JOURNAL OF VIROLOGY, May 1978, p. 485-497 Vol. 26, No. 20022-538X/78/0026-0485$02.00/0Copyright i 1978 American Society for Microbiology Printed in U.S.A.

Virion RNA Species of the Arenaviruses Pichinde, Tacaribe,and Tamiami

A. C. VEZZA, J. P. CLEWLEY, G. P. GARD,t N. Z. ABRAHAM, R. W. COMPANS, ANDD. H. L. BISHOP*

Department ofMicrobiology, The Medical Center, University ofAlabama, Birmingham, Alabama 35294

Received for publication 29 December 1977

The principal RNA species isolated from labeled preparations of the arenavirusPichinde usually include a large viral RNA species L (apparent molecular weight= 3.2 x 106), and a smaller viral RNA species S (apparent molecular weight = 1.6x 106). In addition, either little or considerable quantities of 28S rRNA as well as18S rRNA can also be obtained in virus extracts, depending on the virus stockand growth conditions used to generate virus preparations. Similar RNA specieshave been identified in RNA extracted from Tacaribe and Tamiami arenaviruspreparations. Oligonucleotide fingerprint analyses have confirmed the host ribo-somal Qrigin of the 28S and 18S species. Such analyses have also indicated thatthe Pichinde viral L and S RNA species each contain unique nucleotide sequences.Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfateextraction often have much of their L and S RNA species in the form of aggregatesas visualized by either electron microscopy or oligonucleotide fingerprinting ofmaterial recovered from the top of gels (run by using undenatured RNA prepa-rations). Circular and linear RNA forms have also been seen in electron micro-graphs of undenatured RNA preparations, although denatured viral RNA prep-arations have yielded mostly linear RNA species with few RNA aggregates orcircular forms.

Analyses of the structural components of Pi- as 80S ribosomes of host cell origin (4, 5, 10, 20,chinde virus have established that there is a 21). The function of these ribosomes is unknown.major internal nucleocapsid protein N, (molec- In a recent set of analyses by Rawls and associ-ular weight = 66,000), a large external glycopro- ates, it was shown that Pichinde virus grown intein Gl (molecular weight = 64,000), a smaller cells that have temperature-sensitive ribosomesexternal glycoprotein G2 (molecular weight = yielded progeny virus particles that contained38,000), as well as minor proteins of unknown thermolabile ribosomes (15). These progeny vi-function or location (23, 25). Tacaribe and Tam- ruses were capable of productively infecting cellsiami viruses have a major nucleocapsid protein at temperatures that were nonpermissive for theN (molecular weights = 68,000 and 66,000, re- integrity of those ribosomes.spectively), a single glycoprotein size class G We have recently reported preliminary anal-(molecular weights = 42,000 and 44,000, respec- yses of the RNA species of Pichinde virus prep-tively), as well as some minor protein species at arations grown from high-temperature-pas-least one of which (molecular weights = 79,000 saged, freshly cloned stocks (24, 25). It wasand 77,000, respectively), is associated with the shown that for denatured viral RNA prepara-viral nucleocapsids (11). For all three viruses the tions two major size classes of RNA were pres-purified viral nucleocapsids have a convoluted, ent, L (apparent molecular weight = 3.2 x 106)beaded appearance (11, 25). For Tacaribe virus and S (apparent molecular weight = 1.6 x 106).the nucleocapsids are reported to be circular We did not detect significant quantities of intact(18). 18S rRNA species either in optical scans of theElectron micrographs of ultrathin sections of viral RNA or in preparations of RNA extracted

arenavirus-infected cells have shown that virus from virus grown in cells labeled at various timesparticles characteristically have one or more in- before or after the initiation of infection (25).ternal 20- to 24-nm-diameter granules (1, 16) However, due to the similar, but not identical,that, by a variety of criteria, have been identified electrophoretic mobility of the S RNA to that of

a 28S rRNA marker, although no 28S RNA wast Permanent address: Veterinary Research Station, Glen- detected in virus extracts, no conclusion could

field, N.S.W., Australia. be drawn for the total absence of viral 28S rRNA485

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486 VEZZA ET AL. J. VIROL.

species. Both 28S and 18S rRNA species, as well 25). RNA samples in 0.01 M sodium phosphate-5 mMas a 31S (L), 22S (S) and, in certain circum- EDTA (pH 7.0) were heat denatured at 1000C for 20stances, a 15S viral RNA species have been s and fast cooled in a dry ice-ethanol bath. RNAreported previously for Pichinde virus prepara- species were located on the slab gels by autoradiogra-tions (4, 5, 9, 10). Ribosomal 28S and 18S RNA phy, the bands were cut out, and the RNA was re-as well as 31S and 22S viral RNA species have covered and freed from acrylamide by a modificationas well as poStad vraRNAsprecishav of the procedure described by Jeppesen et al. (14). Gelalso been reported for ruNA preparations ex- pieces in 5 ml of 0.4 M NaCI-50 mM Tris-hydrochlo-tracted from the arenaviruses lymphocytic cho- ride buffer-5 mM EDTA-0.5% sodium dodecyl sulfateriomeningitis virus, and Junin virus (2, 19-22). (pH 7.4), containing 200,ug of Proteinase K (BeckmanThe question of whether 28S and 18S RNA Instruments, Atlanta, Ga.), were homogenized in a 15-content in viral extracts can be varied depending ml siliconized-glass Dounce homogenizer. The mixtureon the conditions of growth, virus stock, etc., has was then centrifuged at 40,000 rpm for 1 h in an SW41been examined in the study reported here. We rotor to aid elution of the RNA from the gel residues.also report analyses of the viral RNA species of The whole slurry (i.e., pellet and supernatant) wasTacaribe and Tamiami viruses. Each virus ap- then mixed and extracted with an equal volume of

contain a large, L, and small, ., viral phenol-m-cresol-chloroform-8-hydroxyquinoline andpears toscies ,a s ana and eithr adjusted to 1% (vol/vol) with respect to,B-mercapto-RNA species, as well as 285 rRNA and either ethanol. The phenol phase, together with the interfacesignificant (Tacaribe) or trace (Tamiami) that contained the majority of the polyacrylamideamounts of 18S rRNA. residues, was then reextracted twice with 0.4 MWe have used oligonucleotide fingerprinting NaCl-50 mM Tris-hydrochloride-5 mM EDTA (pH

to confirm the identity of the viral rRNA species 7.4). The combined aqueous phases were reextractedfor Pichinde and show that its L and S RNA with fresh phenol mixture to further remove polyacryl-species have distinguishable nucleotide se- amide residues and subsequently precipitated with 2.5quences. This observation, if taken to indicate volumes of ethanol at -20°C. The RNA was recoveredquences. Thiso seraio iftakn to indice by centrifugation and dissolved in 1 ml of distilled

gthatithcLi ando

RN si .eahe uique water, 100 ,ug of bacterial tRNA was added, and thegenetic information, is in agreement with our mixture was centrifuged in a Beckman microfuge forrecent report of high-frequency genetic recom- 2 min to remove traces of insoluble acrylamide. Thebination with temperature-sensitive Pichinde supernatant was adjusted to 1% (wt/vol) HC104 andmutants (24). after 15 min at 0°C was centrifuged at 10,000 rpm for

15 min at 4°C in a Sorvall RC5 centrifuge to precipi-

MAITERALUS AND METHODS tate the RNA and leave free acrylamide in solution.The RNA was then dissolved in 0.4 NaCl-50mM Tris-

Viruses. The sources of the cloned and uncloned hydrochloride-5 mM EDTA (pH 7.4) and precipitatedstocks of Pichinde, Tacaribe, and Tamiami viruses with 2.5 volumes of ethanol at -20°C. After centrifu-have been described (11, 24, 25). gation, the RNA was finally dissolved in 15 pl of 0.02

Cell cultures, infection of cells, and virus pu- M Tris-hydrochloride-2 mM EDTA (pH 7.4) andrification. Tacaribe, Tamiami and, on occasions, Pi- stored at -20°C.chinde virus were grown at 370C in roller cultures of Uninfected cell rRNA was obtained as follows. ABHK-21 or Vero cells by using 50 to 80% confluent cell total of 10 large petri dishes of BHK-21 cells at 60%monolayers and the individual growth media described confluency were washed with Eagle minimal essentialpreviously (11, 25). Pichinde virus was usually grown medium, then overlaid with 15 ml of minimal essentialat 35°C in confluent monolayers of BHK-21 cells in medium containing 10% newborn calf serum and 2 mCiBlake bottles (25). The labeling conditions involved of [32P]sodium orthophosphate. After 2 days at 37°C,either 20 MuCi of [3H]uridine (ICN, Irvine, Calif.) per the medium was removed, the cells were washed twiceml of growth medium or 250 MCi of 32P-labeled sodium with ice-cold phosphate-buffered saline, then scrapedorthophosphate (ICN) per ml ofmedium. Except when off the dishes with a rubber policeman and allowed toindicated, the label was usually added with fresh me- swell on ice in 0.01 M Tris-hydrochloride-0.01 Mdium 20 h after the initiation of an infection. Virus was NaCl-1.5 mM MgCl2 (pH 7.4) for 15 min. The cellharvested either 3 or 4 days later. In individual exper- suspension was mixed with Triton N101 (0.5%iments, alternate protocols were employed as indi- (vol/vol), final concentration), and the cells were ho-cated in the text. Tacaribe virus was precipitated from mogenized in a glass Dounce homogenizer. Nucleiclarified cell supernatant fluids by the addition of were removed by centrifugation at 500 x g for 5 min,(NH4)2SO4, whereas Tamiami and Pichinde viruses and the cytoplasmic supernatant was adjusted to 0.15were collected by polyethylene glycol-sodium chloride M NaCl-0.01 M EDTA-1% (wt/vol) sodium dodecylprecipitation (11, 25). Each virus was purified as de- sulfate and extracted with the phenol-m-cresol-chlo-scribed elsewhere (17). roform-8-hydroxyquinoline mixture. The aqueousRNA extraction, melting, and resolution of phase was then adjusted to 0.40 M NaCl and reex-

RNA species by polyacrylamide gel electropho- tracted with the phenol mixture. After precipitationresis. The procedures used to extract RNA from with ethanol, the pellet was resuspended in 0.01 Mpurified virus preparations and resolution of RNA by Tris-hydrochloride (pH 7.4), NaCl was added to give2.0, 2.1, 2.2, or 2.4% (wt/vol) polyacrylamide tube or a 1 M final concentration, and the solution was frozenslab gel electrophoresis have been described (3, 6, 12, in a dry ice-ethanol bath, brought to 00C, and imme-

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VOL. 26, 1978 ARENAVIRUS RNA SPECIES 487

diately spun at 10,000 rpm for 30 min to precipitate virus-infected cells (25; A. C. Vezza, G. P. Gard,single-stranded RNA. The pellet was resuspended in R. W. Compans, and D. H. L. Bishop, In B. W.0.4 M NaCl-0.05 M Tris-hydrochloride-5 mM EDTA J. Mahy and R. D. Barry (ed.), Negative Strand(pH 7.4) containing 1% (wt/vol) sodium dodecyl sul- Viruses and the Host Cell, in press). Whetherfate extracted with phenol, and then precipitated with

aethanol. The RNA pellet was resuspended in 0.02 M any 28S rRNA was present could not be deter-Tris-hydrochloride-0.1 M NaCl-2 mM EDTA-0.1% mined unequivocally due to the proximity of thesodium dodecyl sulfate (pH 7.8), layered onto a 15 to S RNA with the marker 28S rRNA species,30% (wt/vol) preformed sucrose gradient in the same although clearly at most only a small amountbuffer, and centrifuged at 23,000 rpm for 12.5 h at was present (25).20°C. The distribution of radioactivity in the gradient The RNA species of Pichinde virus. Be-was determined, and the 18S and 28S peaks were cause previous analyses that we have conductedpooled and precipitated with ethanol. The RNA was detected no 18S RNA and little 28S RNA inrecovered by centrifugation and after a second ethanol viral RNA preparations obtained from a high-precipitation finally dissolved in 15 A1 of 0.02 M Tris- temperature-passaged, freshly cloned prepara-hydrochloride-2 mM EDTA (pH 7.4). tempichinde , frestion erpreAnnealing and RNase resistance of viral RNA tlon of Pichinde, the question of whether thosespecies. Viral RNA species were annealed and their laboratory manipulations affected the ability ofRNase resistance was determined by using procedures the virus to incorporate rRNA has been inves-described elsewhere (3). tigated by analyzing (i) the RNA species found

Resolution of RNase Ti-derived oligonucleo- in virus grown with the unpassaged Pichindetides by two-dimensional polyacrylamide gel stock, and (ii) the effect of growing the high-electrophoresis. The digestion of 32P-labeled RNA temperature-passaged, cloned Pichinde virus inby RNase T, and the resolution of oligonucleotides by other experimental conditions (other than thosetwo-dimensional polyacrylamide gel electrophoresLs that gave little evidence of 28S and 18S rRNAemployed the procedures developed by De Wachter g25]and Fiers (6, 8). The final positions of the oligonucle- ]J) . .otides were obtained by autoradiography. Our origmal stock of Pichnsdevlrus was re-

Electron microscopy of viral RNA species. cieved from C. Pfau (Rensselaer PolytechnicRNA was spread by a modified Kleinschmidt tech- Institute, Tory, N.Y.), and, without furthernique (7). Support films were prepared from 3.5% treatment (i.e., without high-temperature pas-(wt/vol) Parlodion in amyl acetate on 300-mesh cop- saging or cloning in our laboratory), was used toper grids and used the same day. Approximately 5 pl infect confluent BHK-21 monolayer cultures (inof a solution containing 1 mg of RNA per ml of 0.01 M stationary Blake bottles) in the presence ofsodium phosphate buffer-5 mM EDTA (pH 7.0) was [3H]uridine at an input multiplicity of infectionmixed with 45 pl of a hyperphase of 70% (wt/vol)formamide-0.1 M Tris-hydrochloride-0.01 M EDTA of 0.01 PFU per cell. The progeny virus was(pH 8.2) containing 100 ,g of cytochrome c (horse puried, and its RNA was recovered. When anheart type VI, Sigma Chemical Co., St. Louis, Mo.) unmelted sample of RNA was resolved by poly-per ml. The hyperphase mixture was spread on 15 ml acrylamide gel electrophoresis, the pattern ofof a hypophase of 40% (vol/vol) formamide-0.01 M 3H-labeled viral RNA species shown in Fig. 1ATris-hydrochloride-i mM EDTA (pH 8.2). After 1 was obtained. The profile obtained for a meltedmin the protein film, containing the RNA, was picked viral RNA sample, subjected to coelectropho-up on the Parlodion-coated grids, stained with a solu- resis with 32P-uninfected cell RNA, is shown intion of 0.125 mM uranyl acetate (dissolved in 90% Fig. 1B. In both cases, and in contrast to the[vol/vol] ethanol), and subsequently washed in 90% . ' .(vol/vol) ethanol The samples were then rotary results we obtaed previously, there was a ma-shadowed with platinum-palladium and examined in jor 2vlral RNA species which comigrated withaPiisEM 301 electron microscope. the 3P-labeled 28S cell rRNA. In addition somea Philips EM 301 electron microscope. of the 3H label comigrated with the 18S cell

rRNA species. In both samples viral L and SRESULTS RNA species were also observed. Although the

unmelted RNA sample was subjected to electro-Two principal RNA size classes, L (apparent phoresis in a 2.4% gel and the L RNA only

molecular weight = 3.2 x 106) and S (apparent penetrated a short distance into the gel, it ap-molecular weight = 1.6 x 106), were initially peared that some of the sample remained at thedetected in Pichinde virus preparations grown gel surface. For the melted RNA, which was runin stationary Blake cultures of BHK-21 cell in a 2.2% polyacrylamide gel, essentially none ofmonolayers when a high-temperature-passaged, the viral RNA remained at the gel surface.freshly cloned stock of virus was used (24, 25). Because the data shown in Fig. 1 indicatedOnly trace amounts of labeled 18S rRNA were that there were considerably more 28S and 18Sfound in the viral RNA extracts, although both RNA species present in virus grown from thelabeled 18S and 28S rRNA species were re- original virus stock than from our high-temper-covered in normal proportions from Pichinde ature-passaged, cloned stock grown under iden-

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488 VEZZA ET AL. J. VIROL.

A Pichinde Prototype-Urwnelted B Pichinde Prototype Melted

0 4 6 20 84

15 6-H

DISTANCE~ ~ -0OE(c)D0-NEUOED(m

to -;

NN

i8 55 2-

2 4 6 2 4 6

DISTANCE MOVED (cm) DISTANCE MOVED (cm)

FIG. 1. RNA species extracted from Pichinde virus. I3H]uridine-labeled preparations of Pichinde virusobtained from viral infections initiated with an uncloned virus stock were extracted for RNA and mixed witha 32P-labeled uninfected cell extract and either resolved directly by 2.4% polyacrylamide gel electrophoresis(A) or heat denatured and then resolved on a 2.2%o gel ofpolyacrylamide (B).

tical conditions (25), the effect of alternate in Fig. 1), it appeared that the amount of labeledgrowth conditions on the RNA species obtained 28S and 18S RNA species recovered from Pi-with our high-temperature-passaged, cloned vi- chinde virus preparations could be varied withrus was examined. Pichinde virus was grown, the conditions of growth. Also, it appeared thattherefore, at 370C in roller cultures of BHK-21 under some circumstances aggregates of viralcell monolayers at a multiplicity of infection of RNA, probably not involving the rRNA species,1 PFU per cell. The infection employed 50% were frequently present in RNA extracts. Sub-confluent cell monolayers and utilized the high- sequent experiments involving oligonucleotidetemperature-passaged, cloned virus stock. To fingerprinting indicated that the aggregatedlabel the virus, [3H]uridine was incorporated RNA did indeed contain viral L and S RNAinto the growth medium from the beginning of species but not significant amounts of 28S RNAthe infection timne course. RNA was extracted (see below).from purified virus and, with or without melting, RNA species of Tamiami and Tacariberesolved by polyacrylamide gel electrophoresis viruses. Preparations of [3H]uridine-labeled(Fig. 2A and B). For the melted RNA samples Tacaribe and Tamiami viruses were obtained by(Fig. 2B), viral L and S RNA species were ob- using roller cell cultures of BHK-21 cells, andserved, as well as significant but small quantities the virus was harvested 4 days later. A prepa-of 28S RNA that migrated with the 28S cell ration of32P-labeled Pichinde virus was obtainedribosomal marker. A small amount of 18S RNA from BHK-21 cell monolayers grown in Blakewas also observed. Noteworthy was the fact that bottles.a proportion (about 20%) of the unmelted RNA When the RNA extracted from Tamiami virussample (Fig. 2A) remained at the surface of the was melted and subjected to coelectrophoresisgel, whereas for the melted sample, relative to in a 2.4% polyacrylamide gel with either 32P-the 28S and 18S species, the amounts of L and labeled uninfected cell RNA (Fig. 3A), or "p-S RNA species increased (Fig. 2B). A similar labeled Pichinde RNA (Fig. 3B), two major sizeresult, but with more total 28S RNA, was ob- classes of Tamiami viral RNA were observed.tained for Pichinde virus grown in Vero cells These two RNA species had electrophoretic mo-(Fig. 2C and D). For the unmelted Vero-grown bilities similar to those of the Pichinde L and SRNA sample, the L RNA was too close to the RNA species. No significant quantities oflabeledgel surface to be resolved as a distinct entity, or 18S rRNA was detected in the Tamiami extract,to determine whether (again relative to the although whether 28S RNA was present couldrRNA species) it increased upon melting, how- not be determined due to the similar electropho-ever the S RNA clearly increased upon melting retic mobilities of the 28S cell rRNA speciesrelative to the 28S RNA species. When the with the faster-moving viral S RNA species (Fig.results of these experiments were compared with 3A).the data described previously (25, and those seen Coelectrophoresis of [3H]uridine-labeled Ta-

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VOL. 26, 1978 ARENAVIRUS RNA SPECIES 489

caribe virus with 32P-labeled Pichinde RNA gave cell RNA, and subjected to electrophoresisan RNA profile in which Tacaribe L and S type either directly on 2.2% polyacrylamide gels orRNA species were evident (Fig. 3C). In addition after melting. The data obtained for the 3-daythere were Tacaribe RNA species with an elec- virus harvests are shown in Fig. 4. For bothtrophoretic mobility like that of 18S rRNA spe- viruses a substantial amount of the viral RNAcies. RNA aggregates were also evident at the in the unmelted samples remained at the geltop of the gel (Fig. 30). surface. For Tacaribe virus, some 28S and 18SThe effect of melting the RNA species of RNA species were evident in both the melted

Tacaribe and Tanamia viruses on the resulting and unmelted RNA samples. For Tamiami virus,RNA profile was also examined as a function of a large amount of labeled 28S RNA species, butthe time of virus harvest. Three-day harvests of no 18S RNA species, was present. For both virusboth viruses were obtained, the media were re- samples, relative to the 28S RNA (and 18S RNAplaced with fresh radioactive media, and a sec- for Tacaribe virus), the heat-denatured samplesond virus harvest was collected 2 days later. The gave substantially more radioactivity in the LRNA was extracted from each purified virus and S RNA peaks than the unmelted samples.preparation, mixed with 32P-labeled, uninfected Because a large proportion of the unmelted

A Possoged Pichmnde BHK Rollers B Possoged PichnleBWK Relsve N-15 28!

10 32p 10 60 s

L L

45 45

-o 010 283 K,30. 30.

CL 0.~~~~~~.

91~~~~~~~~~~~~~~~~~1

2 4 6 8 2 4 6DISTANCE MOVED (cm) DISTANCE MOVED (cm)

CPaossged Pchinde VeraRollers D Possoged PichindefVERO Rollers Melted28 L9

ID .10 20-20

3H

.0~~~~~~~ 03 ~~~5~g10. 10.

32F

2 4 6 8 2 4 6DISTANCE MOVED (cm) DISTANCE MOVED km)

FIG. 2. Viral RNA obtained fr-om Pichinde virus grown in roller cultures of BHK-21 or Vero cells. R'H]-uridine-labeled preparations of Pichinde viral RNA were extracted fr-om purified virus obtained fr-om rollercultures ofBHK-21 (A and B) or Vero cells (C and D) that had been infected with a cloned stock ofPichindevirus at 50% cell confluency and harvested 4 days later. The RNA profiles obtained for native (A and C) orheat-denatured (B and D) mixtures of 3H-labeled viral RNA and 32P-labeled uninfected cell RNA wereresolved by electrophoresis in 2.4% (A and C) or 2.2% (B and D) polyacrylamide gels.

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490 VEZZA ET AL. J. VIROL.

A. Tamiami Viral RNA B. Tamiami and Pichinde C Tacaribe and Pichinde45 RNA Coelectrophoresis RNA Coelectrophoresis

28s510 20 20 60 8 40

-48 -b'-15 05?15, 6- 30uDAEM.CD ED0~~~~~~~~~~~~~~H36 L- r1 '_,

labeled Tacrib a 3a0:Pc4v203 ~~~~~~~~~3N-' E 32p, 24~

'I 32p C)

H~~~~~~~~~~~~~~~~~~~~~~~~~~5 12 2 3H101

2 4 2 4 2 4DISTANCE MOVED(cm) DISTANCE MOVED(cm) DISTANCE MOVED(cm)

FIG. 3. Viral RNA species of Tamiami, Tacaribe, and Pichinde viruses. [3H]uridine-labeled preparationsof Tamiami and Tacaribe viruses obtained from roller cultures of BHK-21 cells (see text) were extracted forRNA. A portion of the Tamiami viral RNA was mixed with 32P-labeled uninfected cell RNA and resolved by2.4% polyacrylamide gel electrophoresis (A). Another portion of Tamiami viral RNA was mixed with 32P-labeled Pichinde RNA and then resolved by electrophoresis in a 2.4%polyacrylamide gel (B). A mixture of 'H-labeled Tacaribe and 32P-labeled Pichinde viral RNA was likewise resolved by 2.1% polyacrylamide gelelectrophoresis (C).

RNA for each virus preparation remained at the an oligonucleotide fingerprint analysis of angel surface, this again suggested that in the RNA species having a complexity of 106 daltonsunmelted RNA samples there were aggregates (unpublished data). Consequently, to obtainof both the viral L and S RNA species. enough of the individual viral RNA species forThe melted versus unmelted profiles of RNA Pichinde, we have resorted to slab gel electro-

obtained from Tacaribe (Fig. 5), and Tamiami phoresis to separate and recover the major viral5-day virus harvests (data not shown), gave sim- RNA size classes (Fig. 7). 32P-labeled Pichindeilar data except that many more RNA bands virus RNA obtained from virus grown in roller-were present! The origin or relationships of these bottle cultures was either melted or left unde-RNA species to the viral L and S RNAs, or natured and then resolved on a 2.2% polyacryl-cellular RNA species, is not known. amide slab gel as shown in Fig. 7. Three majorOligonucleotide fingerprint analyses of viral RNA species were recovered from the

BHK-21 28S and 18S rRNA species. 32P-la- melted sample. These corresponded, in order ofbeled 28S and 18S rRNA species were extracted decreasing size, to the L, 28S, and S RNA spe-from uninfected BHK-21 cells after growing 60% cies. A small quantity of 18S RNA was alsoconfluent cell monolayers in the presence of 133 recovered from the gel, as was the aggregated,iCi of [32P]sodium orthophosphate per ml of RNA from the gel surface of an unmelted RNAgrowth medium for 2 days at 37°C. The 28S and sample. Each RNA species was digested with18S rRNA species were recovered (see above) RNase T1, and the resulting oligonucleotidesand, after digestion with RNase T1, the resulting were resolved by two-dimensional polyacryl-oligonucleotides were resolved by two-dimen- amide gel electrophoresis (Fig. 8).sional polyacrylamide gel electrophoresis (Fig. When the fingerprint of the viral 28S RNA6). By comparison with the final positions of the (Fig. 8G) was compared with that of the unin-two dye markers, bromophenol blue (Fig. 6, X, fected cell 28S rRNA (Fig. 6B), it was apparentupper center) and xylene cyanol FF (Fig. 6, X, that the viral 28S RNA was ribosomal in origin.lower left), most of the labeled oligonucleotides Likewise, the 18S viral RNA that was recoveredwere recovered in the upper half of the electro- (Fig. 8H) appeared to contain 18S rRNA. Bothpherograms. samples contained trace amounts of oligonucle-Oligonucleotide fingerprint analyses of otides that appeared to be viral L and S in origin,

the viral RNA species recovered from Pi- presumably originating from degraded or, in thechinde virus. We have found that at least 2 x case of the 28S sample, incompletely separated105 cpm of 32P-labeled viral RNA is needed for viral RNA species.

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A Tacorib Doy 3-Lmed 8 Tocoibe Day 3-MIted25 6 12 8 m20

S5 ~~~~~~~~10L

646124

26

x x~~~~~~~~I0

2 lrACL OE (cm lSjcMOE (m

2 2 4

2

2p2

2 4 6 2 4 6DISTANCE MOVED be) DISTANCE MOVED (cm)

C Tamiem Day 3-I.nmelted D Tom.or Day 3-Melted20 41256 5

I ITAC MVD(mDISTANEMVD(m

10 104asa4

gels.

4~~~~~~~9

4~~~~~~#32p~~~~~~~~X~~~~~~~

2 4 6 2 4 6

DISTANCE MOVED (cm) DISTANCE MOVED (cm)

FIG. 4. Viral RNA obtained fr-om 3-day harvests of Tacaribe and Tamiami viruses. 13H]uridine-labeledTacaribe (A and B) or Tamiami (C and D) virus obtained fr-om 3-day virus harvests grown in roller BHK-21cultures was extracted for RNA, and portions were mixed with UP-labeled uninfected cell RNA and eitherrun directly (A and C) or after heat denaturation (B and D) on 2.2%polyacrylamide gels.

A. Tocoribe Day 5-unmelted B. Tocoribe Day 5-Melted20 .12 4- 105

I ~~~~~~~~~L5 83-

3H ~~~8

6N 2-~ E2

32P~~~~~~~~~~~22 3 2

2 4 6 2 4 6DISTANCE MOVED (cm) DISTANCE MOVED (cm)

FIG. 5. Viral RNA obtained fr-om a 5-day harvest of Tacaribe virus. f'H]uridine-labeled Tacaribe virusobtained as a 3- to 5-day harvest fr-om roller BHK-21 cells was extracted for RNA, mixed with 'P-labeleduninfected cell RNA, and either resolved directly (A) or after heat denaturation (B) on 2.2%polyacrylamide

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492 VEZZA ET AL. J. VIROL.

18s 28s

-T

-~~~~

*41-0*J..

4,

.~~ ~ ~ ~ ~~ I

3r

40

18s + 28 sFIG. 6. Oligonucleotide fingerprints of28S and 18S BHK-21 cell RNA species. "9P-labeled preparation,s of

purified 28S and 18S were obtained as described in the text. Portions of the 18S RNA (top left panel), 28SRNA (top right panel), or mixtures of both species (bottom right panel) were digested with RNase Ti, and theresulting oligonucleotides were resolved by two-dimensional polyacrylamide gel electrophoresis (6, 8). Thepositions of the oligonucleotides were determined by autoradiography. The finalpositions ofthe marker dyes,xylene cyanol FF (X, lower left) and bromophenol blue (X, upper center), are indicated. A schematic compositeof the 18S oligonucleotides (-) and -28S oligonucleotides (O) representing the positions of their respectiveoligonucleotides in the digest of the 28S and 18S mixture (bottom rightpanel) is given in the bottom left panel.The faint spots in the 28S pattern probably represent oligonucleotides derived from contaminating cellularnonribosomal RNA species.

Comparison of the L RNA oligonucleotide and S digests in Fig. 8E). Likewise, there werefingerprint (Fig. 8C) with that of the S RNA several in the L RNA fingerprint (some of which(Fig. 8D) indicated that there were several oli- are indicated by stars in Fig. 8C) that were notgonucleotides (some of which are indicated by present in the S RNA fingerprint. The aggre-arrows in Fig. 8D) in the S RNA that were not gated RNA was found to have both L and S typepresentinthe L fingerprint (see schematic com- oligonucleotides but no 28S oligonucleotidesposite in Fig. 8F and coelectrophoresis of the L (Fig. 8B), whereas the fingerprint of the total

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VOL. 26, 1978 ARENAVIRUS RNA SPECIES 493

viral RNA extract (Fig. 8A) clearly contained r RNA unr meltedboth L, S, and 28S oligonucleotides (certain of TOP Nmethe 28S oligonucleotides being indicated by ar-rows in Fig.8A).-

Oligonucleotide fingerprints have not been ob- 2_tained for Tacaribe and Tamiami viruses due tothe difficulties of obtaining enough of their ra-dioactive RNA for such analyses.Electron microscopy of the arenavirus

RNA samples. When unmelted preparations ofTacaribe, Pichinde, or Tamiami virion RNAswere examined by electron microscopy, many ofthe observed molecules were in the form ofcomplex structures apparently consisting of ag- I 8Sgregates of multiple individual RNA molecules.Some smaller complexes are shown in Fig. 9Aand B; in addition much larger aggregates werefrequently seen. Closed circular molecules (Fig.9A, D, and E) as well as linear and hairpin forms(Fig. 9C) were also observed in unmelted RNApreparations. After melting, the number of largeaggregates was reduced markedly, and mostRNA was observed as individual linear with few FIG. 7. Pichinde viral RNA resolved by slab poly-,

> ,. . ................... .acrylamide gel electrophoresis. 32P-labeled Pichindecircular forms. Both circular and linear mole- a gcules were observed with lengths ranging from virus grown in roller cultures of BHK-21 cells (see1.3 to17

were Howsever, mostlinearrangmgwell Fig. 2) was extracted for RNA. A portion was sub-1.3 to 1.7 ,utm. However, most linear as well as jected to electrophoresis in a 2.27o slab polyacryl-the few residual circular molecules in melted amide gel (center lane). An identical sample was heatpreparations were less than 1.0 ,um in length, denatured and run in the right lane. A portion of32P-suggesting that fragmentation of molecules had labeled uninfected cell RNA was run in the left lane.occurred during preparative procedures. Further After electrophoresis thepositions ofthe various RNAstudies ofthe distribution of contour lengths and species were determined by autoradiography. The topcircular versus linear forms will be worthwhile of the gel and the final positions of the 28S and 18Sifunfragmented RNA preparations are obtained. RNA species are indicated.The RNase resistance of Pichinde, Tacar-

ibe, and Tamiami viral RNA species. With Tamiami viruses two viral RNA species havethe observation of RNA complexes in Tacaribe, been observed (L and S). Frequently, the virusesTamiami (Fig. 9), and Pichinde (data not shown) contain small to large quantities of 28S RNAviral RNA extracts, and aggregates in undena- that, when present in Pichinde preparations, hastured RNA samples (see Fig. 1 through 5), the been shown to have an oligonucleotide finger-question of whether RNA samples contained print identical to that of uninfected cell 28Ssignificant quantities of complementary RNA rRNA. An 18S RNA is also found in some prep-was examined. The RNase resistance of Pi- arations of Tacaribe and Pichinde. Again, atchinde, Tacaribe, and Tamiami viral RNA ex- least for Pichinde virus, the 18S RNA has antracts were determined by using native, heat- oligonucleotide fingerprint like that of unin-denatured and self-annealed samples (Table 1). fected cell 18S rRNA. Most preparations ofAll the native RNA samples had significant Tamiami and Pichinde viruses (but not Tacaribeamounts of RNase-resistant RNA. After heat virus) have less labeled 18S RNA than onedenaturation, much lower levels of residual would predict based on the count ratios ofRNase resistance were obtained. Self-annealed marker 18S and 28S RNA. This has also beenRNA preparations gave for the most part levels noted by comparing the ratios of labeled 28S toof RNase resistance slightly higher than the 18S ratios found in infected cell extracts (A. C.native samples (Table 1). Different preparations Vezza, G. P. Gard, R. W. Compans, and D. H. L.of Tacaribe or Pichinde virus gave RNA samples Bishop, In B. W. J. Mahy and R. D. Barry (ed.),containing quite widely different quantities of Negative Strand Viruses and the Host Cell, inRNase-resistant RNA (Table 1). press). These data could be explained by the

specific exclusion of labeled 18S rRNA fromDISCUSSION assembly into virions. However, this possibility

In all preparations of Pichinde, Tacaribe, and seems unlikely in view of the previous reports

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VOL. 26, 1978 ARENAVIRUS RNA SPECIES 495

concerning the recovery of 80S ribosomes from TABLE 1. RNase resistance of Pichinde, Tacaribe,Pichinde preparations (10, 15). An alternative and Tamiami viral RNA species"explanation could be that 18S RNA in virion % RNase resistanceribosomes is frequently degraded or nicked so Viral RNAthat it is not recovered as intact 18S molecules Native Heat dena- Self-an-after phenol extraction. This possibility is cur- tured nealedrently under investigation. PichindeWe have observed variable recoveries of 28S Sample 1 16 2 20

RNA from Pichinde preparations (25, and com- Sample 2 27 2 29pare the melted RNA profiles in Fig. 1 and 2). Sample 3 23 10 29With our high-temperature-passaged, cloned vi- Tacariberus stock, even when grown in roller-cell cultures Sample 1 8 4 6by using semiconfluent cell monolayers, we re- Sample 2 25 13 24cover much less 28S and 18S RNA than with anuncloned virus stock. One possible reason could Tamiami 5 2 4be that the initial progeny from cloned virus Portions of [3H]uridine-labeled viral RNA extractsstocks lack or have few ribosomes. Another pos- (0.2 to 1 mg/ml, lo-pL samples containing 1 x 104 to 10sibility could be that rRNA species are unstable x 104 cpm in 0.01 M sodium phosphate-5 mM EDTA,in certain virus preparations. These possibilities pH 7.0) were either untreated or diluted with 90 Al ofare also currently under investigation. the same buffer and heat denatured at 100°C for 20 s,

Oligonucleotide fingerprinting has been used or mixed with 10 p1 of 1 M sodium phosphate (pH 7.0)to show that the L and S RNA species of Pi- and self-annealed at 60°C for 2 days. The RNA sam-chinde have distinguishable oligonucleotides ples were then diluted with 1 ml of 0.4 M NaCl-0.01and presumably different genetic information. M Tris-hydrochloride; one half was precipitated withThis result agrees we. with our recent demon- 5% (wt/vol) trichloroacetic acid, and the other halfstis result agrees wel witnour recent demon- was digested with RNase A (10 jg of RNase A, 37"Cstration of high-frequency genetiC recombina- for 30 min), before 5% (wt/vol) trichloroacetic acidtion with temperature-senSitive, conditional-le- precipitation (3). The percent acid-insoluble materialthal Pichinde mutants (24). The observation remaining after RNase treatment is given.that the temperature-sensitive mutants of Pi-chinde could be categorized into two nonover-lapping groups of mutants also agrees well with after sample preparation precludes our drawingthe presence of two viral RNA species for Pi- any conclusion of whether the L and/or S RNAchinde. No oligonucleotide fingerprints have species could be covalently closed or end-hydro-been obtained for the various Tacaribe and gen-bonded structures. Further studies involv-Tamiami viral RNA species due to the difficul- ing analyses of the individual L and S RNAties in obtaining adequate amounts of suitably species eluted from gels to answer this questionlabeled virus. are in progress.Both circular and linearRNA forms have been Aggregates of viral RNA have been observed

observed for Tacaribe and Tamiami viral RNA by both gel electrophoresis of undenatured RNApreparations as well as for Pichinde (data not samples and by electron microscopy. The aggre-shown). Circular RNA species have been ob- gated RNA obtained from the top of gels has aserved for the bunyavirus Uukuniemi virus (13), fingerprint consisting of L and S RNA oligonu-and circular nucleocapsids have been seen in cleotides. RNase resistance analyses indicatepreparations of Tacaribe virus (18). Although that complementary RNA sequences are fre-heat-denatured Tacaribe RNA preparations quently present in the viral RNA extracts, al-lacked aggregated RNA forms and contained though whether these complementary se-mostly linear structures, the reduced lengths quences are present in the same RNA molecule

FIG. 8. Oligonuckeotide fingerprint analyses of the viral RNA species of Pichinde virus. 3P-labeled RNAwas resolved as described in the legend to Fig. 7. The RNA bands corresponding to the L, 28S, S, 18S, andaggregates at the top ofthe gel (see text and Fig. 7) were recovered and extracted for RNA, and the RNase T1 -

derived oligonucleotides were resolved by two-dimensional polyacylamide gel electrophoresis. The finalpositions ofthe oligonucleotides were identified by autoradiography, and thepositions ofthe two dye markersare indicated (see Fig. 6). The oligonucleotide fingerprints of the total viral RNA (A), the aggregates (B), theL RNA (C), the S RNA (D), a mixture of the digests ofL and S RNA (E), a schematic representation of the L(0) and S (0) oligonucleotides in the Epanel (F7, the 28S RNA (G), and 18S RNA (H) are given. The largest28S oligonucleotides in the total RNA digest are indicated by large arrows in panel A. Certain unique Loligonucleotides are indicated by stars inpanels C and E. Certain unique S oligonucleotides are indicated bythin arrows in panels D and E.

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VOL. 26, 1978 ARENAVIRUS RNA SPECIES 497

or are located on separate molecules has not ribosome-like structures from Pichinde virus. J. Gen.been determined. Self-annealing of the purified, Virol. 26:21-31.individual L and S species and mixtures of the 11. Gard, G. P., A. C. Vezza, D. H. L. Bishop, and R. W.Compans. 1977. Structural proteins of Tacaribe andsame will be needed to resolve that question. Tamiami virions. Virology 83:84-96.

ACKNOWLEDGMENTS 12. Hefti, E., and D. H. L. Bishop. 1975. The 5' nucleotidesequence of vesicular stomatitis viral RNA. J. Virol.

This research was supported by Public Health Service 15:90-96.grants AI 13402 and AI 14183 (from the National Institute of 13. Hewlett, M. J., R. F. Pettersson, and D. Baltimore.Allergy and Infectious Diseases), CA-13148 and CA-18611 1977. Circular forms of Uukuniemi virion RNA: an(from the National Cancer Institute), and grant no. VC149B electron microscopic study. J. Virol. 21:1085-1093.from the American Cancer Society. N.Z.A. was supported in 14. Jeppesen, P. G. N., B. G. Barrell, F. Sanger, and A.part by training grant T32 GM07164 from the National Insti- R. Coulson. 1972. Nucleotide sequences of two frag-tute for General Medical Sciences. ments from the coat protein cistron of bacteriophageWe thank Tery Cox and John Fitzpatrick for excellent R17 ribonucleic acid. Biochem. J. 128:993-1006.

technical assistance. 15. Leung, W.-C., and W. E. Rawls. 1977. Virion associated

LITERATURE CITED ribosomes are not required for the replication of Pi-chinde virus. Virology 87:174-176.1. Abelson, H. T., G. H. Smith, H. A. Hoffman, and W. 16. Murphy, F. A., P. A. Webb, K. M. Johnson, S. G.

P. Rowe. 1969. Use of enzyme labeled antibody for Whitfield, and W. A. Chappell. 1970. Arenaviruses inelectron microscope localization of lymphocytic chorio- Vero cells: ultrastructural studies. J. Virol. 6:507-518.meningitis virus antigens in infected cell cultures. J. 17. Obijeski, J. F., A. T. Marchenko, D. H. L. Bishop, B.Natl. Cancer Inst. 42:497-515. W. Cann, and F. A. Murphy. 1974. Comparative

2. Ani6n, M. C., 0. Grau, Z. Martinez Segovia, and M. T. electrophoretic analysis of the virus proteins of fourFranze-Fernandez. 1976. RNA composition of Junin rhabdoviruses. J. Gen. Virol. 22:21-33.virus. J. Virol. 18:833-838. 18. Palmer, E. L., J. F. Obijeski, P. A. Webb, and K. M.

3. Bishop, D. H. L., and P. Roy. 1971. Properties of the Johnson. 1977. The circular, segmented nucleocapsidproduct synthesized by vesicular stomatitis virus parti- of an arenavirus-Tacaribe virus. J. Gen. Virol.cles. J. Mol. Biol. 58:799-812. 36:541-545.

4. Carter, M. F., N. Biswal, and W. E. Rawls. 1973. 19. Pedersen, L. R. 1970. Density gradient centrifugationCharacterization of the nucleic acid of Pichinde virus. studies on lymphocytic choriomeningitis virus and onJ. Virol. 11:61-68. viral ribonucleic acid. J. Virol. 6:414-420.

5. Carter, M. F., F. A. Murphy, J. P. Brunschwig, C. 20. Pedersen, L. R. 1971. Lymphocytic choriomeningitis virusNoonan, and W. E. Rawls. 1973. Effects of actino- RNAs. Nature (London) New Biol. 234:112-114.mycin D and ultraviolet and ionizing radiation on Pi- 21. Pedersen, I. R. 1973. Different classes of ribonucleic acidchinde virus. J. Virol. 12:33-38. isolated from lymphocytic choriomeningitis virus. J.

6. Clewley, J. P., J. Gentsch, and D. H. L. Bishop. 1977. Virol. 11:416-423.Three unique viral RNA species of snowshoe hare and 22. Pedersen, L. R. 1973. LCM virus, its purification and itsLa Crosse bunyaviruses. J. Virol. 22:459-468. chemical and physical properties, p. 13-23. In F. Leh-

7. Davis, R. W., M. Simon, and N. Davidson. 1971. Elec- mann-Grube (ed.), Lymphocytic choriomeningitis virustron microscope heteroduplex methods for mapping and other arenaviruses. Springer, Berlin.regions of base sequence homology in nucleic acids. 23. Ramos, B. A., R. J. Courtney, and W. E. Rawls. 1972.Methods Enzymol. 21:413-428. Structural proteins of Pichinde virus. J. Virol.

8. De Wachter, R., and W. Fiers. 1972. Preparative two- 10:661-667.dimensional polyacrylamide gel electrophoresis of 32P- 24. Vezza, A. C., and D. H. L. Bishop. 1977. Recombinationlabeled RNA. Anal. Biochem. 49:184-197. between temperature-sensitive mutants of the arenavi-

9. Dutko, F. J., E. A. Wright, and C. J. Pfau. 1976. The rus Pichinde. J. Virol. 24:712-715.RNAs of defective interfering Pichinde virus. J. Gen. 25. Vezza, A. C., G. P. Gard, R. W. Compans, and D. H.Virol. 31:417-427. L. Bishop. 1977. Structural components of the arena-

10. Farber, F. E., and W. E. Rawls. 1975. Isolation of virus Pichinde. J. Virol. 23:776-786.

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