Frequent generation of new 3′-defective interfering particles of vesicular stomatitis virus

143, 630-635 (1985)

VIKOLOGY

Frequent

Generation Particles

C. YONG KANG,*~’

of New 3’-Defective

of Vesicular

MANFRED

Stomatitis

SCHUBERT,?.

Interfering Virus

AND ROBERT

A. LAZZARINQ

of Mi~obiology and Immunology, School of Medicine, Faculty of Health Sciences, University ‘Department Ottawa. Ottawa, Onta?%o KIH 8M5, Canada and TLaboratmy of Molecular Genetics, NINCDS, 20205 Natirmal b&it&es of He&h,, Bethesda, Maryland Received October 25, 1084 accepted February

of

18, 1985

We have isolated and partially characterized a number of different genome types of defective interfering (DI) particles newly generated by a highly heat-resistant strain of vesicular stomatitis virus in either Rat(B77) or Vero cells. Northern blot analyses revealed that many of these DI genomes contain N gene sequences and/or sequences of the NS, M, and G genes. One type contains NS sequences without any indication for the presence of either N, M, or G sequences. Another type of DI particle genomes did not contain any detectable sequences of N, NS, M, or G, hut did contain panhandle-type sequences and, thus, most likely resembles the 5’-panhandle-type DI particles. Unlike previously assumed, these data demonstrate that DI genomes which have the 3’-terminal N, NS, M, and G genes or portions of these genes conserved do frequently arise together with 5’-DI particle genomes after serial undiluted passages of the heat-resistant strain of VeSiCUhr StOmatitiS virus. & 1985 Academic Press, Inc

Defective interfering (DI) virus particles contain only a portion of the genomic sequences of the parental standard virus (8, 13, 19, 23). Thus, DI particles cannot self-replicate, but can only be propagated and amplified after eoinfection with competent standard virus as helper. The helper virus provides the missing gene products for the replication of DI particles. The fact that the DI particle genomes are efficiently replicated by the enzymes from the standard helper virus demonstrates that the genomes of the DI particles and standard virus share the minimum essential characteristics for replication. Coinfection of DI particles with standard virions results in inhibition of standard virus

production,

a phenomenon

known

as

autointerference. The molecular mechanism of autointerference is not. yet known. The genetic content of a substantial number of DI particles of negativestrand viruses have been characterized by hybridization, oligonucleotide fingerprint I Author addressed. 004%6822185 Copyright All rights

to whom requests for reprints

$3.00

b 1985 by Academic Press. Inc 01 rernmductlon in any form reserved.

should he 630

analysis, and direct sequencing analysis. These include VSV (6, 11, 13, 14, 16, 17, 28), Sendai virus (15, FZ), and influenza virus (4, 5). It is clear from these studies that the terminal promoter sequences essential for RNA replication are always conserved. Most of the previously isolated DI particles of VSV represent a portion of the 5’ half of the genome with a so-called compound or panhandle-type initiation site at the 3’ terminus of the RNA. In contrast, there is only one well-characterized DI particle of VSV, designated DILT, which contains the genomic information from the 3’ half of the parental genome (6, 14, 20, 28). This DI particle appears to be a true deletion mutant in which most of the polymerase gene (L) has been deleted. The presence of both parental viral RNA termini in this DI particle genome has been demonstrated by hybridization (18), heteroduplex mapping, and by direct sequencing (6). Because the 3’ half of the parental genome is conserved in the DI-LT genome, it can be transcribed by the viral polymerase,

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yielding leader RNA and four functional messenger RNAs (3, 9). This synthetic activity of DI-LT is unique among DI particles and confers special biological properties (1, 2, 21). DI-LT interferes with the replication of standard virus from both Indiana (homotypic interference) and New Jersey (heterotypic interference) serotypes of VSV (21). In contrast, all other DI particle RNAs analyzed to date have retained different portions of the 5’ terminus of the standard virion genome, and they only interfere with the replication of standard virus of their own serotype (21; Kang, unpublished observation). Since these two different types of DI particles originate from the two different halves of the standard virus genome and have distinct biological properties, we were interested in isolating additional 3’-DI particles in order to study the relationships between the genomic sequences retained with respect to homotypic and heterotypic interference. We took the original heat-resistant strain of the Indiana serotype of VSV @I), heated at 45” for 30 min, and made appropriate virus dilutions, infected cells, overlayed with 0.9% agar, and incubated at 40.5” overnight. Plaques arising from this treatment were picked and subsequent plaque isolations were made by repeating the procedure five times and by two additional plaque purifications in cells pretreated with 1 pg/ml actinomycin D. The heat-resistant virus was propagated either in rat cells R(B7’7) cells obtained from Howard Temin, McArdle Laboratory, University of Wisconsin or in Vero cells (American Type Culture Collection). Subsequent high m.o.i. passages with this virus were made until an appropriate concentration of DI particles was detected. After some five 15-hr passages, detectable levels of DI particles were produced. These were appropriately diluted to get large quantities of DI particles in the subsequent passage. The DI particles were partially purified on sucrose gradients. Three distinct size groups of DI particles were produced from R(B7’7) cells. These were designated small (Rat-S), medium (RatM), and large (Rat-L). Another passage

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in the same cell line produced a group of DI particles which we designated Rat-C. The virus passaged in Vero cells produced two different groups of DI particles, the first group sedimenting just above the standard virus particles and the second group sedimenting just above the first. The first group of DI particles produced from Vero cells was designated as VeroG and the other group was designated as Vero-L. Purified DI particles were concentrated by centrifugation and the pellet was resuspended in 0.01 M Tris-HCl, pH 7.3, 0.001 M EDTA, 0.1 MNaCl, and 0.5% SDS. Proteinase K (50 pg/ml) was added and incubated for 30 min at 30”. The RNA was extracted as previously described (10). Prior to electrophoresis, the RNAs were denatured in 0.02 M borate buffer, pH 8.3, 0.2 mM EDTA, 50% deionized formamide, 6% formaldehyde, 10% glycerol, 0.2% bromophenol blue, and 0.2% xylene cyan01 for 3 min at 65”. The RNAs were separated in 1.2% agarose gels, containing 3% formaldehyde in 0.02 M borate buffer, pH 8.3, and 0.2 mM EDTA, electroblotted onto Gene-screen (New England Nuclear), and hybridized according to standard procedures (29) to [a-32P]ATP-labeled, nicktranslated (BRL nick-translation kit) cDNA probes obtained from J. Rose (7, 24). These cDNA probes represent almost the complete sequences of the N gene (pNF4), the NS gene (pNS1’73), the M gene (pM309), and the G gene (pGFl), respectively. In addition, [Lu-32P]GTP-labeled (-) sense DI leader RNA (Fig. 2) was used as a hybridization probe. This 46-nucleotide probe is identical to the 5’-terminal sequences of VSV genomic RNA (25, 27) and was synthesized in an in vitro transcription reaction (3) using [cx-~~P]GTP and DI 011 RNA as a template. Its complementthe panhandle region-is found at the 3’terminal origin of replication of the majority of DI RNAs analyzed to date, DI RNAs derived from the L gene region (13, 16, 19). Unlike the cDNA hybridization probes which can anneal to both (+) and (-) sense VSV sequences, this singlestranded RNA probe hybridizes exclusively to its (+) sense complement. The

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Northern blot hybridizations using the four nick-translated cDNA probes is shown in Fig. 1. The hybridization pattern using the short DI leader RNA is pre-

sented in Fig. 2. With every DI particle RNA preparation, a large number of subgenomic RNAs was detected upon hybridization with the various probes. In fact,

-28s

-MS -

‘L

23456-

FIG. 1. Agarose gel electrophoresis of DI genomic RNAs and hybridization with 32P-labeled VSV-specific cDNA probes. The purified DI particle RNAs were separated in 1.2% agarose gels and transferred onto Gene-screen by electroblotting using the Rio-Rad transblotting instrument. The blotted RNA was hybridized with nick-translated pNF 4, pNS 173, pM 309, and pGF 1. 14Clabeled ribosomal RNAs were used as size markers.

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-28s

4Oll-18s 7-

46mr

DI-Products

FIG. 2. Agarose gel electrophoresis of DI genomic RNAs and hybridization with 32P-labeled DI leader RNA transcript. The purified DI particle RNAs were separated on a 1.2% agarose gel, transblotted onto Gene-screen, and hybridized with [a-=P]GTP-labeled 46-nucleotide DI leader RNA transcript. 14C-labeled ribosomal RNAs were used as size markers.

over 20 different RNA species could be counted using, i.e., DI leader RNA as a probe (Fig. 2). Even a single DI particle band isolated from a sucrose gradient contained multiple subgenomic RNA species. Size selection on a gradient only led to a slight enrichment of a particular size group (compare Rat S, M, and L in Fig. 1). Comparison of the migration of the RNA species relative to the ribosomal 18 S and 28 S size markers demonstrates that the sizes of the DI RNA species cover a broad range from approximately 10 S, roughly 1 kb and well below the 18 S marker, to approximately 30 to 35 S (6 kb) above the 28 S marker. Significant, amounts of any DI RNA species, however, were only detected within a size range from 16 S to 32 S. It is possible that the RNA species below 16 S may represent

633

partial breakdown products of the major RNA species. Their low abundancy may also suggest a minimum length requirement for packaging subgenomic ribonucleocapsids into DI particles. We would like to point out that although we refer to the subgenomic and defective RNAs as DI RNAs the ability of each individual species to interfere with the replication of the parental virus has not been demonstrated. As shown in Fig. 1, many RNA species were detected which hybridized to the N, NS, M, and G specific probes, demonstrating that DI RNAs with genetic information from the 3’ half of the VSV genome were frequently generated in R(B7’7) as well as Vero cells using the heat-resistant strain of VSV. We do not know, whether selection and propagation of the virus at 45” and 40.5”, respectively, may alter the functional properties of the viral polymerase and/or cellular factor(s) which unexpectedly allowed this frequent generation of 3’-DI particles to occur. So far, only two DI particle RNAs have been studied in detail which map in the 3’terminal region. The vast majority of DI RNAs has been mapped in the 5’-terminal L-gene region. With the system described in this communication, DI RNAs derived from the 3’ half of the VSV genome arose frequently after several undiluted passages. Because of the large number of DI RNAs, we cannot rule out the possibility that two or more DI RNA species comigrate on the gel and may hybridize to different probes. This, of course, would affect the interpretation of the hybridization data and, consequently, the mapping of these DI RNAs. In addition, the intensity of the signals by Northern blot is dependent on the concentration of a particular RNA species and also on the size of the portion of the gene that has been conserved. Therefore, instead of referring to individual RNA species, the numerical designations l-7 in Figs. 1 and 2 mark RNA size classes and tentative maps of the DI RNAs are not shown. However, despite these criteria, a close approximation of the structures of a few individual DI RNA species may be made.

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For example, a number of 30 S to 32 S DI RNA species marked 1 and 2 in Fig. 1 seem to hybridize with all of the N, NS, M, and G specific cDNA probes. Smaller RNA species, however, like those of the 16 S size class, marked 7 in Fig. 1, apparently only hybridized with probes derived from the first two genes of the VSV genome, N and NS. Although we did not probe with cDNAs derived from the Lgene region which corresponds to 57% of the total VSV genome (26), the overall sizes of these DI RNAs roughly correspond to the combined sizes of the individual genes that may have been conserved. The fact that more DI RNAs gave positive signals with the N and NS gene clones than with the M and G gene clones is consistent with the idea that genes like N and NS which are positioned near the 3’-terminal origin of replication are more frequently conserved than internal genes such as M and G. However, some DI RNAs seem to contain the NS gene, but neither N, M, nor G (see Rat-C, size class 5, and Rat-L, size class 7), suggesting that a double-copy-choice replication event must have been involved in the generation of these putative double-deletion DI particle RNAs. Some DI particle RNAs did not contain any sequences from the 3’ half of the VSV genome like Vero G size classes 4 and 7 which gave a strong hybridization signal when the 46-nucleotidelong DI product RNA was used as a probe (Fig. 2). These DI genomes may resemble the typical 5’-panhandle DI RNAs which have the 5’ end of the VSV genome and part of the L gene conserved. Size class 2, rat S and Vero G; size classes 4 and ‘7, rat L as well as size class 7, rat M which contain sequences from the 3’ half of the genome may also contain panhandle-type sequences like the more conventional 5’panhandle DI RNAs. These DI RNAs appear to be similar to the compound-type DI-LTe RNA previously described (6, 12, 13,

18).

As pointed out earlier, there are many subgenomic RNAs which hybridize with the DI product probe, but within each RNA preparation only one to three species are present in a relatively high concentration. On the other hand, there are a few

more RNA species which gave strong positive signals with the four cDNA probes (Fig. 1). It appears that the generation of DI particle genomes favors neither genomes which contain the 3’ half nor genomes containing the 5’ half of the VSV genome. We do not know whether this is a characteristic of the heat-resistant strain of VSV. We cannot calculate the molar ratios of these 5’ and 3’ DI RNAs at this time because the various probes differ in length and specific activity, therefore, an evaluation of specific selective advantages of some DI genome structures over others as previously proposed, i.e., for DI-LTz over DI-LT1 (6, 12, 18) has to await further experimentation. In order to test the biological activities of the individual DI particles, it is necessary to establish a technique which will selectively amplify or complement one of these DI particles, i.e., by using cell lines which constitutively express a particular VSV gene. We are currently attempting to obtain pure preparations of the DI particles. This will allow a precise mapping of the RNAs using different restriction fragments of the cDNA clones and will make it possible to test the biological activities of the individual DI particles representing different portions of the 3’ half of the virus genome in respect to homotypic and heterotypic autointerference. ACKNOWLEDGMENTS We thank Dr. John K. Rose for the VSV cDNA clones used in this study. We thank Dr. Lud Prevec and Dr. Ken Dimock for constructive criticism of this manuscript. This study was supported by Grant MA-7696 from the Medical Research Council of Canada. REFERENCES 1. ADACHI, T., and LAZZARINI, R. A., Virology 87, 152-163 (1978). L BAY, P. H. S., and REICHMANN, M. E., J. Irirol. 419, 172-182 (1982). 3. COLONNO, R. J., LAZZARINI, R. A., KEENE, J. D., and BANERJEE, A. K., Proc. N&l. Acad. Sci. USA 74, 1884-1888 (1977). 4. DAVIS, A. R., and NAYAK, D. P., Proc. N&l. Acad. Ski. USA 76, 3092-3096 (1979). 5. DAVIS, A. R., HITI, A. L., and NAYAK, D. P., Proc. N&l. Acad Sci. USA 77, 215-219 (1980).

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