Sendai virus DI RNA species with conserved virus genome termini and extensive internal deletions

VIROLOGY

118,1’7-27

(1982)

Sendai Virus DI RNA Species with Conserved Virus Genome Termini and Extensive Internal Deletions L. S. AMESSE, C. L. PRIDGEN, AND D. W. KINGSBURY’ Diviakm

of Virology, St Jude Children’s Research Hospital, Memphis, Tennessee 98101 Received September 94, 1981; accqded November

90, 1981

Ten species of RNA from Sendai virus DI particles had apparent molecular weights corresponding to 9 to 30% of the virus genome. In every DI RNA, most of the RNase Tlresistant oligonucleotides were from the L gene at the 5’ end of the genome, and they formed an overlapping series, indicating that all of these DI RNA species possess common B-terminal sequences. The unusual feature possessed by nine of the DI RNAs was the presence of one to four oligonucleotides from the 3’ region of the genome, representing the putative leader RNA template and/or part of the adjacent NP gene. No RNA species contained any of the oligonucleotides representing the remaining virus genes (P, M, FO, or HN) between NP and L on the genetic map. Therefore, the majority of Sendai virus DI RNAs are extensively deleted internally, but they retain sequences proximal to (and probably including) the 8 and 5’ termini of the virus genome. We propose the term “fusion DI RNAs” for these species, since 3’ sequences of the NP gene are fused to 5’ sequences of the L gene, erasing all transcription termination signals. This sequence arrangement may explain the replication advantage possessed by these DI RNA species.

INTRODUCTION

The ability of many types of viruses to cause persistent infections is well documented, although the mechanisms involved remain poorly understood. In some cases, viruses may persist by generating defective-interfering (DI) particles which inhibit replication of the standard virus (Huang and Baltimore, 1970). We have been using Sendai virus as a model system to study the generation and replication of DI RNA molecules. This virus is a prototype paramyxovirus, representing a group of negative-strand RNA viruses that includes several human pathogens implicated in chronic diseases. The structures of several types of DI RNAs derived from nonsegmented negative-strand viruses have been characterized. Leppert et al (197’7) discovered that several Sendai virus DI RNAs possess only the 5’ terminus of the virus genome and various portions of the adjacent L gene. All other virus genome sequences, includ‘To whom correspondence and requests for reprints should be addressed.

ing those at the 3’ terminus, were deleted; in their place was a segment of 110-150 nucleotides complementary to the 5’ end of the nondefective genome, providing a novel 3’ terminus. Analogous “copy-back” RNA species, containing only 5’4erminal L gene sequences and their complements, are the most common class described for the rhabdovirus, VSV (Schnitzlein and Reichmann, 1976; Perrault et aL, 1978). However, two other examples of VSV DI RNA have a markedly different structure. A true deletion mutant, LT, retains both genome termini and four of the virus genes; it lacks most of the L gene (Schnitzlein and Reichman, 1976; Colonno et al, 1977). The other DI RNA, LT,, has recently been shown to resemble LT, but it is more complex, due to the addition of a “copyback” 3’-terminal sequence of 70 nucleotides complementary to the 5’ terminus (Keene et CCL,1981). We now describe a series of Sendai virus DI RNAs exhibiting converved terminal sequences and extensive internal deletions, a structure not previously seen among nonsegmented negative-strand RNA viruses. We discuss this structure with regard to previous models of DI par17

0042-6822/82/050017-11$02.00/O Copyright Q 1982 by Academic Praes, Inc. All righta of reproduction in any form reserved.

AMESSE,

18

PRIDGEN,

title generation, survival, and ability to interfere with replication of standard virus. MATERIALS

AND METHODS

DI RNAs. The origins of the DI strains used in this work are as follows: Strain 1 DI particles were present in the original stock of the Enders strain, described by Kingsbury et aL (1970). Strains ‘7 to 12 were derived from nondefective Enders strain plaque isolates by six repeated high multiplicity passages in chick embryo lung cells or embryonated eggs (Kingsbury and Portner, 1970). None of these strains has been passaged more than four times since their original description. Strains R and H were gifts of D. Kolakofsky (Leppert et cd, 1977). To prepare 32P-labeled DI RNA species, CEL cells in monolayer cultures were infected with virus samples containing a 5to 50-fold excess of DI particles (Kingsbury et al, 1970) at a multiplicity of about 10 infectious units per cell. r&P]Orthophosphate was added 24 hr later and virus was harvested after 48 hr further incubation (Amesse and Kingsbury, 1982). Virus was collected from the medium by centrifugation, suspended in 0.005 M TrisHCl, 0.001 M EDTA, 0.1 M NaCl (pH 7.4), made 1% in SDS and 0.05% in Proteinase K, heated for 15 min at 56”, extracted twice with phenol, and precipitated with ethanol. After electrophoresis in an acidurea-agarose gel (1.25% in agarose), bands identified by rapid autoradiography of the wet gels were cut out and extracted with phenol as described by Freeman et al. (1979). Ribonuclease Tl-resistant oligonucleotides in these RNAs were separated by two-dimensional polyacrylamide gel electrophoresis (Amesse and Kingsbury, 1982). Second-digestion analysis of Tl-oligonucleotides. Oligonucleotides were located in wet polyacrylamide gels by autoradiography. The spots were cut out with a cork borer, crushed in a Dounce homogenizer, extracted with 0.001 M EDTA (pH 7.4), concentrated by lyophilization, and analyzed by PEI-cellulose chromatography after RNase A digestion, as described by Volckaert and Fiers (1974).

AND KINGSBURY

RESULTS

Sizes of DI RNAs The DI particles used in this study contained a variety of RNA species of different sizes. Fig. 1 displays the electrophoretie separation of glyoxal-denatured RNA molecules from eight separately passaged DI strains. Six of these strains, bearing numerical designations, are from our collection. The alphabetically labeled strains, H and R (Leppert et al, 1977), were provided by D. Kolakofsky. The DI RNA species are designated by lower case letters, so that the single DI RNA in our strain 11 is designated lla, and the two DI RNA species in strain R are designated Ra and Rb, etc. Apparent molecular weights of the RNAs ranged from a minimum of 4 X 10’ (lla) to a maximum of 1.4 X lo6 (la). Taking the molecular weight of the nondefective virus genome as 4.8 X lo6 (Kolakofsky et al, 1974), these RNAs correspond to 9 to 31% of the size of the genome (Table 1). In several cases, multiple DI RNAs were resolved: a maximum of four was seen in strain 12, strains 1 and 7 each had three, and strain R had two (Fig. 1). Strain 1 contains the spontaneously occurring DI particles that we described in 1970 (Kingsbury et al, 1970). At that time, we had resolved two RNA species by sucrose gradient centrifugation and electrophoresis in nondenaturing gels; these are represented by bands la and lc in Fig. 1. An additional band, lb, has now been resolved. When multiple DI RNA species were seen within a strain, they differed in relative abundance. Kolakofsky (1979) has shown that these abundances vary from passage to passage, for unknown reasons. Genome-size (50 S) RNA was represented in variable amounts, relative to DI RNAs, in different preparations (Fig. 1). The apparent molecular weight of the genome calculated from its electrophoretic mobility was 4.5 X 106,a value within 10% of the molecular weight obtained by electron microscopy of genome-antigenome duplexes (Kolakofsky et d, 1974). Also seen were variable amounts of 28 S and 18 S ribosomal RNAs. which are

SENDAI VIRUS DI RNAs

19

FIG. 1. Sizes of Sendai virus DI RNAs. Virus particles labeled in tivo with [32P]orthophosphate were disrupted with SDS and Proteinase K and the RNA was denatured with glyoxal (McMaster and Carmichael, 1977) before electrophoresis in an acid-urea-agarose gel (1.25% in agarose) (Freeman et o& 1979). At the top of each lane is the designation of each DI particle strain. Lower case letters represent DI RNA species and the numbers 18 and 29 designate ribosomal RNAs.

designated numerically. These cellular RNA species appear to be included within virions in the budding process (Kolakofsky and Bruschi, 1975), providing convenient built-in molecular weight markers. DI RNAs 9a and 7b migrated close to 18 S ribosomal RNA in glyoxal gels, but in the less stringent denaturing conditions of the acid-urea gels used to prepare RNAs for oligonucleotide mapping, these viral RNA species were well-separated from ribosomal RNA. L Gene Oligmucleotides in DI RNAs Several of the RNA species shown in Fig. 1 were labeled in viva with [32P]orthophosphate and isolated from

acid-urea-agarose gels (Freeman et al., 1979). Their ribonuclease Tl-resistant oligonucleotides are shown in Figs. 2 through 5. None of the RNA species in strain 12 has been examined yet. Instead, the single DI RNA found in strain 10, which had an apparent molecular weight identical to RNA Ha (Table l), is shown (Fig. 2). The oligonucleotides assigned to each DI RNA species are listed in Table 2. This list has been arranged in order of increasing representation of oligonucleotides previously identified in the L gene of the nondefective genome (Amesse and Kingsbury, 1982). Most of the characteristic oligonucleotides in each DI RNA are L gene-specific and they form an overlapping series. Since 5’terminal nucleotide sequences are commonly conserved in negative-strand virus

AMESSE, PRIDGEN, AND KINGSBURY

20 TABLE 1

APPARENTMOLECULARWEIGHTSOF SENDAIVIRUS DI RNAS’

RNA species

MW x 10-s

Percentage of genomg

lla 10 Ha 9a 7b 3a lb Rb Ra la

0.40 0.49 0.49 0.63 0.70 0.75 0.91 0.92 1.13 1.44

3.7 10.7 10.7 14.8 15.2 16.3 19.8 20.0 24.6 31.3

oligonucleotide 7, which we believe is derived from the 3’-terminal template of viral leader RNA (Amesse and Kingsbury,

“Molecular weights relative to ribosomal RNA species under denaturing conditions (Fig. 1). The molecular weight of 28 S ribosomal RNA was taken as 1.75 X 10s and 18 S ribosomal RNA as 0.68 X lo6 (McMaster and Carmichael, 1977). bThe genome molecular weight was taken as 4.8 x 106(Kolakofsky et al., 1974).

DI RNAs (Perrault et d, 1978), and since Leppert et al. (1977) have explicitly demonstrated the presence of B-terminal L gene sequences in RNAs Ha and Ra (Kolakofsky’s H14S and R20S), this overlapping pattern appears to represent the sequence order of L gene oligonucleotides. (Groups of oligonucleotides that were not divided between different DI RNA species in this series are enclosed within parentheses, since they cannot be ordered). RNA la had the largest number of L gene-specific oligonucleotides (17), representing about two-thirds of the total oligonucleotides identified in this gene. At the other extreme, RNA 8a possessed only five Lspecific oligonucleotides, less than 20% of the oligonucleotides in this gene and less than one-third of those seen in RNA la. S’-Specific Seqtmnca in DI RNAs Of the 10 RNA species in this series, only Rb lacked oligonucleotides from the 3’ region of the viral genome. All seven of the RNAs from our collection contained

FIG. 2. Ribonuclease Tl-resistant oligonucleotides in DI RNAs 10, lla, and Ha. v-labeled RNA species were isolated by electrophoresis as described under Materials and Methods. Two-dimensional electrophoresis of oligonucleotides in polyacrylamide gels has been described (Amesse and Kingsbury, 1982). The horizontal dimension (left to right) separates mainly by base composition and the vertical dimension (bottom to top) mainly by size. Spots are numbered as described for the virus genome (Amesse and Kingsbury, 1982). Arrows point to oligonucleotides not seen in the virus genome.

SENDAI VIRUS DI RNAs

FIG. 3. Tl-oligonucleotides in Sendai virus DI RNA species 8a, 9a, and ‘7b. Conditions are summarized in the legend of Fig. 2.

1932). Four of the DI RNAs, 8a, lla, 10, and la, contained oligonucleotides from the NP gene, which is adjacent to the leader. In addition, two RNA species from Kolakofsky’s collection, Ha and Ra, contained NP-specific oligonucleotides (Table 2). Indeed, Ra possessed the complete set of four oligonucleotides found in the NP gene, more than any other RNA in the series. Additional oligonucleotide spots, not previously seen in our analyses of Sen-

21

dai virus genomes, were found in 10, lla, Ha, Ra, and lc (arrows, Figs. 2, 4, and 5). These may represent mutated sites, or fusions of smaller oligonucleotides generated by the deletion of genetic information. In no case did we encounter any oligonucleotides representing the four genes in the interior of the virus genome (P, M, FO,and HN). Thus, although we cannot rule out retention of parts of these genes, it is likely that they have been completely deleted from all of the DI RNAs. The NP-specific oligonucleotides formed an overlapping sequence analogous to that formed by L-specific oligonucleotides (Table 2). If it is assumed that the first oligonucleotide to appear in the sequence, number 10, is closest to the 3’ terminus, then, by analogy to the reasoning used to order the L oligonucleotides, a partial sequence of NP oligonucleotides can be determined (Table 2). At first glance, the presence of NP-specific sequencies in Ha and Ra, indicating conservation of a region close to the 3’ terminus of the virus genome, seems anomalous, since oligonucleotide 7, thought to represent the 3’-terminal leader template, was not found in these RNA species. However, the R strain of Sendai virus that gave rise to the R DI RNA species differed from the Enders strain parent of our collection, lacking oligonucleotide 7 (Amesse and Kingsbury, 1982). The same is true of the nondefective H strain parent of the H DI RNA (Amesse and Kingsbury, unpublished data). Therefore, we consider it likely, by analogy with the DI strains derived from the Enders strain parent, that Ha and Ra have retained the leader template sequence between oligonucleotide 10 and the 3’ terminus of the genome. It is also possible that Rb has conserved leaderterminal sequences, despite the loss of most or all of the NP gene. To verify the identifications of oligonucleotides from DI RNAs corresponding to 3’-terminal sequences, we extracted these oligonucleotides from two-dimensional gels, digested them with pancreatic RNase A, and separated the digestion products on PEI-cellulose plates (Volckaert and Fiers, 1977). Comparisons of

22

AMESSE,

PRIDGEN,

AND KINGSBURY

to

FIG. 4. Tl-oligonucleotides of RNA species la and lc. The arrow present in RNA lc that was not seen in the virus genome.

the second-digestion patterns of oligonucleotides 7 and 10 taken from the nondefective genome and RNAs 8a and la indicate identity (Fig. 6). These comparisons

indicates

an oligonucleotide

were also made with NP-specific oligonucleotides 2,20, and 21 from RNA Ra, with analogous results (data not shown). Thus, most, if not all, of the DI RNAs that we

SENDAI

FIG. 5. Tl-oligonucleotides strain genome is indicated

VIRUS DI RNAs

of RNA species Ra and Rb. An oligonucleotide by the arrow.

have examined have retained various amounts of 3’ terminus- and 5’ terminus-

23

not seen in the R-

proximal sequences and appear to have lost all of the intervening genetic infor-

24

AMESSE, PRIDGEN, AND KINGSBURY TABLE 2 Tl-OLIGONUCLEOTIDES IN SENDAI VIRUS DI RNAs

DI RNA 8a lla 10 Ha Ra 9a

3 (leader) (7) (7) (7)

5'

-(NP

(L Gene)

gene) -

(10) (21)

(3, 14, 28, (24 (3, 14.w (15) w (3, 14.w (15) (24) (3, 14, 28,

00) ('1 (10) (') (10) (') (10)

7b 1C

(7) (7) (7)

(‘)

Rb la

(7)

(10)

(21)(2920)(')

31, 43) 31, 43) 31.43) 31, 43)

(30, 37, 38) (15) (24) (3, 14, 28, 31,43) (17, 27) (30, 37, 38) (15) (24) (3, 14, 28, 31, 43) (17, 27) (30, 37, 38) (15) (24) (3, 14, 28, 31, 43) (17, 27) (30, 37, 38) (15) (24) (3, 14, 28, 31, 43) (9, 16) (17, 27) (30, 37, 38) (15) (24) (3, 14, 28, 31, 43) (13, 36, 42) (9, 16) (17, 27) (30, 37, 38) (15) (24) (3, 14, 28, 31, 43)

’ (*) represents an oligonucleotide not seen in the 50 S genome.

mation, including the four internal genes and inner portions of the 3’- and 5’-terminal genes. DISCUSSION

Our results suggest that Sendai virus DI RNAs commonly have conserved terminal sequences and extensive internal deletions. This is a novel arrangement among

the DI RNAs that have been described for nonsegmented negative-strand RNA viruses. Only two previous examples, LT and LTz of VW, possess both genome termini. However, these DI RNAs also retain most of the virus genes (Schnitzlein and Reichmann, 1976; Colonno et al, 1977). It is to the segmented negative-strand RNA virus, influenza virus, that one must turn to find DI RNAs with extensive internal dele-

FIG. 6. Second digestion of selected Tl-oligonucleotides from nondefective and DI RNAs of Sendai virus. Oligonucleotides 7 and 10 were extracted from two-dimensional fingerprinting gels, digested with pancreatic RNase A, and chromatographed in two dimensions on PEI-cellulose plates (Materials and Methods). In each panel, the origin is at the lower left and migration in the first dimension is toward the right. The three vertical groups of spots represent, from left to right, (Ap).U, (Ap),G, and (Ap).C oligonucleotides (Volckaert and Fiers, 1977).

SENDAI VIRUS DI RNAs

25

MODELS OF FUSION DT RNA GENERATION ND-GENOME 0 NP Fo-HN

ND-ANTIGENOME @ NP

L

Fo-tiN

L

1

5’e3’

a

3’D5’

@

0

DI ANTIGENOME

DI GENOME

FIG. ‘7.Fusion DI RNAs may be generated from either nondefective (ND) minus-strand genomes or plus-strand antigenomes. The small open circle designates the viral RNA polymerase which, after copying a portion of its template, may reattach close to the other end of the template, skipping most of the virus genome sequences in the process. The final RNA product in each case is a template for the complementary DI RNA.

tions (Nayak, 1930). But this is not really a good parallel, since each influenza virus RNA segment represents a discrete gene, and no deletion can cross a gene boundary. Until we have sequenced the nucleotides at the 3’ and 5’ termini of our DI RNA species and compared them to the termini of the nondefective genome, we cannot be sure that the very ends of the genome have been conserved in our DI series. However, it seems likely that all of the 5’ termini in the DI RNAs are identical to the 5’ terminus of the genome, since this has been the case in every previous instance (Leppert et &, 19’7’7;Keene et aL, 1981). At the 3’ end, previously described DI RNA species have either saved the genome terminus or they possess surrogate termini that work as well or better as templates for initiating RNA synthesis: the copy-back type of DI RNA has a 3’ terminus that is precisely complementary to its 5 terminus, so that the viral RNA polymerase sees an identical sequence, whether it starts on a plus- or minus-strand template. Indeed, that particular sequence on a negativestrand template may give the polymerase a faster than normal start and confer a selective advantage on the replication of DI genomes (Leppert et al, 1977). Even though practically all of our DI RNAs possess sequences from the 3’ end

of the genome, the 3’-terminal nucleotides might still consist of a copy-back complementary sequence. A case in point is the LTc DI RNA of VSV, which has both a copy-back 3’ terminus and, in tandem, downstream, the original genomic 3’-terminal sequence. Such an arrangement might well apply to some Sendai virus DI RNAs, since Kolakofsky (1976) and Leppert et al. (1977) had evidence for self-complementary terminal sequences 110 to 115 nucleotides long at the termini of the RNA species which they designated R20S and H14S, corresponding to RNA species Rb and Ha that we have analyzed. Rb is the only RNA in our series that yielded no oligonucleotides characteristic of the 3’proximal part of the genome, and it may be a true copy-back DI RNA. Ha contained oligonucleotide 10 from the NP gene, so that its structure must be more complex, if it possessesa copy-back 3’ terminus. We are currently sequencing the 3’ ends of our DI RNAs to address these questions. Since most of our DI RNAs possess only parts of the NP and L genes linked in tandem, we propose to call them “fusion DIs.” Possessing no termination signals for transcription, owing to the deletion of all intervening genetic material, they must act exclusively as templates for replication. In this respect, they are functionally

26

AMESSE, PRIDGEN, AND KINGSBURY COPY-BACK

131 RNA FROM FUSION DI RNA

FUSION DI ANTIGENOME

5’

w 1 COPY-BACK DI GENOME

3’ -

0

5’

FIG. 8. A copy-back DI RNA can be generated from a fusion DI antigenome if the RNA polymerase (small circle), after copying part of the L gene, chooses the nascent strand, still attached, as its template. This will generate a 3’ terminus (filled box) that is an exact complement of the 5’ terminus (open box). For the analogous scheme with a nondefective plus-strand genome template, see Leppert et al (1977).

equivalent to the copy-back DI RNAs, which also lack all internal transcription termination signals (Leppert et d, 1977). It remains to be seen whether the replicative advantage of these types of DI RNAs over the nondefective genome depends more on the nature of their 3’ termini, on their inability to be used as templates for transcription, or on their small size. Figure 7 shows how fusion DIs might be generated at either step in RNA replication, using a plus-strand or a minusstrand nondefective RNA template. Existing models of copy-back DI generation do not envision the participation of a minusstrand template (Leppert et uL, 19’77; Huang, 1977). Therefore, there seems to be an increased opportunity for the generation of fusion DIs. This would explain their abundant representation in our series, but it raises the question of why they have not been described before. Perhaps the answer lies more in the selection pressures that operate on DI RNAs after their generation than in the generation process itself. For example, if a copy-back DI RNA competes with the nondefective genome more effectively than a fusion DI RNA, an overall rapid rate of viral RNA replication and multiple passages would select the former. We may have failed to select copyback DIs because Sendai virus grows more

slowly than VSV and our DI RNA clones have short passage histories (Materials and Methods). It should also be considered that generation of a fusion DI RNA may be a step in the evolution of a copy-back DI RNA, since it contains some dispensable genetic information at its 3’ end. Figure 8 illustrates how a fusion DI RNA might serve just as well as the nondefective genome as the template for generation of a copy-back DI RNA. ACKNOWLEDGMENTS Elizabeth Ann Lyne was a dedicated and skillful technical assistant. This work was supported by Research Grant RG1142from the National Multiple Sclerosis Society, by Basic Science Training Grant CA99346 and Cancer Center Support Grant CA21765 from the National Cancer Institute, and by American Lebanese Syrian Associated Charities.

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SENDAI VIRUS DI RNAs and amplification of Sendai virus defective-interfering genomes. Virdogy 93, 589-593. KOLAKOFSKY,D., BOY DE LA TOUR,E., and BRUSCHI, A. (1974). Self-annealing of Sendai virus RNA. J. Viral 14, 33-39. KOLAKOFSKY,D., and BRUSCHI, A. (1975). Antigenomes in Sendai virions and Sendai virus-infected cells. Virdogy 66, 185-191. LEPPERT,M., KORT, L., and KOLAKOFSKY,D. (1977). Further characterization of Sendai virus DI-RNAs: A model for their generation. Cell 12.539-552. MCMASTER, G. K., and CARMICHAEL,G. G. (1977). Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acidine orange. Proc Nat. Acad Sci USA 74,4835-4838.

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