JOURNAL OF VIROLOGY, Aug. 1970, p. 243-252Copyright ( 1970 American Society for Microoiology

Vol. 6, No. 2Pri,tted in U.S.A.

Morphogenesis of the Nucleoprotein of VesicularStomatitis Virus

BARBARA A. ZAJAC AND KLAUS HUMMELER

Divisionz of Experimenttal Pathology, Thle C'hildreii's Hospital of Plhiladelphia, Department of Petliatrics,Medical School, University of Pennlzsylvaniia, Plhiladelphia, PennsYlvanlia 19146

Received for publication 12 June 1970

Accumulation of the nucleoprotein of vesicular stomatitis virus (VSV) in thecytoplasm of BHK-21 cells and in two of four human cell lines was demonstrated.Appearance and progression of the nucleoprotein inclusions paralleled developmentof virus-specific immunofluorescence and production of virus progeny. The inclu-sions appeared early as discrete foci of filamentous material which eventually in-creased in size to form large masses which replaced normal cytoplasmic constitu-ents. The filamentous strands were found in close proximity to budding virions. Theinclusion material was extracted from infected cells and purified in cesium chloridegradients. The isolated filaments resembled the ribonucleoprotein isolated frompurified virions. They incorporated 3H-uridine, exhibited virus-specific complement-fixing activity, had a buoyant density of 1.32 g/cm3, and appeared as single wavy

strands the width of which varied from 2.5 to 8.5 nm, depending on the angle ofviewing.

The morphology of vesicular stomatitis virus(VSV) has been investigated extensively (6, 8, 10,14, 17). The ribonucleoprotein of the virus, whichis contained in the virion in helical form, has alsobeen analyzed for its morphological, physical, andchemical properties (2, 13, 15, 19, 21). Little isknown, however, about the morphogenesis of theribonucleoprotein of this virus.

Studies concerned with the morphogenesis ofthe ribonucleoprotein of rabies, a virus morpho-logically similar to VSV, have shown that accumu-lation of the ribonucleoprotein as cytoplasmicinclusions is a regular occurrence. It has also beenfound that the size of these inclusions dependedon the strain of rabies virus employed and thetype of host cell used for infection (11). The find-ing of "rabies-like" inclusions in VSV-infectedcalf kidney cells (17) suggested that the morpho-genesis of the ribonucleoprotein of VSV alsomight be readily investigated, depending on thehost cell used.The present studies were undertaken, therefore,

to investigate the morphogenesis of the ribo-nucleoprotein of the Indiana strain of VSV inseveral human cell lines as well as BHK-21 cells.The results presented show that cytoplasmic in-clusions consisting of filaments, identified as theribonucleoprotein of VSV, can be produceddepending on the host cell used for infection.

MATERIALS AND METHODSTissue culture. A BHK-21 line of Syrian hamster

kidney cells was grown on monolayers in Blakebottles in a previously described medium (16), whichconsisted of a mixture of basal medium Eagle (BME,2X) in Hanks balanced salt solution (HBSS), 10%fetal calf serum (FCS), and 10¾( tryptose phosphatebroth.Human amnion cells (AV3) were purchased from

Flow Laboratories, Inc., Rockville, Md, and humanforeskin fibroblasts (HFS) were obtained throughthe courtesy of K. Paucker. Both lines were main-tained as described for BHK-21 cells.

The RPMI-6410 (12) and SK-LI (3) lympho-blastoid cell lines were grown and maintained as sus-pension cultures in medium RPMI 1640 supple-mented with 10%S fetal calf serum as previouslydescribed (23).

Virus. Plaque purified stocks of the Indiana strainof VSV were derived from infected BHK-21 cells.These preparations had titers ranging from 5 X 108to 2 X 109 plaque-forming units (PFU)/ml.

Infection of cells with virus. Monolayers of cells(2 X 106) in 30-mi plastic tissue culture flasks wereinfected at a multiplicity of 10 in the medium describedabove. Virus was allowed to adsorb for 1 hr at 37 C,the monolayers were rinsed twice with HBSS, andfresh medium was added. The cultures were incubatedat 37 C and harvested at given intervals.

For infection of lymphoblastoid cells, 10 X 106cells were sedimented and resuspended in I ml of viruscontaining an input multiplicity of 10. After incuba-

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tion, with intermittent shaking for 1 hr at 37 C, thecells were sedimented, washed twice with HBSS,resuspended in growth medium to a concentrationof 106 cells per ml, and incubated at 37 C. Cultureswere harvested 18 hr after infection.

Immunofluorescence assay. Monolayers of cells in60-mm petri dishes, containing four cover slips (6 by30 mm), were infected as described above. The coverslips were harvested at given intervals after infection.Preparation and staining of the lymphoblastoid cells,as well as of infected cover-slip cultures, have beendescribed (9). All smears were stained with a fluoresceinisothiocyanate-conjugated rabbit anti-VSV globulin.

Infectivity assay. Plastic petri dishes (60 mm) wereseeded with BHK-21 cells in the above medium. Theywere incubated at 37 C in a 5% CO2 atmosphere.Confluent monolayers were apparent after 24 hr. Atthis time, the medium was drained and the cells wereinoculated with 0.2 ml of the various dilutions ofvirus. After an adsorption period of 1 hr at 37 C, theinfected monolayers of cells were overlaid with 5 mlof medium supplemented with 1.2%/o Difco agar andneutral red at a final dilution of 1:40,000. The ap-parent plaques were enumerated 48 hr later, and titerswere expressed as the number of plaque-forming unitsper milliliter.

Nucleoprotein extraction. A modified proceduresimilar to that described by Compans and Choppin(4) was used. Host cell ribonucleic acid (RNA) syn-thesis was inhibited by treating monolayers of BHK-21 cells with 1 /g of actinomycin D (Merck & Co.,Inc, West Point, Pa.) per ml for 4 hr at 37 C (18).The monolayers were then rinsed and infected withVSV as described. After virus adsorption for 1 hr at37 C, the cultures were refed with fresh medium con-taining uridine-5-3H (Schwarz BioResearch, Orange-burg, N.Y.; specific activity, 20 Ci/mmole) at aconcentration of 1 uCi/ml and incubated at 37 C.Sixteen hours after infection, the cells were harvestedby scraping with a rubber policeman, pelleted by low-speed centrifugation, rinsed twice with phosphatebuffered saline (7), resuspended in distilled water,and left at 4 C for 1 hr. The supernatant, after cen-trifugation at 900 X g for 20 min, was treated withdeoxyribonuclease (20 jg/ml) for 30 min at roomtemperature, in the presence of Mg'+, and thenclarified by low-speed centrifugation. CsCl (2.0 g)was added to 4.5 ml of the resulting supernatant, andthe solution was centrifuged at 140,000 X g for 24 hrin a Spinco SW 50 L rotor. Fractions were collectedfrom the bottom in 0.17-ml amounts. The nucleo-protein-containing fractions were located by measur-ing radioactivity and optical density. For measuringradioactivity, 25-,uliter samples were dispensed intovials containing Bray's solution (1) and counted in aTri-Carb liquid scintillation spectrometer. Opticaldensities were determined in a DB spectrophotometer.Before examination in the electron microscope, frac-tions were dialyzed overnight at 4 C against 0.13 M

NaCI, 0.05 M Tris (NT) buffer (pH 7.8).Complement fixation. Complement fixation (CF)

tests, were performed through the courtesy of S.Mazzur. Mouse ascites-fluids hyperimmune to VSVwere kindly supplied by F. A. Murphy. The mouse

hyperimmune fluids reacted both with coat and inter-nal components of the virus but not with BHK-21cell antigens. Titers were expressed as the reciprocal ofthe highest dilution of antigen showing complement-fixing activity, against 2 units of the hyperimmunefluids.

Electron microscopy. At the predetermined inter-vals, the medium was decanted from the cultures, thecells were washed with phosphate-buffered saline,and 1% phosphate-buffered osmic acid was added.After 5 min, cells were scraped into the fixative bvmeans of a rubber policeman and were pelleted. Thecell pellet was dehydrated in ethyl alcohol andembedded in epoxy resin. Thin sections were stainedwith lead citrate and uranyl acetate and examined ina Siemens Elmiskop at a magnification of X 10,500.

For examination by negative contrast, a drop ofthe dialyzed material was transferred to a carbon-coated Formvar grid by means of a platinum loop. Adrop of pho3photungstic acid (pH 6.8) was added tothis and the excess fluid removed by filter paper. Thepreparation was transferred into the electron micro-scope, while still moist, and viewed at a magnificationof X53,500.

RESULTS

Preliminary screening of cells. Four human celllines, two monolayer, and two suspension cul-tures, as well as BHK-21 cells were infected withVSV at a multiplicity of 10. Table 1 summarizesthe results obtained. Infectivity titrations, im-munofluorescence, and thin-section electronmicroscopy were carried out 18 hr after infection.Although all cell lines tested produced viralantigens and infectious progeny, cytoplasmicinclusions were found only in SK-L1, RPMI-6410,and BHK-21 cells. Figure 1 is representative of theappearance of the inclusions 18 hr after infectionwith VSV. The major portion of the cytoplasmhas been replaced by filamentous material. Nor-mal cytoplasmic organelles were displaced and thefilaments were found throughout the cytoplasmand in proximity to membranes from which viruscould be seen budding.Growth of VSV in BHK-21 and RPMI-6410

TABLE 1. Screeninig of cells for inclusion formation18 hr after injection with VSV

C 0

Cell Plaque-forming 0 C6units/mi

HFS 50.0 X 101 40 -

AV3 30.0 X 106 70 -

SK-L1 5.0 X 106 40 +RPMNI-6410 8.5 X 106 70 +BHK-21 1,260.0 X 106 95 +

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VOL. 6, 1970 245

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FIG. 2. Development of inifectious virus and virus-specific immunofluorescence in BHK-21 and RPMI-6410 cells infected with VSV.

cells. A comparison of replication of VSV in one

human cell line and in BHK-21 cells was investi-gated before studying the development of theinclusions. The viral replication was followed byinfectivity titrations and immunofluorescence.The data presented in Fig. 2 show that in BHK-21cells the first significant increase in virus infectivityand number of cells containing virus-specificimmunofluorescence occurred between 3 to 4 hrafter infection. Subsequently, a gradual increasein infectivity titer and number of fluorescent cellswere observed, until maximum titers were reached18 to 24 hr after infection. The replication of VSVRPMI-6410 cells was less efficient than in BHK-21cells. Release of infectious progeny was slowerand the amount of infectious virus produced wasconsiderably less. Cells containing virus-specificimmunofluorescence were not evident until 5 hrafter infection, at which time only 4% of the cellswere stained. The number of fluorescing cells andthe production of infectious progeny did notincrease significantly until 8 hr after infection.Maximum infectivity titers were reached at 18 hr.The greater permissiveness of the BHK-21 cellsrendered them more suitable for the electronmicroscopic study. Subsequent studies on thedevelopment and nature of the inclusions werecarried out therefore with BHK-21 cells.Development of inclusions in BHK-21 cells. The

morphological development of the cytoplasmic

inclusions was studied in cell cultures harvestedat hourly intervals from 3 to 8 hr after infection.The results paralleled those found using immuno-fluorescence (Fig. 2). In thin sections of cells 3 hrafter infection, no inclusion formation nor virusreproduction were found. At 4 hr after infection,discrete foci of granular material (Fig. 3) could beseen in about 20 to 30% of the cells. The earlyinclusions appeared as areas of increased electrondensity readily distinguishable from the surround-ing normal cytoplasm. At 5 to 6 hr after infection,a considerable number of cells contained theseinclusions. Homogeneous masses composed offilamentous material could be found throughoutthe cytoplasm (Fig. 4); the inclusions were mod-erately electron dense, of varying size, andreplaced the normal cytoplasmic constituents.Clusters of membrane-bound virus particles werefound within or in close proximity to the inclu-sions. Virus particles also were seen budding fromthe surface membranes or into cytoplasmicvacuoles with filaments of the inclusions in closeproximity (Fig. 5). A suggested relationship ofthe filamentous strands to the budding virusparticles can be seen in Fig. 6. From 6 to 8 hrafter infection, the morphological changes wereenhanced and characterized by the formation inthe cytoplasm of large areas of these filamentousmasses which replaced normal cytoplasmic con-stituents, similar to that seen at 18 hr after infec-tion (Fig. 1). The inclusions were not limited bymembranes.

Neutralization of VSV with rabbit anti-VSVserum, before infection of cells, prevented forma-tion of inclusions and production of infectiousvirus, as tested 8 and 18 hr after infection.

Isolation and identification of inclusion material.To relate the structures observed on thin sectionsof infected cells to the ribonucleoprotein of VSV,the material was extracted from the cells and sub-jected to various procedures for identification.BHK-21 cells treated with 1 jig of actinomycin

D per ml, infected with VSV, and labeled with3H-uridine were treated as previously described.After equilibrium centrifugation of the extractedmaterial in CsCI for 24 hr, fractions werecollected, and absorbancy and total counts weredetermined. The fraction containing the coinci-dent peak of absorbency at 260 nm and radio-activity was dialyzed overnight at 4 C against NTbuffer. Examination of this fraction by negative-contrast staining revealed the presence of single-stranded filaments. These filaments were furtherpurified by recentrifugation to equilibrium inCsCl. Fractions were collected, the optical den-sity at 260 nm, total radioactive counts, CF ac-tivity, and the density are shown in Fig. 7. Thepeak of absorbency (fraction 11) coincided with

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FIG. 3. Inclusions (I) in BHK cells 4 hr after infection with VSV. X 30,500.

FIG. 4. Appearance of inclusions (1) in the cytoplasm of BHK-21 cells 5 to 6 hr after infection. Filaments canbe seen in the homogeneous masses antd virus particles (V) bound by membranes are within or in close proximityto the inclusions. X 30,500.

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fr t I z - i i *w; ..................................................... * . ifiMrjeS,,-.

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FIG. 5. Filaments of the inclusions (I) underlying the cell membranes of BHK-21 cells next to virus particles(V). X 52,500.

that of the radioactivity incorporated into thefilamentous component. A small contribution toabsorbency and radioactivity was also seen infractions 10 and 12, respectively. The buoyantdensity of the isolated strands (fraction 11) was1.32 g/cm3, as determined in repeated experi-ments. The virus-specific nature of the isolatedstrands was demonstrated by complement fixa-tion by using specific anti-VSV mouse ascitesfluids. The peak CF activity of 256 units/1.0 ml(fraction 11) coincided with the peak of absor-bency and radioactivity. Fractions 10 and 12 eachdisplayed 16 units of CF activity, whereas allother fractions were negative.Examination of fractions 10, 11, and 12 by

negative-contrast staining revealed the presenceof single-stranded filaments. The strands isolatedin fraction 11 are illustrated in Fig. 8A. Whetherexamined after the first or second banding inCsCl, they were always found as single strands.These were either loosely coiled or completelyunwound and occasionally looped back on them-selves. In none of the preparations examined weretightly coiled helices found. Occasionally, singlestretched strands could be found that had anundulating appearance (Fig. 8B), the width ofwhich varied from 2.5 to 8.5 nm, depending onthe angle of viewing. Size variations were prob-ably due to the geometry of the subunits whichmay be either rectangular or elliptical.

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FIG. 6. Filaments (arrows) in proximity to virus particles budding from the plasmalemma of a BHK-21 celL.X 73,500.

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FIG. 7. Equilibrium centrifugation in CsCI ofnucleoprotein isolated from VSV-infected BHK-21cells. Fractions were collected, the density (solid line),and total counts (0) are showit in the upper panel, aswell as the optical densities at 260 nm (0) which wereread after dilution of the fractions with 0.4 ml of NTbuffer. The CF activity (0) of thle fractions is presentedin the lower panel.

The ultraviolet-adsorption spectrum of thefilamentous strands is shown in Fig. 9. The profile

represented is indicative of the nucleoproteinnature of the material.

DISCUSSIONWagner et al. (22) recently reported that the

core protein (N) of VSV was synthesized in thecytoplasm of L cells in large intracellular poolsThis N protein subsequently aggregated (pre-sumably with progeny RNA) to form a sediment-able nucleocapsid. These aggregates were foundin the cells from 2 hr after infection onward. Thestudies presented here demonstrate morphologi-cally that cytoplasmic inclusions, consisting offilaments of viral ribonucleoprotein, were foundin VSV-infected cells. The appearance of theinclusions was closely related to the developmentof virus-specific antigen as demonstrated byimmunofluorescence. The filaments isolated fromthe inclusions contained ribonucleic acid and pro-tein, demonstrated serological VSV-related ac-tivity, and had similar physical properties as thestrands isolated from purified virions (15, 19, 21),i.e., a buoyant density of 1.32 g/cm3 and theappearance of undulating single strands whichvaried in width from 2.5 to 8.5 nm.The nucleoprotein of VSV appeared to be much

less structured than that of the tightly coiledhelices of SV5 nucleocapsids isolated from in-fected cells (4). However, it was morphologicallysimilar to rabies virus nucleocapsids (20). Simp-son and Hauser (19) and Nakai and Howatson(15) have shown that as the nucleoprotein com-ponent of VSV was released from the virion thehelix unwound to form an undulatory strand. Theresults suggested that the bonds which link the

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250 ZAJAC AND HUMMELER J. VIROL.

FIG. 8. Nucleoprotein component isolated from VSV infected BHK cells after CsCI density gradient centrifu-gation. (a) Nucleoprotein showing different degrees of uncoiling. Part of a strand has looped back on itself(arrow). X 160,500. (b) Completely uncoiled nucleoprotein component. Arrows point out the difference observedin the width of the strand due to the angle of viewing. X 267,500.

successive helical turns are more labile than the the cytoplasmic inclusions. This was emphasizedbonds which stabilize paramyxovirus nucleo- by the fact that the diameter of the strands whencapsids. The finding of only unwound strands in measured in thin secVon was approximately 9 nm,our preparations, derived from cells, indicated whereas the in situ helical nucleocapsid of VSVthat the nucleoprotein did not exist as helices in has a diameter of approximately 50 nm. These

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FIG. 9. Ultraviolet absorptiont spectrum of purifiedVSV-nzucleoproteini. Solvenit: NT buffer; light pat/i:1 cm; proteini conttenit: 40 ,ug/ml.

data suggest that the highly organized helicalnature of the ribonucleoprotein component of theVS virion is acquired as the nucleocapsid is fedinto the budding virion and does not exist as sucha helical structure in the cytoplasm of infectedcells.The formation of the nucleoprotein inclusions

was influenced by the host cell used. The reasonsfor the lack of inclusion formation in two of thecell lines and the mechanisms leading to the ac-

cumulation of the material in the other cell linesremain unclear. It has been shown with para-influenza virus SV5 (5) that the accumulation ofviral ribonucleoprotein was dependent on the hostcell. When morphogenesis of the virus was an

efficient process, the synthesis of nucleoproteinoccurred at a rate equivalent to its incorporationinto virions, and no accumulation occurred;however, minimal morphological differentiationof virus particles on the cell membranes resultedin nucleoprotein accumulation. This latter condi-tion seems to be a reasonable explanation inthe case of the lymphoblastoid RPMI-6410 cell.For this cell, production of infectious virus was

delayed and less virus was produced per fluores-cent cells as compared to BHK-21 cells. The de-velopment of infectious progeny in BHK-21 cells,however, did not differ in the time and amountproduced as compared to permissive cells of otherorigins in which no accumulation of nucleopro-tein had been observed (8). Virus particle differ-

entiation, furthermore, did occur on all availablecell membranes. Thus, the accumulation of large

amounts of the nucleoprotein in the cytoplasm ofBHK-21 cells did not seem to be a cell membrane-mediated phenomenon. The excess production ofnucleoprotein in the fully permissive hamster cellsmay be caused rather by some alteration of themechanism(s) responsible for the synthesis of thisviral component.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Serviceresearch grant Al-04911 and training grant AI-00104 from theNational Institute of A!lergy and Infectious Diseases.

The competent technical assistance of M. Cassel and N.Petruzzi is gratefully acknowledged.

LITERATURE CITED

1. Bray, G. A. 1960. A simplU, efficient liquid scintillator forcounting aqueous solution in a liquid scintillation counter.

Anal. Bioclhem. 1:279-285.2. Cartwright, B., C. J. Smale, and F. Brown. 1970. Dissection-

of vesicular stomatitis virus into infective ribonucleopro-tein and immunizing components. J. Gen. Virol. 7:19-32.

3. Clarkson, B. D., A. Strife, and E. de Harven. 1967. Continu-ous culture of seven new cell lines (SK-L1 to 7) from pa-

tients with acute leukemia. Cancer 20:926-947.4. Compans, R. W., and P. W. Choppin. 1967. Isolation and

properties of the helical nucleocapsid of the parainfluenzavirus SV5. Proc. Nat. Acad. Sci. U.S.A. 57:949-956.

5. Compans, R. W., K. V. Holmes, S. Dales, and P. W. Choppin.1966. An electron microscopic study of moderate and viru-lent virus-cell interactions of the parainfluenza virus SV5.Virology 30:411-426.

6. David-West, T. S., and N. A. Labzoffsky. 1968. Electronmicroscopic studies on the development of vesicularstomatitis virus. Arch. Gesamte Virusforsch. 23:105-125.

7. Dulbecco, R., and M. Vogt. 1954. Plaque formation andisolation of pure lines with poliomyelitis virus. J. Exp.Med. 99:167-182.

8. Hackett, A. M., Y. C. Zee, F. L. Schaffer, and L. Talens.1968. Electron microscopic study of the morphogenesis ofvesicular stomatitis virus. J. Virol. 2:1154-1162.

9. Henle, G., and W. Henle. 1966. Immunofluorescence in cellsderived from Burkitt's lymphoma. J. Bacteriol. 91:1248-1256.

10. Howatson, A. F., and G. F. Whitmore. 1962. The develop-ment and structure of vesicular stomatitis virus. Virology16:466-478.

11. Hummeler, K., and H. Koprowski. 1969. Investigating therabies virus. Nature 221:418-421.

12. Iwakata, S., and J. T. Grace, Jr. 1964. Cultivation in vitroof myeloblasts from human leukemia. N.Y. State J. Med.64:2279-2282.

13. McCombs, R. M., M. Benyesh-Melnick, and J. P. Brunschwig.1966. Biophysical studies of vesicular stornatitis virus.J. Bacteriol. 91:803-812.

14. Mussgay, M., and J. Weibel. 1963. Electron microscopicstudies on the development of vesicular stomatitis virusin K.B. cells. J. Cell Biol. 16:119-129.

15. Nakai, T., and A. F. Howatson. 1968. The fine structure ofvesicular stomatitis virus. Virology 35:268-281.

16. Russell, W. C. 1962. A sensitive and precise plaque assayfor herpes virus. Nature 195:1028-1029.

17. Schulze, P., and H. Liebermann. 1966. Elektronenmikro-skopische Untersuchungen zur Morphologie and Ent-wicklung des Virus der Stomatitis Vesicularis in Kalber-

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nierenzellkulturen. Arch. Exp. Veterinaermed. 20:713-729.

18. Sedwick, W. D., and F. Sokol. 1970. Nucleic acid of rubellavirus and its replication in hamster kidney cells. J. Virol.5:478-489.

19. Simpson, R. W. and R. E. Hauser. 1966. Structural compo-

nents of vesicular stomatitis virus. Virology 29:654-667.20. Sokol, F., H. D. Schlumberger, T. J. Wiktor, H. Koprowski,

and K. Hummeler. 1969. Biochemical and biophysical

studies on the nucleocapsid and on the RNA of rabiesvirus. Virology 38:651-655.

21. Wagner, R. R., T. C. Schnaitman, R. M. Snyder, and C. A.Schnaitman. 1969. Protein composition of the structuralcomponents of vesicular stomatitis virus. J. Virol. 3:611-618.

22. Wagner, R. R., R. M. Snyder, and S. Yamazaki. 1970. Pro-teins of vesicular stomatitis virus: kinetics and cellularsites of svnthesis. J. Virol. 5:548-558.

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