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Studies on the generation of vesicular stomatitis virus (Indiana serotype) defective interfering particles
VIROLOGY
84,142-182 WW
Studies
on the Generation of Vesicular Stomatitis Virus (Indiana Serotype) Defective Interfering Particles
C. YONG KANG,** l THOMAS GLIMP,* JON P. CLEWLEY,t DAVID H. L. BISHOPt
AND
* Department of Microbiology, The University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas 75235, and t Department of Microbiology, University of Alabama Medical Center, Birmingham, Alabama 35294 Accepted August 19,1977 Using freshly cloned vesicular stomatis virus (Indiana serotype) (VSV,,) serial undiluted high-multiplicity passage in four different cell lines has produced different size classes of defective interfering (DI) particles at different passage numbers. The same clonal isolates of VSV in a given cell type appear to produce identical patterns of particles, although different clonal isolates produce different size classes of DI particles. Thus, the classes of DI particles generated during serial passages appear to relate in some way to the clone of virus and to the type of host cell used. All the DI particles generated in different cell types are capable of homologous interference in any cell type. Oligonucleotide sequence analyses of five DI particle RNAs indicate that they are all generated from a specific region of the standard B virion genome and contain only negative-stranded genomic RNA.
particles are readily separable from the standard virus particles. Cooper and BelMany carefully studied animal virus lett (1959) first described the autointerfersystems have been shown to contain not ence phenomenon of VSV, and Hackett only the standard infectious virions but (1964) identified small, truncated defective also defective virus particles which interparticles in the preparation of the virus. fere with the replication of their related Huang and Wagner (1966) showed that standard infectious viruses. the truncated defective particles are reIn general, defective interfering (DI) sponsible for the homologous viral interparticles contain all the viral structural . proteins but only a part of the genome of ference Most DI particles of VSV contain RNA the standard virus. DI particles cannot sequences representing varied portions of self-replicate in infected cells, and thus the cistron for L protein. An exception is require a helper function which is provided the long DI particle generated from the by the standard infectious virus particles. heat-resistant strain of VSV,,, which repDI particles normally are generated during serial undiluted high-multiplicity pas- resents cistrons for N, NS, M, and G sages of standard infectious virus. How- proteins (Bishop and Roy, 1971; Leamnson and Reichmann, 1974; Stamminger ever, very little is known about the inducand Lazzarini, 1974). Under certain expertion of DI particles upon infection by stan- imental conditions one can obtain DI prepdard virus particles (Huang, 1973). Vesicular stomatitis virus (VSV) pro- arations which contain partially doublevides an excellent model system to study stranded RNA (Roy et al., 1973; Lazzarini al., 1975; Perrault, 1976). DI particles of the induction of DI particles and the mech- et VSV contain all the proteins found in anism of viral interference since the DI standard virions (Kang and Prevec, 1969), 1 Author to whom reprint requests should be including a functional RNA polymerase (Emerson and Wagner, 1972). addressed. INTRODUCTION
142 0042-6822/78/0841-0142$2.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
GENERATION
OF VSV DI PARTICLES
DI particles of VSV can be generated from a plaque-purified clone of standard virus by serial undiluted passages of virus (Stampfer et al., 1971). Using repeated clonal isolates of a temperature-sensitive mutant, Reichmann et al. (1971) observed the generation of DI particles all of which contained the same size classes of RNA. Schincariol and Howatson (1970) also observed production of a consistent type of DI particles by the heat-resistant strain of the Indiana serotype of VSV (VSV,,,). In contrast, Holland et al. (1976) and Roy et al. (1973) observed generation of different sizes of DI particles by different clonal isolates propagated in one cell type or by the same clonal isolates propagated in different hosts. Thus, it appears that the generation of different size classes of DI particles is related in some way to the clone of virus used and also to the host cells in which the DI particles are generated. We do not know what host-dependent functions determine either the rate of DI generation or the selection of different size classes of DI particles. This report describes the generation of different size classes of DI particles in four different host cells at different numbers of undiluted passages using monoclonal isolates of the prototype Indiana strain of VSV. The rate of generation of the DI particles appears to be determined by the host cell. Oligonucleotide fingerprint analysis of the DI particles isolated from the different host cells reveals that DI particles arise from the same region of the standard B virion genome. MATERIALS
AND
METHODS
Cells and viruses. Growing cultures of the B77 strain of avian sarcoma virustransformed rat cells [R(B77)] were obtained from Dr. Howard Temin, McArdle Laboratory, University of Wisconsin. Mouse L, cells were kindly provided by Dr. Kathryn Holmes, Uniformed Services University Medical School. Baby hamster kidney cell clone 21 (BHK,,) was purchased from the American Type Culture Collection. Baby hamster kidney cell clone 53 (BHK,,) was provided by Dr. D. H. L. Bishop, University of Alabama, and the
143
human cell line established by double infection with Rous sarcoma virus (RSV) and simian virus 40 (SV40) which is designated as RSa were the kind gifts of Dr. T. Kuwata, Chiba University, Japan (Kuwata et al., 1976). We have cloned these cells at least once in our laboratory. None of these cells produces any detectable amounts of the endogenous viruses. R(B77) cells and L, cells were grown as monolayer cultures in Dulbecco modified minimal essential medium (DMEM), supplemented with 5% heat-treated fetal calf serum (FCS). BHK,, cells were grown in DMEM supplemented with 10% FCS and RSa cells were grown in Temin modified minimal essential medium (TMEM) supplemented with 10% FCS and 10% tryptose phosphate broth. All the media, serum, and antibiotics were purchased from Grand Island Biological Company, Grand Island, N.Y., except the TMEM which was purchased from International Scientific Industries, Cary, Ill. The prototype strain of the Indiana serotype of vesicular stomatitis virus (VSV,,,) used in this paper has been described previously (Kang and Prevec, 1969; Clewley et al., 1977). Virus stocks of VSV,,, clones 1 and 2 and stocks of four other clones diverged at the first plaque isolation from the original stock virus and stocks of each clone were prepared after six additional independent plaque purifications followed by one lowmultiplicity passage using 0.01 PFU/cell in L, cells. Virus was grown for 18 hr, at which time the virus fluid was centrifuged at 600 g for 15 min to remove cellular debris. Three-milliliter aliquots of virus fluid were stored at -75”. The titer of stock virus was approximately 2 x 10y PFU/ml and no detectable DI particles were present in the stock virus when it was analyzed by sucrose gradient centrifugation. Successive undiluted passages of VSV. One-hundred-millimeter culture dishes containing approximately 1 x lo7 cells in monolayers were infected with 0.5 ml of the stock virus to give a multiplicity of infection at the first passage of about 100 PFU/cell; 0.5 ml/lOO-mm dish of undiluted virus from the previous passages was used
144
KANG
for the subsequent passages. The virus was adsorbed for 45 min at 37”, 7 ml of DMEM supplemented with 5% FCS was added, and the dishes were incubated at 37” for 7 hr in a CO, incubator with saturated humidity. The virus titer reached the maximum level at 7 hr of infection under these conditions (Kang and Prevec, 1969). The medium containing virus was harvested and centrifuged at 600 g for 15 min to remove cellular debris. The cellfree virus fluids were stored at -75” until use. Quantitution of virus. The plaque assay technique used for determining the titer of standard infectious virus has been described previously (Kang and Prevec, 1969). We used BHK,, cells in all plaque assays. The amount and type of virus particles produced in each passage were monitored by the following procedure. The medium from infected cells was harvested and centrifuged at 600g for 15 min at 5” to remove cell debris. The supernatant was centrifuged at 81,000 g for 75 min in a Spinco SW 27 rotor to pellet the virus. The virus pellets were resuspended in 0.3 ml of phosphate-buffered saline (PBS) and layered on a linear 5-30% sucrose gradient made in PBS. After centrifugation at 110,OOOgfor 35 min at 5” in a Spinco SW 41 rotor, the gradients were collected from the t,op using a Buchler Auto-Densi-Flow IIC . The presence and distribution of virus in the gradient were monitored continuously by passing the effluent through an LKB Uvicord II, the absorbance at 280 nm being continuously recorded on a Fisher Recordall 5000.
ETAL. pended in approximately 0.3 ml of 0.02 M Tris-hydrochloride buffer (pH 7.3) containing 0.1 M NaCl and 0.001 M EDTA (TSE). The concentrated virus sample was layered on a linear 5-30% sucrose gradient made in TSE and centrifuged at 110,000g for 35 min. The visible bands of DI-1, DI2, and DI-3 were collected separately and [32PlRNAs were purified by the method described previously (Clewley et d., 1977). The purified 32P-labeled viral RNAs were digested with T, ribonuclease and the oligonucleotides were separated by the twodimensional gel-electrophoresis systems of de Watcher and Fiers (1972) as described previously (Clewley et al., 1977). RESULTS
Yield
of VSV in Four Different Hosts during Serial Undiluted Passages of a Clone of Standard Virion
To determine host effects on the generation of DI particles during serial undiluted high-multiplicity passages of VSV, a monoclonal isolate (plaque purified) of VSV was propagated in human (RSa), hamster (BHK& rat lR(B7711, and mouse (LJ cell lines. The yield of standard infectious virions was measured by plaque assay. Figure la demonstrates that clone No. 1 of VSVINDpassaged in the four different cell lines yields different amounts of infectious B virion in different serial passages. Similar patterns of reduction in production of the infectious viruses were observed when we used another clone (clone No. 2) (Fig. lb). RSa cells produce a lo-fold reduction of infectious VSV in each passage and an approximately three-log reduction from the original titer in yield of virus at the Preparation of ?P-labeled DI particle fourth passage. In contrast L, cells produce RNAs and oligonucleotide fingerprint analysis. Confluent monolayers of 1 X 10’ a constant amount of virus up to the sixth R(B77) cells were infected with 0.5 ml per to seventh passages and then show rapid dish of fourth passage VSV (see Fig. 2) to decline in the subsequent passages. The produce three distinct populations of DI BHK,, cells and R(B77) cells behave simiparticles. After 45 min of absorption, 7 ml larly. Five to six passages were required of DMEM containing 200 $Z!i of 32Pper ml before the yield of virus was reduced apwas added. After 7 hr, culture fluids con- proximately three logs from the initial taining 32P-labeled virus were collected titer. To determine whether the reduction of and centrifuged at 600 g for 15 min. The supernatant fluid was then centrifuged at titer after the serial undiluted passages of 81,000 g for 75 min in a Spinco SW 27 VSV is the result of the generation of DI rotor. The pelleted virus was resus- particles, the viruses from each passage
GENERATION 0
OF VSV DI PARTICLES
145
VSV,ND Clone I
0511123456789 ’ I ’ I ’ I ’ ’ Number
of
undiluted
passages
FIG. 1. Yield of standard B virion during successive undiluted passages of two clonal isolates of VSViND in four different hosts. The initial stock virus was prepared by the consecutive plaque isolation method as described in Materials and Methods. Confluent monolayer cultures of approximately lo7 cells per loo-mm culture dish were infected with 1 x 10s plaque-forming units of the stock virus for the first passage. After 45 min of absorption at 37”, 7 ml of prewarmed DMEM supplemented with 5% FCS was added to each culture dish and the dishes were incubated at 37” in CO2 for 7 hr. Cell-free virus fluids were harvested after 7 hr of growth and were centrifuged at 600 g for 15 min. The subsequent passages were made by infecting 0.5 ml of undiluted previous-passage virus onto 10’ cells/dish. At the end of each passage, the yield of infectious virus was measured by plaque assay on BHK,, cells.
were analyzed in sucrose gradient centrifugation. Figure 2 demonstrates that the production of DI particles directly correlates with the titer of virus shown in Fig. la. The RSa cells produce detectable amounts of DI particles at the second undiluted passage. The DI particles produced in the RSa cells are rather heterogeneous in size. The BHK,, cells produce a detectable amount of DI particles at the third undiluted passage and we could detect at least two distinct sizes of DI particles produced in BHK,, cells. In contrast, R(B77) cells produce three different size classes of DI particles which were not detectable until the fourth passage. Lz cells take seven to eight passages to produce detectable amounts of DI particles. There are three different sizes of DI particles produced in the Lz cells (Fig. 2). It is clear from these data that different host cells generate different numbers of size classes of DI particles after different numbers of passages. These results strongly suggest
that a host function determines the type of DI particles produced since different host cells produced multiple size classes of DI particles even though a monoclonal isolate was used to initiate the infections. We have repeated these experiments using the same clone of virus in the same host cells and found the results are reproducible as long as we use the same clonal isolate of virus with the same cell type (data not shown). Both Roy et al. (1973) and Holland et al. (1976) demonstrated that different clones of VSV generated different sixes of DI particles in the same host. To determine whether different clones of VSV produce repeatedly different patterns of DI particles, we prepared five other stocks of VEX,,, from five individual plaques from the original stock virus followed by six consecutive independent plaque isolations as described in Materials and Methods. We made serial undiluted passages of clone No. 2 (Fig. lb) and analyzed the
146
KANG ET AL. vsVl,,n
VSVIND
CiOtW 1 RSa
‘OP
D~stonce
of sedimentation
(Cm)
B%!
I
Clone 2 RlB77)
L2
bottom
FIG. 2. Analysis of total virus particles from the continuous undiluted passages of clone 1 of VSV,,. Samples of lysate from four culture dishes of each passage illustrated in Fig. la were centrifuged at 600 g for 15 min to remove cellular debris. The virus particles were pelleted from the supernatant by centrifugation at 81,000 g for 75 min in a Spinco SW 27 rotor, resuspended in 0.3 ml of PBS, and layered on a linear 530% sucrose gradient made in PBS. After centrifugation for 110,000g at 5” for 35 min in a Spinco SW 41 rotor the gradients were analyzed by the technique described in Materials and Methods. The clones of VSV, types of cells, passage numbers, and positions of the standard B virion (STD) and of the defective interfering particles (DI) are indicated. Electron microscopic examination of material in fractions sedimenting just in advance of the virus revealed only clumps of particles identical to B virions.
FIG. 3. Analysis of total virus particles from the continuous undiluted passages of clone 2 of VSViND. Samples of lysate from four culture dishes of each passage illustrated in Fig. lb were analyzed in the sucrose gradient. See legend to Fig. 2.
classes in L, cells. In contrast, clone No. 1 produces three different size classes of DI particles in both R(B77) cells and LZ cells. Clone No. 2 consistently generates the same pattern of DI particles in the same host cell during serial undiluted passages. In addition, we found four other clones generated the same pattern of the DI particles as that shown with clone No. 1 (data not shown). It is clear that different hosts produce different size classes of DI particles at different numbers of undiluted passages. Thus, it appears that the production of DI particles by serial undiluted passages of vesicular stomatitis B virion is dependent on (i) the clonal isolates of the virus and (ii) the host cells.
virus particles produced during undiluted passages. Figure 3 demonstrates that the number of passages required to generate a detectable amount of DI particles correlates well with the titer of the infectious virion (Fig. lb). However, the pattern of of Biological Activities of DI particles produced by clone No. 2 (Fig. Determination DI Particles Generated during Undi3) is different from that produced by clone 1uted Passages No. 1 (Fig. 2). There are distinct differWe demonstrated previously that DI ences between clone No. 1 and clone No. 2 propagated in R(B77) cells and in L, cells. particles of VSV can serve as templates by helper infecClone No. 2 produces only .one type of DI for their multiplication particles in R(B77) cells and two size tious B virions (Prevec and Kang, 1970)
and that reduction of infectious B virion synthesis occurred when DI particles were mixedly infected with standard B virions. To test the biological activities of DI particles generated by multiple passages, we infected cells with purified DI particles mixed with an early passage of VSV, which did not give rise to a detectable amount of DI particles. These experiments are shown in Fig. 4. We used DI particles from the fifth passage of clone No. 1 which was grown in R(B77) cells and purified by sucrose gradient centrifugation. Figure 4a shows the types of virus particles produced in this passage. Fractions 8-10 (designated DI-l), fractions 12-14 (designated DI-2), and fractions 16-18 (designated DI-3) were pooled separately and used for mixed infection with the first passage of VSV (Fig. 4). When partially purified DI-1 particles were mixed with infectious standard B virions and grown in three different hosts,
h
2 ‘OP
4
6
147
OF VSV DI PARTICLES
GENERATION
8 31stonce
predominantly DI-1 was produced while the production of infectious virus particles was reduced (Figs. 4c, g, and k). In addition, the BHK,, and R(B77) cells produced significant quantities of DI-2 (Figs. 4g and k). This may well be the result of contaminating DI-2 in the DI-1 preparation and of preferential replication of DI-2 in the BHK,, and R(B77) cells, as compared to RSa cells. Mainly DI-2 species were produced when the DI-2 preparation was mixedly infected with infectious standard B virions (Figs. 4d, h, and 11,and mainly DI-3 species were produced when we mixedly infected with the preparation containing DI-3 particles (Figs. 4e, i, and m). In all cases we observed that the yield of standard B virions was reduced as a result of mixed infection with DI particles. It is obvious that the level of infectious standard B virion produced in three different host cells varied. We found no detectable amount of virus production when we in-
n
2 of
4 6 8 sedtmentofton !CT)
2
4
6 bottk
FIG. 4. Effects of DI particles on the total particle yield in three different hosts mixedly infected with the standard B virion. DI-1, DI-2, and,DI-3 were prepared from lysates of the fifth passage of VSV,,, clone l-infected R(B77) cells (Figs. 2 and 4a). Confluent-culture dishes containing about 1 x 10’ cells were infected with 5 x lo8 PFU of first-passage VSV iND clone 1 with or without addition of 0.1 ml of DI particle preparation as indicated. After absorption for 45 min at 37” the prewarmed culture medium was added and the dishes were incubated for 7 hr at 37” in a CO, incubator. Samples of lysate from four culture dishes of each combination were analyzed on the sucrose gradient described in the legend to Fig. 2.
148
KANG
ET AL.
fected cells with purified DI particles alone from BHK,, (the heavier DI particle (data not shown). Therefore, we conclude shown in Fig. 2, BHK,,-P,) also have an that all three DI particles generated in identical oligonucleotide fingerprint patR(B77) cells by serial undiluted passages tern (data not shown). Therefore, all five of clone No. 1 of VSV,,, (Fig. 4a) are DI particle RNA sequences represent a indeed defective and interfering particles. common region of RNA sequence in the We performed similar experiments using standard B virion genome and represent DI particles isolated from BHK,, cells and only the negative-stranded genomic RNA. L cells. We found that the two DI particles DISCUSSION generated in BHK,, cells and the three DI The patterns of DI particles generated particles generated in Lz cells by clone during serial undiluted passages of clonal No. 1 (Fig. 2) are all defective in their infectious B virions of VSV have been replication and are capable of interference. studied in several laboratories (Holland et Thus, we conclude that all small truncated al., 1976; Reichmann et al., 1971; Schincaparticles generated in the four different riol and Howatson, 1970; Stampfer et al., cells are defective interfering particles. 1971). These reports brought to our attention the question of whether the processes Oligonucleotide Fingerprint Analysis of in the generation of DI particles are ranDI Particle RNAs dom or directed. We have shown here that We were interested in determining the the generation of DI particles during highnucleotide sequences of DI particle RNAs multiplicity passages is dependent upon generated in different cell types using the both the clonal isolate of the virus and the monoclonal isolate of the virus. Figure 5 host cell. One clonal isolate generates difillustrates the oligonucleotide fingerprint ferent patterns of DI particles in different of the standard B virion RNA. The nomen- cells. Thus, the host plays a role in conclature was arrived at by comparing the trolling the generation of DI particles. In radioautograms of samples run for differ- addition, we have shown that a different ent lengths of time in the first or second clone may generate different patterns of dimension to produce slightly better reso- DI particles in the same host. Thus, in lution of some of the groups of oligonucle- some way the clone of infectious B virions otides. Although we found that some of also contains a determining factor for DI the fingerprint patterns showed better sep- particle synthesis. We have observed, aration of spots between 18 and 23, the however, that a given clonal isolate of B oligonucleotide fingerprint pattern shown virions always generates the same pattern of DI particles in a given cell type. This is in Fig. 5 was best for overall resolution. Figure 6 shows the oligonucleotide finger- consistent with the observations of Reichprints of RNAs from the three DI particles mann et al. (1971) and Schincariol and generated from R(B77) cells (see Figs. 2 Howatson (1970) who respectively generand 4a). All the identifiable oligonucleo- ated particular-size DI particles with partides of DI-1 RNA are present in DI-2 ticular temperature-sensitive mutants of RNA and all the oligonucleotides present VSV, or with a heat-resistant strain of in DI-2 RNA are also present in DI-3 VSV,,. A part of our data is also consistRNA. These results strongly suggest that ent with the observation of Holland et al. the DI-3 RNA contains all the nucleotide (1976) that different clonal isolates genersequences of DI-2 and PI-1 RNAs, and ate different patterns of DI particles when that DI-2 RNA contains all the nucleotide propagated in the same host or that the sequences of DI-1 RNA as illustrated in same clonal isolate generates different Fig. 7. In addition, we found that DI-2 patterns of DI particles in different hosts passages. HowRNA from R(B77) cells and DI-1 RNA during high-multiplicity from BHK,, (the lighter DI particle shown ever, Holland et al. (1976) used BHK,, in Fig. 2, BHK,,-P,) share identical pat- cells to amplify their virus before the final terns of oligonucleotide fingerprint. The analysis which may have added other varDb3 RNA from R(B77) cells and the DI-2 iables.
GENERATION
,--I : . i. 4. ; : .. .. .
@@ P.. .. . .
OF VSV DI PARTICLES
150
KANG
ET AL.
GENERATION
DI-2
11
DI-311
11
11
I 11
II
II
III)
151
OF VSV DI PARTICLES
8 10I 1411 36 36I 3711 44 66II67 11 6 11
I I I\ 11
I]]/
IIII
.
1 III]
/ III] 24 d 53
. or 29 or 31
. . or52
FIG. 7. Hypothetical
scheme for mapping the DI-1, DI-2, and DI-3 RNAs using oligonucleotide fingerprint analysis. All 14 identifiable oligonucleotides in DI-1 RNA are present in DI-2 RNA and all 14 oligonucleotides of DI-1 RNA and the additional 10 oligonucleotides in DI-2 RNA are present in DI-3 RNA. The DI-3 RNA contains five additional oligonucleotides which are not present in either DI-1 or DI-2 RNA. All the oligonucleotides present in all three DI particle RNAs are present in the standard B virion RNA as shown in Fig. 6.
Holland et al. (1976) suggested that the generation of DI particles during the successive undiluted high-multiplicity passages of VSV is a random process and that a selective amplification process during the additional passages determines the visible quantities of different size classes of DI particles. Accordingly, they have further predicted that the infectious B virion is not genetically predisposed to generate particular DI particles. Our results suggest that there may be some genetic determinant in infectious B virions which in conjunction with certain host factors generate consistent patterns of DI particles. DI particles of VSV have been detected as early as the third 7.5-hr passage in Chinese hamster ovary cells using a sensitive detection method (Stampfer et d., 1971). Holland et al. (1976) observed that rabies virus generated large quantities of DI particles at the first passage of the virus, whereas Cole et al. (1971) had to make 18 undiluted passages to detect DI particles of poliovirus. We found that the number of passages required for a visible amount of DI particle synthesis, at least in the case of VSV, is largely dependent on the host cells. Two different virus clones passaged in four different hosts produced DI particles at the same number of passages in given cell types. This observation suggests that the initial induction and subsequent amplification of DI particles in various hosts is different. It appears that the rate of induction determines the overall production of DI particles since the
yield of standard B virion during the first passage in these four different hosts did not vary significantly. We found that the defective particles we generated were also interfering particles and that the DI particles generated in one cell type are capable of interfering with the production of infectious particles in different hosts. However, the degree of interference in different hosts varies significantly as determined by standard B virion production. This observation is consistent with data previously reported (Potter and Stewart, 1976). Surprisingly, the RNAs from the five different DI particles share completely overlapping nucleotide sequences in spite of the fact that two are generated in BHK,, cells and three are generated in R(B77) cells. They represent a constant region of the infectious negative-stranded B virion genome and no positive-stranded RNA sequences were detected. We do not understand yet how a deletion takes place during the replication of VSV. However, direct evidence is being sought showing that the host cell plays a role in the induction of DI particles. ACKNOWLEDGMENTS We are very grateful to Rae Allen and Jan Reed for their excellent technical support. We thank Drs. J. Bednarz-Prashad, B. Ozanne, and J. Shadduck for stimulating discussions and for their detailed reviews of this manuscript. This investigation was supported by Public Health Service Grants CA 16479 and CA 20012 from the National Cancer Institute and by Grants AI 13402, AI 12070, and AI 10984 from the National Institute of Allergy and Infectious Diseases.
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KANG REFERENCES
BISHOP,D. H. L., and ROY, P. (1971). Properties of the product synthesized by vesicular stomatitis virus particles. J. Mol. Biol. 58, 799-814. COOPER,P. D., and BELLET, A. J. (1959). A transmissible interfering component of vesicular stomatitis virus preparations. J. Gen. Microbial. 21, 485 497. CLEWLEY, J. P., BISHOP, D. H. L., KANG, C. Y., COFFIN, J., SCHNITZLEIN,W. M., REICHMANN, M. E., and SHOPE, R. E. (1977). Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the vesicular stomatitis virus subgroup. J. Viral. in press. COLE, C. N., SMOLAR,D., WIMMER, E., and BALTIMORE,D. (1971). Defective interfering particles of poliovirus. I. Isolation and physical properties. J. Viral. 7, 478-485. DE WATCHER, R., and FIERS, W. (1972). Preparative
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LEAMNSON, R. N., and REICHMANN, M. E. (1974). The RNA of defective vesicular stomatitis particles in relation to viral cistrons. J. Mol. Biol. 85, 551-568. PERRAULT,J. (1976). Cross-linked double-stranded RNA from a defective vesicular stomatitis virus particle. Virology 70, 360-371. POTTER,K. N., and STEWART,R. B. (19761.Comparison of vesicular stomatitis virus defective interfering particle synthesis in chick embryo and L cells. Cunad. J. Microbial. 22, 1458-1463. PREVEC, L., and KANG, C. Y. (1970). Homotypic and heterotypic interference by defective particles of vesicular stomatitis virus. Nature (London) 228,25-27. REICHMANN, M. E., PRINGLE, C. R., and FOLLETT, E. A. C. (1971). Defective particles in BHK cells
infected with temperature-sensitive mutants of vesicular stomatitis virus. J. Virol. 8, 154-160. ROY, P., REPIK, P., HEFTI, E., and BISHOP, D. H. L. (1973). Complementary RNA species isolated from vesicular stomatitis (HR strain) defective virions. J. Virol. 11,9X-925. SCHINCARIOL,A. L., and HOWATSON,A. F. (1970). Replication of vesicular stomatitis virus. I. Viral specific RNA and nucleoprotein in infected L cells. Virology 42, 732-734. STAMMINGER, G., and LAZZARINI, R. A. (1974). Analysis of the RNA of defective VSV particles. Cell 3, 85-93.
STAMPFER,M., BALTIMORE, D., and HUANG, A. S. (1971). Absence of interference during high-multiplicity infection by clonally purified vesicular stomatitis
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84,142-182 WW
Studies
on the Generation of Vesicular Stomatitis Virus (Indiana Serotype) Defective Interfering Particles
C. YONG KANG,** l THOMAS GLIMP,* JON P. CLEWLEY,t DAVID H. L. BISHOPt
AND
* Department of Microbiology, The University of Texas Health Science Center, Southwestern Medical School, Dallas, Texas 75235, and t Department of Microbiology, University of Alabama Medical Center, Birmingham, Alabama 35294 Accepted August 19,1977 Using freshly cloned vesicular stomatis virus (Indiana serotype) (VSV,,) serial undiluted high-multiplicity passage in four different cell lines has produced different size classes of defective interfering (DI) particles at different passage numbers. The same clonal isolates of VSV in a given cell type appear to produce identical patterns of particles, although different clonal isolates produce different size classes of DI particles. Thus, the classes of DI particles generated during serial passages appear to relate in some way to the clone of virus and to the type of host cell used. All the DI particles generated in different cell types are capable of homologous interference in any cell type. Oligonucleotide sequence analyses of five DI particle RNAs indicate that they are all generated from a specific region of the standard B virion genome and contain only negative-stranded genomic RNA.
particles are readily separable from the standard virus particles. Cooper and BelMany carefully studied animal virus lett (1959) first described the autointerfersystems have been shown to contain not ence phenomenon of VSV, and Hackett only the standard infectious virions but (1964) identified small, truncated defective also defective virus particles which interparticles in the preparation of the virus. fere with the replication of their related Huang and Wagner (1966) showed that standard infectious viruses. the truncated defective particles are reIn general, defective interfering (DI) sponsible for the homologous viral interparticles contain all the viral structural . proteins but only a part of the genome of ference Most DI particles of VSV contain RNA the standard virus. DI particles cannot sequences representing varied portions of self-replicate in infected cells, and thus the cistron for L protein. An exception is require a helper function which is provided the long DI particle generated from the by the standard infectious virus particles. heat-resistant strain of VSV,,, which repDI particles normally are generated during serial undiluted high-multiplicity pas- resents cistrons for N, NS, M, and G sages of standard infectious virus. How- proteins (Bishop and Roy, 1971; Leamnson and Reichmann, 1974; Stamminger ever, very little is known about the inducand Lazzarini, 1974). Under certain expertion of DI particles upon infection by stan- imental conditions one can obtain DI prepdard virus particles (Huang, 1973). Vesicular stomatitis virus (VSV) pro- arations which contain partially doublevides an excellent model system to study stranded RNA (Roy et al., 1973; Lazzarini al., 1975; Perrault, 1976). DI particles of the induction of DI particles and the mech- et VSV contain all the proteins found in anism of viral interference since the DI standard virions (Kang and Prevec, 1969), 1 Author to whom reprint requests should be including a functional RNA polymerase (Emerson and Wagner, 1972). addressed. INTRODUCTION
142 0042-6822/78/0841-0142$2.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
GENERATION
OF VSV DI PARTICLES
DI particles of VSV can be generated from a plaque-purified clone of standard virus by serial undiluted passages of virus (Stampfer et al., 1971). Using repeated clonal isolates of a temperature-sensitive mutant, Reichmann et al. (1971) observed the generation of DI particles all of which contained the same size classes of RNA. Schincariol and Howatson (1970) also observed production of a consistent type of DI particles by the heat-resistant strain of the Indiana serotype of VSV (VSV,,,). In contrast, Holland et al. (1976) and Roy et al. (1973) observed generation of different sizes of DI particles by different clonal isolates propagated in one cell type or by the same clonal isolates propagated in different hosts. Thus, it appears that the generation of different size classes of DI particles is related in some way to the clone of virus used and also to the host cells in which the DI particles are generated. We do not know what host-dependent functions determine either the rate of DI generation or the selection of different size classes of DI particles. This report describes the generation of different size classes of DI particles in four different host cells at different numbers of undiluted passages using monoclonal isolates of the prototype Indiana strain of VSV. The rate of generation of the DI particles appears to be determined by the host cell. Oligonucleotide fingerprint analysis of the DI particles isolated from the different host cells reveals that DI particles arise from the same region of the standard B virion genome. MATERIALS
AND
METHODS
Cells and viruses. Growing cultures of the B77 strain of avian sarcoma virustransformed rat cells [R(B77)] were obtained from Dr. Howard Temin, McArdle Laboratory, University of Wisconsin. Mouse L, cells were kindly provided by Dr. Kathryn Holmes, Uniformed Services University Medical School. Baby hamster kidney cell clone 21 (BHK,,) was purchased from the American Type Culture Collection. Baby hamster kidney cell clone 53 (BHK,,) was provided by Dr. D. H. L. Bishop, University of Alabama, and the
143
human cell line established by double infection with Rous sarcoma virus (RSV) and simian virus 40 (SV40) which is designated as RSa were the kind gifts of Dr. T. Kuwata, Chiba University, Japan (Kuwata et al., 1976). We have cloned these cells at least once in our laboratory. None of these cells produces any detectable amounts of the endogenous viruses. R(B77) cells and L, cells were grown as monolayer cultures in Dulbecco modified minimal essential medium (DMEM), supplemented with 5% heat-treated fetal calf serum (FCS). BHK,, cells were grown in DMEM supplemented with 10% FCS and RSa cells were grown in Temin modified minimal essential medium (TMEM) supplemented with 10% FCS and 10% tryptose phosphate broth. All the media, serum, and antibiotics were purchased from Grand Island Biological Company, Grand Island, N.Y., except the TMEM which was purchased from International Scientific Industries, Cary, Ill. The prototype strain of the Indiana serotype of vesicular stomatitis virus (VSV,,,) used in this paper has been described previously (Kang and Prevec, 1969; Clewley et al., 1977). Virus stocks of VSV,,, clones 1 and 2 and stocks of four other clones diverged at the first plaque isolation from the original stock virus and stocks of each clone were prepared after six additional independent plaque purifications followed by one lowmultiplicity passage using 0.01 PFU/cell in L, cells. Virus was grown for 18 hr, at which time the virus fluid was centrifuged at 600 g for 15 min to remove cellular debris. Three-milliliter aliquots of virus fluid were stored at -75”. The titer of stock virus was approximately 2 x 10y PFU/ml and no detectable DI particles were present in the stock virus when it was analyzed by sucrose gradient centrifugation. Successive undiluted passages of VSV. One-hundred-millimeter culture dishes containing approximately 1 x lo7 cells in monolayers were infected with 0.5 ml of the stock virus to give a multiplicity of infection at the first passage of about 100 PFU/cell; 0.5 ml/lOO-mm dish of undiluted virus from the previous passages was used
144
KANG
for the subsequent passages. The virus was adsorbed for 45 min at 37”, 7 ml of DMEM supplemented with 5% FCS was added, and the dishes were incubated at 37” for 7 hr in a CO, incubator with saturated humidity. The virus titer reached the maximum level at 7 hr of infection under these conditions (Kang and Prevec, 1969). The medium containing virus was harvested and centrifuged at 600 g for 15 min to remove cellular debris. The cellfree virus fluids were stored at -75” until use. Quantitution of virus. The plaque assay technique used for determining the titer of standard infectious virus has been described previously (Kang and Prevec, 1969). We used BHK,, cells in all plaque assays. The amount and type of virus particles produced in each passage were monitored by the following procedure. The medium from infected cells was harvested and centrifuged at 600g for 15 min at 5” to remove cell debris. The supernatant was centrifuged at 81,000 g for 75 min in a Spinco SW 27 rotor to pellet the virus. The virus pellets were resuspended in 0.3 ml of phosphate-buffered saline (PBS) and layered on a linear 5-30% sucrose gradient made in PBS. After centrifugation at 110,OOOgfor 35 min at 5” in a Spinco SW 41 rotor, the gradients were collected from the t,op using a Buchler Auto-Densi-Flow IIC . The presence and distribution of virus in the gradient were monitored continuously by passing the effluent through an LKB Uvicord II, the absorbance at 280 nm being continuously recorded on a Fisher Recordall 5000.
ETAL. pended in approximately 0.3 ml of 0.02 M Tris-hydrochloride buffer (pH 7.3) containing 0.1 M NaCl and 0.001 M EDTA (TSE). The concentrated virus sample was layered on a linear 5-30% sucrose gradient made in TSE and centrifuged at 110,000g for 35 min. The visible bands of DI-1, DI2, and DI-3 were collected separately and [32PlRNAs were purified by the method described previously (Clewley et d., 1977). The purified 32P-labeled viral RNAs were digested with T, ribonuclease and the oligonucleotides were separated by the twodimensional gel-electrophoresis systems of de Watcher and Fiers (1972) as described previously (Clewley et al., 1977). RESULTS
Yield
of VSV in Four Different Hosts during Serial Undiluted Passages of a Clone of Standard Virion
To determine host effects on the generation of DI particles during serial undiluted high-multiplicity passages of VSV, a monoclonal isolate (plaque purified) of VSV was propagated in human (RSa), hamster (BHK& rat lR(B7711, and mouse (LJ cell lines. The yield of standard infectious virions was measured by plaque assay. Figure la demonstrates that clone No. 1 of VSVINDpassaged in the four different cell lines yields different amounts of infectious B virion in different serial passages. Similar patterns of reduction in production of the infectious viruses were observed when we used another clone (clone No. 2) (Fig. lb). RSa cells produce a lo-fold reduction of infectious VSV in each passage and an approximately three-log reduction from the original titer in yield of virus at the Preparation of ?P-labeled DI particle fourth passage. In contrast L, cells produce RNAs and oligonucleotide fingerprint analysis. Confluent monolayers of 1 X 10’ a constant amount of virus up to the sixth R(B77) cells were infected with 0.5 ml per to seventh passages and then show rapid dish of fourth passage VSV (see Fig. 2) to decline in the subsequent passages. The produce three distinct populations of DI BHK,, cells and R(B77) cells behave simiparticles. After 45 min of absorption, 7 ml larly. Five to six passages were required of DMEM containing 200 $Z!i of 32Pper ml before the yield of virus was reduced apwas added. After 7 hr, culture fluids con- proximately three logs from the initial taining 32P-labeled virus were collected titer. To determine whether the reduction of and centrifuged at 600 g for 15 min. The supernatant fluid was then centrifuged at titer after the serial undiluted passages of 81,000 g for 75 min in a Spinco SW 27 VSV is the result of the generation of DI rotor. The pelleted virus was resus- particles, the viruses from each passage
GENERATION 0
OF VSV DI PARTICLES
145
VSV,ND Clone I
0511123456789 ’ I ’ I ’ I ’ ’ Number
of
undiluted
passages
FIG. 1. Yield of standard B virion during successive undiluted passages of two clonal isolates of VSViND in four different hosts. The initial stock virus was prepared by the consecutive plaque isolation method as described in Materials and Methods. Confluent monolayer cultures of approximately lo7 cells per loo-mm culture dish were infected with 1 x 10s plaque-forming units of the stock virus for the first passage. After 45 min of absorption at 37”, 7 ml of prewarmed DMEM supplemented with 5% FCS was added to each culture dish and the dishes were incubated at 37” in CO2 for 7 hr. Cell-free virus fluids were harvested after 7 hr of growth and were centrifuged at 600 g for 15 min. The subsequent passages were made by infecting 0.5 ml of undiluted previous-passage virus onto 10’ cells/dish. At the end of each passage, the yield of infectious virus was measured by plaque assay on BHK,, cells.
were analyzed in sucrose gradient centrifugation. Figure 2 demonstrates that the production of DI particles directly correlates with the titer of virus shown in Fig. la. The RSa cells produce detectable amounts of DI particles at the second undiluted passage. The DI particles produced in the RSa cells are rather heterogeneous in size. The BHK,, cells produce a detectable amount of DI particles at the third undiluted passage and we could detect at least two distinct sizes of DI particles produced in BHK,, cells. In contrast, R(B77) cells produce three different size classes of DI particles which were not detectable until the fourth passage. Lz cells take seven to eight passages to produce detectable amounts of DI particles. There are three different sizes of DI particles produced in the Lz cells (Fig. 2). It is clear from these data that different host cells generate different numbers of size classes of DI particles after different numbers of passages. These results strongly suggest
that a host function determines the type of DI particles produced since different host cells produced multiple size classes of DI particles even though a monoclonal isolate was used to initiate the infections. We have repeated these experiments using the same clone of virus in the same host cells and found the results are reproducible as long as we use the same clonal isolate of virus with the same cell type (data not shown). Both Roy et al. (1973) and Holland et al. (1976) demonstrated that different clones of VSV generated different sixes of DI particles in the same host. To determine whether different clones of VSV produce repeatedly different patterns of DI particles, we prepared five other stocks of VEX,,, from five individual plaques from the original stock virus followed by six consecutive independent plaque isolations as described in Materials and Methods. We made serial undiluted passages of clone No. 2 (Fig. lb) and analyzed the
146
KANG ET AL. vsVl,,n
VSVIND
CiOtW 1 RSa
‘OP
D~stonce
of sedimentation
(Cm)
B%!
I
Clone 2 RlB77)
L2
bottom
FIG. 2. Analysis of total virus particles from the continuous undiluted passages of clone 1 of VSV,,. Samples of lysate from four culture dishes of each passage illustrated in Fig. la were centrifuged at 600 g for 15 min to remove cellular debris. The virus particles were pelleted from the supernatant by centrifugation at 81,000 g for 75 min in a Spinco SW 27 rotor, resuspended in 0.3 ml of PBS, and layered on a linear 530% sucrose gradient made in PBS. After centrifugation for 110,000g at 5” for 35 min in a Spinco SW 41 rotor the gradients were analyzed by the technique described in Materials and Methods. The clones of VSV, types of cells, passage numbers, and positions of the standard B virion (STD) and of the defective interfering particles (DI) are indicated. Electron microscopic examination of material in fractions sedimenting just in advance of the virus revealed only clumps of particles identical to B virions.
FIG. 3. Analysis of total virus particles from the continuous undiluted passages of clone 2 of VSViND. Samples of lysate from four culture dishes of each passage illustrated in Fig. lb were analyzed in the sucrose gradient. See legend to Fig. 2.
classes in L, cells. In contrast, clone No. 1 produces three different size classes of DI particles in both R(B77) cells and LZ cells. Clone No. 2 consistently generates the same pattern of DI particles in the same host cell during serial undiluted passages. In addition, we found four other clones generated the same pattern of the DI particles as that shown with clone No. 1 (data not shown). It is clear that different hosts produce different size classes of DI particles at different numbers of undiluted passages. Thus, it appears that the production of DI particles by serial undiluted passages of vesicular stomatitis B virion is dependent on (i) the clonal isolates of the virus and (ii) the host cells.
virus particles produced during undiluted passages. Figure 3 demonstrates that the number of passages required to generate a detectable amount of DI particles correlates well with the titer of the infectious virion (Fig. lb). However, the pattern of of Biological Activities of DI particles produced by clone No. 2 (Fig. Determination DI Particles Generated during Undi3) is different from that produced by clone 1uted Passages No. 1 (Fig. 2). There are distinct differWe demonstrated previously that DI ences between clone No. 1 and clone No. 2 propagated in R(B77) cells and in L, cells. particles of VSV can serve as templates by helper infecClone No. 2 produces only .one type of DI for their multiplication particles in R(B77) cells and two size tious B virions (Prevec and Kang, 1970)
and that reduction of infectious B virion synthesis occurred when DI particles were mixedly infected with standard B virions. To test the biological activities of DI particles generated by multiple passages, we infected cells with purified DI particles mixed with an early passage of VSV, which did not give rise to a detectable amount of DI particles. These experiments are shown in Fig. 4. We used DI particles from the fifth passage of clone No. 1 which was grown in R(B77) cells and purified by sucrose gradient centrifugation. Figure 4a shows the types of virus particles produced in this passage. Fractions 8-10 (designated DI-l), fractions 12-14 (designated DI-2), and fractions 16-18 (designated DI-3) were pooled separately and used for mixed infection with the first passage of VSV (Fig. 4). When partially purified DI-1 particles were mixed with infectious standard B virions and grown in three different hosts,
h
2 ‘OP
4
6
147
OF VSV DI PARTICLES
GENERATION
8 31stonce
predominantly DI-1 was produced while the production of infectious virus particles was reduced (Figs. 4c, g, and k). In addition, the BHK,, and R(B77) cells produced significant quantities of DI-2 (Figs. 4g and k). This may well be the result of contaminating DI-2 in the DI-1 preparation and of preferential replication of DI-2 in the BHK,, and R(B77) cells, as compared to RSa cells. Mainly DI-2 species were produced when the DI-2 preparation was mixedly infected with infectious standard B virions (Figs. 4d, h, and 11,and mainly DI-3 species were produced when we mixedly infected with the preparation containing DI-3 particles (Figs. 4e, i, and m). In all cases we observed that the yield of standard B virions was reduced as a result of mixed infection with DI particles. It is obvious that the level of infectious standard B virion produced in three different host cells varied. We found no detectable amount of virus production when we in-
n
2 of
4 6 8 sedtmentofton !CT)
2
4
6 bottk
FIG. 4. Effects of DI particles on the total particle yield in three different hosts mixedly infected with the standard B virion. DI-1, DI-2, and,DI-3 were prepared from lysates of the fifth passage of VSV,,, clone l-infected R(B77) cells (Figs. 2 and 4a). Confluent-culture dishes containing about 1 x 10’ cells were infected with 5 x lo8 PFU of first-passage VSV iND clone 1 with or without addition of 0.1 ml of DI particle preparation as indicated. After absorption for 45 min at 37” the prewarmed culture medium was added and the dishes were incubated for 7 hr at 37” in a CO, incubator. Samples of lysate from four culture dishes of each combination were analyzed on the sucrose gradient described in the legend to Fig. 2.
148
KANG
ET AL.
fected cells with purified DI particles alone from BHK,, (the heavier DI particle (data not shown). Therefore, we conclude shown in Fig. 2, BHK,,-P,) also have an that all three DI particles generated in identical oligonucleotide fingerprint patR(B77) cells by serial undiluted passages tern (data not shown). Therefore, all five of clone No. 1 of VSV,,, (Fig. 4a) are DI particle RNA sequences represent a indeed defective and interfering particles. common region of RNA sequence in the We performed similar experiments using standard B virion genome and represent DI particles isolated from BHK,, cells and only the negative-stranded genomic RNA. L cells. We found that the two DI particles DISCUSSION generated in BHK,, cells and the three DI The patterns of DI particles generated particles generated in Lz cells by clone during serial undiluted passages of clonal No. 1 (Fig. 2) are all defective in their infectious B virions of VSV have been replication and are capable of interference. studied in several laboratories (Holland et Thus, we conclude that all small truncated al., 1976; Reichmann et al., 1971; Schincaparticles generated in the four different riol and Howatson, 1970; Stampfer et al., cells are defective interfering particles. 1971). These reports brought to our attention the question of whether the processes Oligonucleotide Fingerprint Analysis of in the generation of DI particles are ranDI Particle RNAs dom or directed. We have shown here that We were interested in determining the the generation of DI particles during highnucleotide sequences of DI particle RNAs multiplicity passages is dependent upon generated in different cell types using the both the clonal isolate of the virus and the monoclonal isolate of the virus. Figure 5 host cell. One clonal isolate generates difillustrates the oligonucleotide fingerprint ferent patterns of DI particles in different of the standard B virion RNA. The nomen- cells. Thus, the host plays a role in conclature was arrived at by comparing the trolling the generation of DI particles. In radioautograms of samples run for differ- addition, we have shown that a different ent lengths of time in the first or second clone may generate different patterns of dimension to produce slightly better reso- DI particles in the same host. Thus, in lution of some of the groups of oligonucle- some way the clone of infectious B virions otides. Although we found that some of also contains a determining factor for DI the fingerprint patterns showed better sep- particle synthesis. We have observed, aration of spots between 18 and 23, the however, that a given clonal isolate of B oligonucleotide fingerprint pattern shown virions always generates the same pattern of DI particles in a given cell type. This is in Fig. 5 was best for overall resolution. Figure 6 shows the oligonucleotide finger- consistent with the observations of Reichprints of RNAs from the three DI particles mann et al. (1971) and Schincariol and generated from R(B77) cells (see Figs. 2 Howatson (1970) who respectively generand 4a). All the identifiable oligonucleo- ated particular-size DI particles with partides of DI-1 RNA are present in DI-2 ticular temperature-sensitive mutants of RNA and all the oligonucleotides present VSV, or with a heat-resistant strain of in DI-2 RNA are also present in DI-3 VSV,,. A part of our data is also consistRNA. These results strongly suggest that ent with the observation of Holland et al. the DI-3 RNA contains all the nucleotide (1976) that different clonal isolates genersequences of DI-2 and PI-1 RNAs, and ate different patterns of DI particles when that DI-2 RNA contains all the nucleotide propagated in the same host or that the sequences of DI-1 RNA as illustrated in same clonal isolate generates different Fig. 7. In addition, we found that DI-2 patterns of DI particles in different hosts passages. HowRNA from R(B77) cells and DI-1 RNA during high-multiplicity from BHK,, (the lighter DI particle shown ever, Holland et al. (1976) used BHK,, in Fig. 2, BHK,,-P,) share identical pat- cells to amplify their virus before the final terns of oligonucleotide fingerprint. The analysis which may have added other varDb3 RNA from R(B77) cells and the DI-2 iables.
GENERATION
,--I : . i. 4. ; : .. .. .
@@ P.. .. . .
OF VSV DI PARTICLES
150
KANG
ET AL.
GENERATION
DI-2
11
DI-311
11
11
I 11
II
II
III)
151
OF VSV DI PARTICLES
8 10I 1411 36 36I 3711 44 66II67 11 6 11
I I I\ 11
I]]/
IIII
.
1 III]
/ III] 24 d 53
. or 29 or 31
. . or52
FIG. 7. Hypothetical
scheme for mapping the DI-1, DI-2, and DI-3 RNAs using oligonucleotide fingerprint analysis. All 14 identifiable oligonucleotides in DI-1 RNA are present in DI-2 RNA and all 14 oligonucleotides of DI-1 RNA and the additional 10 oligonucleotides in DI-2 RNA are present in DI-3 RNA. The DI-3 RNA contains five additional oligonucleotides which are not present in either DI-1 or DI-2 RNA. All the oligonucleotides present in all three DI particle RNAs are present in the standard B virion RNA as shown in Fig. 6.
Holland et al. (1976) suggested that the generation of DI particles during the successive undiluted high-multiplicity passages of VSV is a random process and that a selective amplification process during the additional passages determines the visible quantities of different size classes of DI particles. Accordingly, they have further predicted that the infectious B virion is not genetically predisposed to generate particular DI particles. Our results suggest that there may be some genetic determinant in infectious B virions which in conjunction with certain host factors generate consistent patterns of DI particles. DI particles of VSV have been detected as early as the third 7.5-hr passage in Chinese hamster ovary cells using a sensitive detection method (Stampfer et d., 1971). Holland et al. (1976) observed that rabies virus generated large quantities of DI particles at the first passage of the virus, whereas Cole et al. (1971) had to make 18 undiluted passages to detect DI particles of poliovirus. We found that the number of passages required for a visible amount of DI particle synthesis, at least in the case of VSV, is largely dependent on the host cells. Two different virus clones passaged in four different hosts produced DI particles at the same number of passages in given cell types. This observation suggests that the initial induction and subsequent amplification of DI particles in various hosts is different. It appears that the rate of induction determines the overall production of DI particles since the
yield of standard B virion during the first passage in these four different hosts did not vary significantly. We found that the defective particles we generated were also interfering particles and that the DI particles generated in one cell type are capable of interfering with the production of infectious particles in different hosts. However, the degree of interference in different hosts varies significantly as determined by standard B virion production. This observation is consistent with data previously reported (Potter and Stewart, 1976). Surprisingly, the RNAs from the five different DI particles share completely overlapping nucleotide sequences in spite of the fact that two are generated in BHK,, cells and three are generated in R(B77) cells. They represent a constant region of the infectious negative-stranded B virion genome and no positive-stranded RNA sequences were detected. We do not understand yet how a deletion takes place during the replication of VSV. However, direct evidence is being sought showing that the host cell plays a role in the induction of DI particles. ACKNOWLEDGMENTS We are very grateful to Rae Allen and Jan Reed for their excellent technical support. We thank Drs. J. Bednarz-Prashad, B. Ozanne, and J. Shadduck for stimulating discussions and for their detailed reviews of this manuscript. This investigation was supported by Public Health Service Grants CA 16479 and CA 20012 from the National Cancer Institute and by Grants AI 13402, AI 12070, and AI 10984 from the National Institute of Allergy and Infectious Diseases.
152
KANG REFERENCES
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infected with temperature-sensitive mutants of vesicular stomatitis virus. J. Virol. 8, 154-160. ROY, P., REPIK, P., HEFTI, E., and BISHOP, D. H. L. (1973). Complementary RNA species isolated from vesicular stomatitis (HR strain) defective virions. J. Virol. 11,9X-925. SCHINCARIOL,A. L., and HOWATSON,A. F. (1970). Replication of vesicular stomatitis virus. I. Viral specific RNA and nucleoprotein in infected L cells. Virology 42, 732-734. STAMMINGER, G., and LAZZARINI, R. A. (1974). Analysis of the RNA of defective VSV particles. Cell 3, 85-93.
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