated particles might be due to breaks in the virus capsid and possible breaks in the viral genome. RNA extracted from such particles could lead to the heterogeneous, skewed type of radioactive profile observed. The heterogeneity of rhinovirus types 2 and 14 RNA has been reported recently (Nair and Lonberg-Holm, 1971). Our estimate of the molecular weight of rhinovirus type 20 RNA using the empirical relationship derived by Spirin (1963) is 2.2 X lo6 daltons. This value is a rough approximation falling slightly below the 2.4 to 2.8 X lo6 daltons range found for rhinovirus type 2 (Brown et aZ., 1970) and other picornaviruses (Burness, 1970; Wild and Brown, 1970; Tannock et al., 1970). It is in agreement with McGregor and Mayor’s (1971) recent results for rhinovirus type 14 obtained by chemical and hydrodynamic methods.

SCHWERDT ACKNOWLEDGMENTS We are indebted to Miss Nancy Pleibel for checking repeatedly our HeLa cell line for mycoplasma contamination and to Dr. King F. Tam for performing the electron microscopy of purified virus preparations. REFERENCES BALTIMORE, D. (1966). Purification and properties of poliovirus double-stranded ribonucleic acid. J. Mol. Biol. 18, 421428. BALTIMORE, D. (1968). Structure of the poliovirus replicative intermediate RNA. J. Mol. Biol. 32, 359-368. BALTIMORE, D., BECKER, Y., and DARNELL, J. E. (1964). Virus-specific double-stranded RNA in poliovirus-infected cells. Science 143, 1034-1036 BALTIMORE, D., and GIRARD, M. (1966). An intermediate in the synthesis of poliovirus RNA. Proc. Nat. Acad. Sci. U. S. 56, 741-748. BALTIMORE, D., GIRARD, M., and DARNELL, J. E. (1966). Aspects of synthesis of poliovirus RNA and the formation of virus particles. virology 29, 179-189. BILLETER, M. A., WEISSMANN, C., and WARNER, R. C. (1966). Replication of viral ribonucleic acid. IX. Properties of double-stranded RNA from Escherichia coli infected with bacteriophage MS2. J. Mol. Biol. 17, 145-173. BISHOP, J. ICI., and KOCH, G. (1969). Plaque assay for poliovirus and poliovirus specific RNAs. In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp, 131-145. Academic Press, New York. BISHOP, J. M., SUMMERS, D. F., and LEVINTOW, L. (1965). Characterization of ribonuclease-resistant RNA from poliovirus-infected HeLa cells. Proc. Nat. Acad. Sci. U. 8. 54, 1273-1281. BRAY, G. A. (1960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-285. BROWN, F., NEWMAN, J. F. E., and STOTT, E. J. (1970). Molecular weight of rhinovirus ribonucleic acid. J. Gen. Viral. 8, 145-148. BURNESS, A. T. H. (1970). Ribonucleic acid content of encephalomyocarditis virus. J. Gen. Viral. 6, 373-380. CHAPPEL, P. J., and HARRIS, W. J. (1966). Biophysical studies of a rhinovirus: Ultracentrifugation and electron microscopy. Nature (London) 209, 790-792. COOPER, P. D. (1961). An improved agar cellsuspension plaque assay for poliovirus: Some factors affecting efficiency of plating. virology 13. 1533157. DALGARNO, . L., and MARTIN, E. M. (1965). St.udies

RHINOVIRUS

RIBONUCLEIC

01: ET&C viral RNA synthesis and its localization in infected Krehs ascites cells. Virology 26, 450405 DASS, P. E., FORSYTH, B. R., and CEANOCK, R. XI. (1966). Density of infectious virus and complement-fixing antigens of two rhinovirus strains. J. Bacterial. 91, X05-1611. DI,MXOCIC, N. J. (1966). Extraction and assay of infectious ribonucleic acid. Xature (London) 209, 792-794. FILL,\; >I., and SAL~~YN, B. (1969). Enhancement of the infectivity of rhinovirus ribonucleic acid by diethylaminoethyl dextran. Appl. Microbial. 17, 190-191. FRASHLIN, R. i%. (196G). Purification and properties of the replicative intermediate of the RNA bacteriophage Rl’i. PTOC. Xnt. Acad. Sci. U S 55, 1504-15!1. FRANII. (1969). fn aitro synthesis of poliovirus ribonucleie acid: Role of the replicative intermediate. J. V
ACID

G$$g

for determining the sedimentation behavior of to protein mixtures. enzymes : iipplication J. Biol. Chew 236, 1372-2379. MONTAGXIER, IA., and SANDERS, F. K. (1963). Replicative form of encephalomyocarditis virus ribonueleic acid. -WatTire (Lcndon) IF?, 664-667’. NAIR, C. N., and LONBERG-HOLII?, K. K. (1971). Infectivity and sedimentation of rhinoviri?s ribonucleic acid. J. viral. 7, 278-280.

PERRY, R. P., and KELLEY,

D. F,. (1968). Per-

sistent synthesis of 5 S RNA when production of 28 S and 18 S ribosoma.! RNA is inhibited by low doses of actinomycin D. J. Ceil. Physiol. 72, 235-245 ” Poss, M. (1964). Infectious double-stranded poliovirus RNA. Virology; 24, 46’7-473. SETHI, S. K., and SCMWERDT, C. IC. (1970). Improved method of the plaque assay of rhinoviruses. Bacterial. Proc. pi 178 (Abstract). SPIRIS, A. S. (1963). Some problems concernkg the macromoliecular structure of ribonueleic acids. Progr. Nucl. .4&l. Res. Mol. 0%. 1, 301-345. TANXOCIC, G. A., GIBBS, A.J., and COOPER, P.1). (1970). A reexamination of the molecular weight of poliovirus RNA. Biochem. Bioph,ys Res Commun. 38, 298-304. TYRRELL, D. A. J., and PSRSOXS, R. (1968) Some virus isolations from common colds. III. Cytopathic effects in tissue culture. Lancet b, 239s.242. VINOGRAD, J., and HEARST, J. E. (1962). EquiLibrium sedimentation of macromolecules and viruses in a density gradient. Fortsclrr. Che,n. Org. Naturst. 20, 372-422. WATAN.LBE, Y. (1965). A double stranded. R-lu’A of poliovirus-infected IZeLa cells : Thermal denaturation and annealing. Biochim. BiopiLys. iicta 95, 515-618. WILO, T. F., and BP,OWW, F. (1970). Replication of foot-and-mouth disease virus ribonucleic acid. j, Gen. vi’irol. 7, P-PI. ZIM~\IIERXAS, E. F., HEETER, M., a.nd !~;~RKFM., J. E. (1963). RNA synthesis in poliovirus infected cells. ~&ZOQ?/ 19, 400-408.