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Defective interfering particles of human parainfluenza virus 3
VIROLOGY
158,439-443
(1997)
Defective Interfering Particles of Human Parainfluenza Virus 3 DONALD
G. MURPHY, KENNETH DIMOCK, AND C. YONG KANG’
Department of Microbiology and Immunology, University of Ottawa School of Medicine, Ottawa, Ontario, Canada K IH 8M5 Received September 1I, 1986; accepted February 6, 1987 A cyclic pattern of virus production was observed when human parainfluenza virus 3 (HPIV3) was serially passaged nine times in LLC-MK2 cells. Viruses produced from serial passages 8 and 9 interfered with the replication of standard HPIV3. Three subgenomic RNA species (DI-1, DI-2, and DI-3) and virus genomic RNA were detected in the progeny virions produced from cells mixedly infected with standard virus and virus from either serial passages 5 or 8. Northern blot analysis with probes representing all six HPIVB structural protein genes revealed that DI-1 and DI-2 RNAs contain sequences from the 5’ end of the standard virus genome. DI-1 RNA contains L, HN, and F specific sequences, while DI-2 RNA contains only L and HN sequences. DI-3 RNA did not hybridize with any of the probes used. The possibility that DI-3 RNA contains sequences from the 5’ end of the standard virus genome is discussed. These results demonstrate that 5’ defective interfering particles are generated during serial passage of HPIV3. o 1997 Academic PWSS, IIIC.
Defective interfering (DI) particles have been observed in most animal virus systems (7, 2). DI particles are usually produced during serial high-multiplicity passage of the standard virus (3). These particles have retained only a portion of the standard viral genome and are unable to replicate in the absence of standard virus. DI particles not only interfere with the replication of the standard virus, but are also enriched at its expense. DI particles are also involved in the establishment and maintenance of viral persistent infections in cultured cells (4, 6). The genome of human parainfluenza virus 3 (HPIVB), a member of the paramyxovirus group, is a singlestranded RNA of negative sense with a molecular weight of approximately 4.5-5.0 X 1O8(6). In infected cells, this negative-sense genome serves as template for the transcription of six messenger RNA species which encode the viral structural proteins (6- 74). These include the polymerase (L), the polymerase-associated phosphoprotein (P), the nucleocapsid protein (NP), the matrix protein (M), the hemagglutinin-neuraminidase (HN), and the fusion protein (F). In addition, a seventh viral protein, the C protein, encoded within the P mRNA, has been identified (15- 77). The HPIV3 gene order has been established and found to be 3’-NP-P+C-M-F-HNL-5’ (14). We have previously reported the cloning and coding assignments of HPIVB mRNA sequences (7). In the present study, we report the generation of HPIV3 DI particles and describe the genetic content of the DI RNA genomes using cloned sequences specific for each of the six HPIV3 structural protein genes. To produce DI particles, HPIV3 strain 47885 was
serially passaged nine consecutive times in LLC-MK2 cells following an initial m.o.i. of 10 PFUkell. The infectious virus titer of the progeny virus from each of the passages is shown in Fig. 1. In the first three passages, a gradual decline in titer was observed. This was then followed by a burst in infectious virus production at the fourth passage. In the next two successive passages, the titer gradually declined again only to be followed by a second burst in infectious virus production at passage seven. Similarly, the titer gradually declined in the last two successive passages. This cyclic production of infectious virus is characteristic of DI particle production (78). To investigate whether the virus present in the later passages contained interference activity, we infected LLC-MK2 cells with a mixture of standard virus and virus from passages 8 or 9. After 48 hr, the culture fluid was assayed for infectious virus. Table 1 shows that as the inoculum from passages 8 or 9 was reduced, the yield of infectious virus increased. Culture fluid from passage 9 had less interfering activity than that of passage 8. These results indicate that virus from passages 8 and 9 interfered with the replication of standard HPIV3. Since paramyxovirus particles are pleomorphic, it has been very difficult (19) or even impossible (20) to separate DI virions from standard virions by rate zonal centrifugation. We have analyzed genomic RNAs from passages 5 and 8 to detect subgenomic RNAs which may represent DI particle genomes (79,27). To amplify the DI particles, virus from passages 5 and 8 was mixed with standard virus and each mixture was used to infect LLC-MK2 cells. The RNA species obtained from both virions and virus-infected cells are shown in Fig. 2.
’ To whom requests for reprints should be addressed. 439
0042-6822/87 $3.00 Copyright 0 1997 by Academic Press. Inc. All rights of reproduction in any form resewed.
SHORT COMMUNICATIONS
440
-m-1
-DI-2
10-v
23455789
FIG. 1. Yield of infectious HPIV3 upon serial undiluted passage. For the first passage LLC-MK2 monolayer cultures were infected with 0.25 ml of virus stock (1.5 X lo* PFWml, m.o.i. 10 PFWcell). For the subsequent passages 0.25 ml of culture fluid from the previous passage was used as inoculum. After 48 hr at 37” the virus was harvested. The culture fluid from each passage was clarified and frozen at -80’ until assayed for infectious virus by plaque formation in LLC-MK2 monolayer cultures. Appropriate serial dilutions were performed and 0.1 ml was used to infect cells. Infected cells were overlayed with 0.8% agar and incubated for 4 days at 37”. Two milliliters of a 1:8000 dilution of neutral red in phosphate-buffered saline was then added to each dish. The dishes were incubated at 37” and the plaques were counted the next day.
Three subgenomic RNA species, in addition to the standard virus genomic RNA, were detected in the progeny virions resulting from the mixed infections. The TABLE 1 ABILITYOFIATE SERIALPASSAGEIJ HPIV3 TO REDUCE THEYIELD0~ INFECTIOUS VIRUS~ lnocuIumb STD (0.1 ml) STD (0.1 ml) + SP8 (0.1 ml) STD (0.1 ml) + SP8 (0.05 ml) STD (0.1 ml) + SP8 (0.025 ml) STD (0.1 ml) + SP9 (0.1 ml) STD (0.1 ml) + SP9 (0.05 ml) STD (0.1 ml) + SP9 (0.025 ml)
Yield (PFU/ml)
Reduction (%)
1.1 x 108 10’ 10’ 10’
-
3.6 X 6.3 X 6.8 x
67 43 38
10’ 9.2x 10’ 1.1 x lo8
42
6.4X
16 0
a LLC-MK2 cells were inoculated with virus as indicated. At 48 hr postinfection, the culture fluid was assayed for infectious virus as described in Fig. 1. STD, standard virus (1.5 X 10’ PFUlml). SP8, serial passage 8 virus (1.O X 10’ PFU/ml); SP9, serial passage 9 virus (8.7 X 1O6PFU/ml). b All inoculum volumes are 0.2 ml.
FIG. 2. Virion RNA and intracellular RNA in LLC-MK2 cells coinfected with standard and serially passaged virus. Confluent LLC-MK2 monolayer cultures were treated 15 hr after infection with 1 @/ml of actinomycin D. [5,6-3H]Uridinewas added 1 hr afterthe actinomycin D treatment. At 40 hr postinfection, the virus was concentrated by centrifugation and the pellet was disrupted with 0.5% SDS in the presence of 400 cg/ml of proteinase K at 37” for 30 min (6). The virion RNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. For extraction of intracellular RNA, infected cells were disrupted in buffer containing 1% Triton X100 and 1% sodium deoxycholate (22). After Dounce homogenization the nuclei were removed by centrifugation. The cellular extract was treated with 0.5% SDS and digested with 400 &ml of proteinase K at 37” for 30 min. The intracellular RNA was extracted and precipitated as described above. The RNA was denatured with glyoxsl and then separated on a 1% agarose gel (23). RNA bands were detected by fluorography (24). (A) Virion RNA from passage 1 virus, standard virus plus passage 5 virus coinfection, and standard virus plus passage 8 virus coinfection, respectively. (6) intracellular RNAfrom standard virus plus passage 5 virus coinfection and standard virus plus passage 8 virus coinfection, respectively. [3H]Uridine-labeled ribosomal RNAs were used as size markers.
same three subgenomic RNA species were also present in the virus-infected cells. These three subgenomic RNA species were not detected in standard virus-infected cells (data not shown). We have designated these three subgenomic RNA species as DI-1, U-2, and DI-3. The smallest subgenomic RNA, DI-3, was most abundant and was amplified more efficiently in the cells mixedly infected with the standard virus plus passage 8 virus. DI-1 RNA was present in approximately the same amount as the standard virus, while DI-2 RNA was present only in minor amounts. To determine the genetic contents of DI-1 , DI-2, and DI-3 subgenomic RNA species, total cytoplasmic RNA from cells coinfected with standard virus plus passage 8 virus was hybridized with cloned DNA sequences specific for each of the six HPIV3 structural protein
SHORT COMMUNICATIONS 3’ mRNA Clones
NP 1 P*C 47
a
1M ( 2
F
1 HN
1
14
to
L
1 5’
922
Genomic RNA Clone
FIG. 3. Genetic map of HPIV3 genome and sequences covered by
the cDNA clones.
genes (7). Figure 3 shows the map positions of each of the virus-specific inserts. The cDNAs represent at least 80% of the NP (pPl47) gene, 50% of the P+C (pP128)gene, 95% of the M (pPl3) gene, 80% of the F (pPI14) gene, 50% of the HN (pPI10) gene, and 33% of the L (mpPlg22) gene. Figure 4A shows the results of the Northern blot hybridizations obtained from standard virus-infected cells. All of the clones hybridized to the standard virus genomic RNA, thus confirming viral specificity. In addition, all the clones hybridized to their corresponding mRNAs. The L mRNA (Fig. 4A, lane Lb) was identified on the basis of its size, approximately 6500 nucleotides, analogous to other paramyxoviruses (25-29). From the published gene sequences (legend of Table 2) and the approximate size of the L mRNA, the HPIV3 genome is estimated to be 15,000 nucleotides in length. Only the HN and L clones hybridized to a single species of HPIV3 mRNA. All of the other clones hybridized to larger transcripts as well as to mRNAs. These are most likely
441
polytranscripts obtained by readthrough transcription as observed for other paramyxoviruses (30-33). Clones representing the M and F mRNAs both hybridized to an abundant M-F dicistronic transcript (74). The P+C clone hybridized to a second transcript which is possibly a P+C-M dicistronic transcript. This polytranscript runs approximately in the same position as the M-F dicistronic transcript. Thus, a band unique to the P+CM dicistronic transcript was not observed following hybridization with the M clone. Both the NP and P+C clones hybridized weakly to the NP-P+C dicistronic transcript (13). The Northern blot analysis of the intracellular RNA from the standard virus plus passage 8 virus coinfection is shown in Fig. 4B. DI-1 RNA hybridized to the L, HN, and F probes, while DI-2 RNA hybridized only to the L and HN probes. Therefore, DI RNAs 1 and 2 contain genetic information from the 5’ half of the HPIV3 genome. None of the clones hybridized to DI-3 RNA. However, 01-3 RNA may well be derived from the 5’ end of the HPIV3 genome since the L-specific cDNA does not cover the 5’-terminal sequences of the HPIV3 genome (Fig. 3). The L mRNA could not be identified in a longer exposure of Fig. 48, lane L. The L mRNA runs slightly ahead of DI-2 RNA. The molecular weights of the DI RNAs were estimated using the HPIV3 genomic RNA and mRNAs as size markers (Table 2). DI-
A NP
P*c
M
F
B HN
L.
L.
NP
P+c
M
F
HN
L *i
*
-Ill-l -DI-2
-u-3
FIG. 4. Hybridization of 32P-labeled HPlV3 CONASto Northern blots of total cytoplasmic RNA. Total cytoplasmic RNAwas isolated from HPIVBinfected LLC-MKP cells and electrophoresed as described in Fig. 2. The intracellular RNA was transferred onto GeneScreen (NEN) and the blots were cut into 4-mm-wide longitudinal strips. The individual strips were hybridized to nick-translated pPl47 (NP), pP128 (P+C), pPl3 (M), pPI 14 IF), pPI 10 (HN), and mpPlg22 (L) cDNAs, respectively. The cDNAs used to probe the RNA blots are indicated at the top of each lane. (A) Total cy-toplasmic RNA isolated from standard virus-infected cells. Lane La is a 3-hr exposure, while lane Lb is an S-hr exposure of lane La. (S) Total cytoplasmic RNA isolated from standard virus plus passage 8 virus-infected cells. The locations of RNAs DI-1, DI-2, and DI-3 (determined by direct labeling) are indicated.
SHORT COMMUNICATIONS
442 TABLE2
PREDICTED MOLECULAR WEIGHTS OF HPlV3 DI RNA@ DI RNA
Estimated molecular weight*
% of total genomeC
DI-1
3.15x 106
64.8
DI-2
2.50X 10’ 2.4X lo6
51.4 4.9
DI-3
a As determined by glyoxal denaturation, electrophoresis on a 1% agarose gel, and Northern blot hybridization (Fig. 48). The molecular weight markers were HPIVB genomic RNA (15,000 nucleotides) (see text) and mRNAs: NP mRNA (1642 nucleotides) (34,35); P + C mRNA (2009 nucleotides) (15-17); M mRNA (1150 nucleotides) (74, unpublished data); F mRNA (1845 nucleotides) (36, 37); and HN mRNA (1882 nucleotides) (14, Storey et al., submitted for publication). The average length of a mRNA poly(A) tail was taken as 300 nucleotides (73).
*The average molecular weight of a ribonucleotide was taken as 324.25.
‘The genome molecular weight was taken as 4.86 X 10’.
1, DI-2, and DI-3 RNAs correspond to 65, 51, and 5% of the size of the standard virus genome, respectively. A common feature of all Sendai virus and vesicular stomatitis virus (VW) DI particle RNAs analyzed to date is that they have conserved the 5’terminus of the standard genome. The majority of Sendai virus DI RNA species have extensive internal deletions with conserved terminal sequences (38, 39). These “fusion” type DI RNA species have the 3’-proximal sequences of the NP gene fused to the 5’-proximal sequences of the L gene. Another kind of DI RNA that has been described for both Sendai virus and VSV is the copy-back or panhandle type (39-42). These RNAs have retained the 5 end of the standard genome but have a 3’ terminus that is complementary to the genomic 5’ terminus. Complex copy-back DI RNAs which contain the 3’-terminal sequences of the standard genome, internal to the 3’ copy-back sequence, have been identified, but rarely. Such is the case for VSV DI RNA LT2 (43) and also possibly for Sendai virus DI RNAs 11a and Ha (39). On the basis of our hybridization data, it seems likely that DI-1 and DI-2 RNAs have retained the 5’-terminal sequences of the standard genome. Furthermore, the HPIVB DI RNAs do not appear to have retained the 3’ terminus of the standard genome. However, they could possess 3’ sequences of a length insufficient to be detected by hybridization. If DI-1 and DI-2 RNAs have retained the 3’ end of the standard genome, then the internal deletion would not be as extensive as observed for the “fusion” type DI RNA species of Sendai virus (38). Although we did not detect “fusion” type DI RNAs in these experiments, we have observed them in LLCMK2 cells persistently infected with HPIV3, using a similar hybriditation protocol (unpublished data). Thus,
the “fusion” type DI RNAs seem to be a general property of paramyxoviruses. It remains to be established if the HPIV3 DI RNAs analyzed in this report have conserved the 3’ terminus of the standard genome or if they have a copy-back sequence at the 3’ end. ACKNOWLEDGMENTS This study was supported by the Medical Research Council of Canada, the Natural Science and Engineering Research Council of Canada, and the World Health Organization. K.D. is the recipient of an Ontario Ministry of Health Career Scientist Award. We thank M.-J. C&B and D. G. Storey for preparation of the cDNA clones.
REFERENCES 1. HUANG,A. S.. and BALTIMORE, D., In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, Eds.), Vol. 10, p. 73. Plenum, New York, 1977. 2. PERFZAULT, J., Curr. Top. Microbial. Immunol. 93, 151-207 (1981). 3. HUANG,A., Annu. Rev. Microbial. 27, 101-l 17 (1973). 4. HOLLAND,J. J., and VILLAREAL, L. P.. Proc. Narl. Aced. Sci. USA 71,2956-2960 (1974). 5. Roux, L., and HOLLAND,1. J., Virology 93, 91-103 (1979). 6. STOREY,D. G., DIMOCK,K., and KANG,C. Y., /. L&o/. 52, 761766 (1984). 7. DIMOCK,K., STOREY,D. G., C&k M.-J., and KANG,C. Y., ln “The Biology of Negative Strand Viruses” (D. Kolakofsky and B. W. J. Mahy, Eds.), Elsevier Biomedical, in press. 8. SANCHEZ, A., and BANERJEE, A. K., Virology 143, 45-54 (1985). 9. WECHSLER, S. L., LAMBERT,D. M., GALINSKI,M. S., and PONS, M. W., 1. viral. 54, 661-664 (1985). 10. JAMBOU,R. C., ELANGO,N., and VENKATESAN, S., J. Viral. 56,298302 (1985). 11. RAY,R., BROWN,V. E., and COMPANS,R. W., J. Infect. Dis. 152, 1219-1230(1985). 12. WECHSLER, S. L., LAMBERT,D. M., GALINSKI,M. S., HEINEKE,B. E., and PONS,M. W., Virus. Res. 3,339-351 (1985). 13. SANCHEZ, A.,and BANERJEE, A. K., Virology 147,177-l 86 (1985). 14. SPRIGGS, M. K., and COLLINS,P. J., J. Wol. 59, 646-654 (1986). 15. LUK, D., SANCHEZ,A., and BANERJEE, A. K., Virology 153, 318325 (1986). S. L., 16. GALINSKI,M. S., MINK, M. A., LAMBERT,D. M., WECHSLER, and PONS,M. W., Virology 155, 46-60 (1986). 17. SPRIGGS, M. K., and COLLINS,P. L., J. Gen. Viral. 67, 2705-2719 (1986). 18. PALMA,E. L., and HUANG,A., 1. Infect. Dis. 129,402-410 (1974). D. W., PORTNER, A., and DARLINGTON, R. W., Virology 19. KINGSBURY, 42,857-871( 1970). 20. ROMAN,J. M., and SIMON,E. H., Virology 69, 298-303 (1976). 21. KOLAKOFSKY. D., Cell 8, 547-555 (1976). 22. DIMOCK,K., HARNISH,D. G., SISSON,G., LEUNG,W.-C., RAWLS, W. E., J. Viral. 43, 273-283 (1982). 23. MCMASTER,G. K., and CARMICHAEL, G. G., Proc. Nat/ Acad. Sci. USA 74,4835-4838 (1977). 24. BONNER,W. M., and LASKEY,R. A., Eur. 1. Biochem. 46, 83-88 (1974). K., and SHIBUTA,H., Nucleic Acids Res. 14, 25. SHIODA,T., IWASAKA, 1545-1563 (1986). K. M., Virology 154, 31-40 26. MORGAN,E. M., and RAKESTRAW, (1986). 27. WILDE,A., MCQUAIN,C., and MORISSON, T., Virus Res. 5,77-95 (1986).
SHORT COMMUNICATIONS 28. RIMA, B. K., BACZKO, K., CLARKE, D. K., CURRAN. M. D., MARTIN, S. J., BILLETER, M. A., and TER MEULEN. V., J. Gen. Viral. 67, 1971-1978(1986). 29. DICKENS, L. E., COLLINS, P. L., and WERIZ, G. W., J. Viral. 52, 364-369(1984). 30. GUPTA, K. C., and KINGSBURY,D. W., Vifo/ogy 141, 102-109 (1985). 37. WILDE, A., and MORRISON,T., J. Viral. 51, 71-76 (1984). 32. PATERSON,R. G., HARRIS,T. J. R., and LAMB, R. A., Virology 138, 310-323( 1984). 33. COLLINS, P. L., and WERTZ, G. W., Proc. Natl. Acad. Sci. USA 80, 3208-3212(1983). 34. SANCHEZ, A., BANEFUEE,A. K., FURUICHI. Y., and RICHARDSON, M. A., Virology 152, 171-180(1986). 35. JAMBOU, R. C., ELANGO, N., VENKATESAN,S., and COLLINS, P. L., 1. Gen. Vifol. 67, 2543-2548 (1986).
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36. SPRIGGS,M. K., OLMSTEAD, R. A., VENKATESAN,S., COLIGAN, J. E., and COLLINS, P. L., Virology 152, 241-251 (1986). 37. CUT& M.-J., STOREY,D. G., KANG, C. Y., and DIMOCK, K., J, Gen. Viral., in press. 38. AMESSE. L. S., PRIDGEN,C. L., and KINGSBURY,D. W., Virology 118, 17-27 (1982). 39. RE, G. G., GUPTA, K. C., and KINGSBURY,D. W., J. l&o/. 46, 659664 (1983). 40. RE, G. G., GUPTA, K. C., and KINGSBURY,D. W., !&o/ogy30,390396 (1983). 4 7. LEPPERT,M., KORT, L., and KOLAKOFSKY,D., Cell 12, 639-662 (1977). 42. IXZARINI, R. A., KEENE,J. D., and SCHUBERT,M., Cell 26, 146154(1981). 43, KEENE,1. D., CHIEN, I. M., and LAZZ~RINI,R. A., Pm. NH/, Acad. Sci. USA 78,2090-2094 (1981).
158,439-443
(1997)
Defective Interfering Particles of Human Parainfluenza Virus 3 DONALD
G. MURPHY, KENNETH DIMOCK, AND C. YONG KANG’
Department of Microbiology and Immunology, University of Ottawa School of Medicine, Ottawa, Ontario, Canada K IH 8M5 Received September 1I, 1986; accepted February 6, 1987 A cyclic pattern of virus production was observed when human parainfluenza virus 3 (HPIV3) was serially passaged nine times in LLC-MK2 cells. Viruses produced from serial passages 8 and 9 interfered with the replication of standard HPIV3. Three subgenomic RNA species (DI-1, DI-2, and DI-3) and virus genomic RNA were detected in the progeny virions produced from cells mixedly infected with standard virus and virus from either serial passages 5 or 8. Northern blot analysis with probes representing all six HPIVB structural protein genes revealed that DI-1 and DI-2 RNAs contain sequences from the 5’ end of the standard virus genome. DI-1 RNA contains L, HN, and F specific sequences, while DI-2 RNA contains only L and HN sequences. DI-3 RNA did not hybridize with any of the probes used. The possibility that DI-3 RNA contains sequences from the 5’ end of the standard virus genome is discussed. These results demonstrate that 5’ defective interfering particles are generated during serial passage of HPIV3. o 1997 Academic PWSS, IIIC.
Defective interfering (DI) particles have been observed in most animal virus systems (7, 2). DI particles are usually produced during serial high-multiplicity passage of the standard virus (3). These particles have retained only a portion of the standard viral genome and are unable to replicate in the absence of standard virus. DI particles not only interfere with the replication of the standard virus, but are also enriched at its expense. DI particles are also involved in the establishment and maintenance of viral persistent infections in cultured cells (4, 6). The genome of human parainfluenza virus 3 (HPIVB), a member of the paramyxovirus group, is a singlestranded RNA of negative sense with a molecular weight of approximately 4.5-5.0 X 1O8(6). In infected cells, this negative-sense genome serves as template for the transcription of six messenger RNA species which encode the viral structural proteins (6- 74). These include the polymerase (L), the polymerase-associated phosphoprotein (P), the nucleocapsid protein (NP), the matrix protein (M), the hemagglutinin-neuraminidase (HN), and the fusion protein (F). In addition, a seventh viral protein, the C protein, encoded within the P mRNA, has been identified (15- 77). The HPIV3 gene order has been established and found to be 3’-NP-P+C-M-F-HNL-5’ (14). We have previously reported the cloning and coding assignments of HPIVB mRNA sequences (7). In the present study, we report the generation of HPIV3 DI particles and describe the genetic content of the DI RNA genomes using cloned sequences specific for each of the six HPIV3 structural protein genes. To produce DI particles, HPIV3 strain 47885 was
serially passaged nine consecutive times in LLC-MK2 cells following an initial m.o.i. of 10 PFUkell. The infectious virus titer of the progeny virus from each of the passages is shown in Fig. 1. In the first three passages, a gradual decline in titer was observed. This was then followed by a burst in infectious virus production at the fourth passage. In the next two successive passages, the titer gradually declined again only to be followed by a second burst in infectious virus production at passage seven. Similarly, the titer gradually declined in the last two successive passages. This cyclic production of infectious virus is characteristic of DI particle production (78). To investigate whether the virus present in the later passages contained interference activity, we infected LLC-MK2 cells with a mixture of standard virus and virus from passages 8 or 9. After 48 hr, the culture fluid was assayed for infectious virus. Table 1 shows that as the inoculum from passages 8 or 9 was reduced, the yield of infectious virus increased. Culture fluid from passage 9 had less interfering activity than that of passage 8. These results indicate that virus from passages 8 and 9 interfered with the replication of standard HPIV3. Since paramyxovirus particles are pleomorphic, it has been very difficult (19) or even impossible (20) to separate DI virions from standard virions by rate zonal centrifugation. We have analyzed genomic RNAs from passages 5 and 8 to detect subgenomic RNAs which may represent DI particle genomes (79,27). To amplify the DI particles, virus from passages 5 and 8 was mixed with standard virus and each mixture was used to infect LLC-MK2 cells. The RNA species obtained from both virions and virus-infected cells are shown in Fig. 2.
’ To whom requests for reprints should be addressed. 439
0042-6822/87 $3.00 Copyright 0 1997 by Academic Press. Inc. All rights of reproduction in any form resewed.
SHORT COMMUNICATIONS
440
-m-1
-DI-2
10-v
23455789
FIG. 1. Yield of infectious HPIV3 upon serial undiluted passage. For the first passage LLC-MK2 monolayer cultures were infected with 0.25 ml of virus stock (1.5 X lo* PFWml, m.o.i. 10 PFWcell). For the subsequent passages 0.25 ml of culture fluid from the previous passage was used as inoculum. After 48 hr at 37” the virus was harvested. The culture fluid from each passage was clarified and frozen at -80’ until assayed for infectious virus by plaque formation in LLC-MK2 monolayer cultures. Appropriate serial dilutions were performed and 0.1 ml was used to infect cells. Infected cells were overlayed with 0.8% agar and incubated for 4 days at 37”. Two milliliters of a 1:8000 dilution of neutral red in phosphate-buffered saline was then added to each dish. The dishes were incubated at 37” and the plaques were counted the next day.
Three subgenomic RNA species, in addition to the standard virus genomic RNA, were detected in the progeny virions resulting from the mixed infections. The TABLE 1 ABILITYOFIATE SERIALPASSAGEIJ HPIV3 TO REDUCE THEYIELD0~ INFECTIOUS VIRUS~ lnocuIumb STD (0.1 ml) STD (0.1 ml) + SP8 (0.1 ml) STD (0.1 ml) + SP8 (0.05 ml) STD (0.1 ml) + SP8 (0.025 ml) STD (0.1 ml) + SP9 (0.1 ml) STD (0.1 ml) + SP9 (0.05 ml) STD (0.1 ml) + SP9 (0.025 ml)
Yield (PFU/ml)
Reduction (%)
1.1 x 108 10’ 10’ 10’
-
3.6 X 6.3 X 6.8 x
67 43 38
10’ 9.2x 10’ 1.1 x lo8
42
6.4X
16 0
a LLC-MK2 cells were inoculated with virus as indicated. At 48 hr postinfection, the culture fluid was assayed for infectious virus as described in Fig. 1. STD, standard virus (1.5 X 10’ PFUlml). SP8, serial passage 8 virus (1.O X 10’ PFU/ml); SP9, serial passage 9 virus (8.7 X 1O6PFU/ml). b All inoculum volumes are 0.2 ml.
FIG. 2. Virion RNA and intracellular RNA in LLC-MK2 cells coinfected with standard and serially passaged virus. Confluent LLC-MK2 monolayer cultures were treated 15 hr after infection with 1 @/ml of actinomycin D. [5,6-3H]Uridinewas added 1 hr afterthe actinomycin D treatment. At 40 hr postinfection, the virus was concentrated by centrifugation and the pellet was disrupted with 0.5% SDS in the presence of 400 cg/ml of proteinase K at 37” for 30 min (6). The virion RNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. For extraction of intracellular RNA, infected cells were disrupted in buffer containing 1% Triton X100 and 1% sodium deoxycholate (22). After Dounce homogenization the nuclei were removed by centrifugation. The cellular extract was treated with 0.5% SDS and digested with 400 &ml of proteinase K at 37” for 30 min. The intracellular RNA was extracted and precipitated as described above. The RNA was denatured with glyoxsl and then separated on a 1% agarose gel (23). RNA bands were detected by fluorography (24). (A) Virion RNA from passage 1 virus, standard virus plus passage 5 virus coinfection, and standard virus plus passage 8 virus coinfection, respectively. (6) intracellular RNAfrom standard virus plus passage 5 virus coinfection and standard virus plus passage 8 virus coinfection, respectively. [3H]Uridine-labeled ribosomal RNAs were used as size markers.
same three subgenomic RNA species were also present in the virus-infected cells. These three subgenomic RNA species were not detected in standard virus-infected cells (data not shown). We have designated these three subgenomic RNA species as DI-1, U-2, and DI-3. The smallest subgenomic RNA, DI-3, was most abundant and was amplified more efficiently in the cells mixedly infected with the standard virus plus passage 8 virus. DI-1 RNA was present in approximately the same amount as the standard virus, while DI-2 RNA was present only in minor amounts. To determine the genetic contents of DI-1 , DI-2, and DI-3 subgenomic RNA species, total cytoplasmic RNA from cells coinfected with standard virus plus passage 8 virus was hybridized with cloned DNA sequences specific for each of the six HPIV3 structural protein
SHORT COMMUNICATIONS 3’ mRNA Clones
NP 1 P*C 47
a
1M ( 2
F
1 HN
1
14
to
L
1 5’
922
Genomic RNA Clone
FIG. 3. Genetic map of HPIV3 genome and sequences covered by
the cDNA clones.
genes (7). Figure 3 shows the map positions of each of the virus-specific inserts. The cDNAs represent at least 80% of the NP (pPl47) gene, 50% of the P+C (pP128)gene, 95% of the M (pPl3) gene, 80% of the F (pPI14) gene, 50% of the HN (pPI10) gene, and 33% of the L (mpPlg22) gene. Figure 4A shows the results of the Northern blot hybridizations obtained from standard virus-infected cells. All of the clones hybridized to the standard virus genomic RNA, thus confirming viral specificity. In addition, all the clones hybridized to their corresponding mRNAs. The L mRNA (Fig. 4A, lane Lb) was identified on the basis of its size, approximately 6500 nucleotides, analogous to other paramyxoviruses (25-29). From the published gene sequences (legend of Table 2) and the approximate size of the L mRNA, the HPIV3 genome is estimated to be 15,000 nucleotides in length. Only the HN and L clones hybridized to a single species of HPIV3 mRNA. All of the other clones hybridized to larger transcripts as well as to mRNAs. These are most likely
441
polytranscripts obtained by readthrough transcription as observed for other paramyxoviruses (30-33). Clones representing the M and F mRNAs both hybridized to an abundant M-F dicistronic transcript (74). The P+C clone hybridized to a second transcript which is possibly a P+C-M dicistronic transcript. This polytranscript runs approximately in the same position as the M-F dicistronic transcript. Thus, a band unique to the P+CM dicistronic transcript was not observed following hybridization with the M clone. Both the NP and P+C clones hybridized weakly to the NP-P+C dicistronic transcript (13). The Northern blot analysis of the intracellular RNA from the standard virus plus passage 8 virus coinfection is shown in Fig. 4B. DI-1 RNA hybridized to the L, HN, and F probes, while DI-2 RNA hybridized only to the L and HN probes. Therefore, DI RNAs 1 and 2 contain genetic information from the 5’ half of the HPIV3 genome. None of the clones hybridized to DI-3 RNA. However, 01-3 RNA may well be derived from the 5’ end of the HPIV3 genome since the L-specific cDNA does not cover the 5’-terminal sequences of the HPIV3 genome (Fig. 3). The L mRNA could not be identified in a longer exposure of Fig. 48, lane L. The L mRNA runs slightly ahead of DI-2 RNA. The molecular weights of the DI RNAs were estimated using the HPIV3 genomic RNA and mRNAs as size markers (Table 2). DI-
A NP
P*c
M
F
B HN
L.
L.
NP
P+c
M
F
HN
L *i
*
-Ill-l -DI-2
-u-3
FIG. 4. Hybridization of 32P-labeled HPlV3 CONASto Northern blots of total cytoplasmic RNA. Total cytoplasmic RNAwas isolated from HPIVBinfected LLC-MKP cells and electrophoresed as described in Fig. 2. The intracellular RNA was transferred onto GeneScreen (NEN) and the blots were cut into 4-mm-wide longitudinal strips. The individual strips were hybridized to nick-translated pPl47 (NP), pP128 (P+C), pPl3 (M), pPI 14 IF), pPI 10 (HN), and mpPlg22 (L) cDNAs, respectively. The cDNAs used to probe the RNA blots are indicated at the top of each lane. (A) Total cy-toplasmic RNA isolated from standard virus-infected cells. Lane La is a 3-hr exposure, while lane Lb is an S-hr exposure of lane La. (S) Total cytoplasmic RNA isolated from standard virus plus passage 8 virus-infected cells. The locations of RNAs DI-1, DI-2, and DI-3 (determined by direct labeling) are indicated.
SHORT COMMUNICATIONS
442 TABLE2
PREDICTED MOLECULAR WEIGHTS OF HPlV3 DI RNA@ DI RNA
Estimated molecular weight*
% of total genomeC
DI-1
3.15x 106
64.8
DI-2
2.50X 10’ 2.4X lo6
51.4 4.9
DI-3
a As determined by glyoxal denaturation, electrophoresis on a 1% agarose gel, and Northern blot hybridization (Fig. 48). The molecular weight markers were HPIVB genomic RNA (15,000 nucleotides) (see text) and mRNAs: NP mRNA (1642 nucleotides) (34,35); P + C mRNA (2009 nucleotides) (15-17); M mRNA (1150 nucleotides) (74, unpublished data); F mRNA (1845 nucleotides) (36, 37); and HN mRNA (1882 nucleotides) (14, Storey et al., submitted for publication). The average length of a mRNA poly(A) tail was taken as 300 nucleotides (73).
*The average molecular weight of a ribonucleotide was taken as 324.25.
‘The genome molecular weight was taken as 4.86 X 10’.
1, DI-2, and DI-3 RNAs correspond to 65, 51, and 5% of the size of the standard virus genome, respectively. A common feature of all Sendai virus and vesicular stomatitis virus (VW) DI particle RNAs analyzed to date is that they have conserved the 5’terminus of the standard genome. The majority of Sendai virus DI RNA species have extensive internal deletions with conserved terminal sequences (38, 39). These “fusion” type DI RNA species have the 3’-proximal sequences of the NP gene fused to the 5’-proximal sequences of the L gene. Another kind of DI RNA that has been described for both Sendai virus and VSV is the copy-back or panhandle type (39-42). These RNAs have retained the 5 end of the standard genome but have a 3’ terminus that is complementary to the genomic 5’ terminus. Complex copy-back DI RNAs which contain the 3’-terminal sequences of the standard genome, internal to the 3’ copy-back sequence, have been identified, but rarely. Such is the case for VSV DI RNA LT2 (43) and also possibly for Sendai virus DI RNAs 11a and Ha (39). On the basis of our hybridization data, it seems likely that DI-1 and DI-2 RNAs have retained the 5’-terminal sequences of the standard genome. Furthermore, the HPIVB DI RNAs do not appear to have retained the 3’ terminus of the standard genome. However, they could possess 3’ sequences of a length insufficient to be detected by hybridization. If DI-1 and DI-2 RNAs have retained the 3’ end of the standard genome, then the internal deletion would not be as extensive as observed for the “fusion” type DI RNA species of Sendai virus (38). Although we did not detect “fusion” type DI RNAs in these experiments, we have observed them in LLCMK2 cells persistently infected with HPIV3, using a similar hybriditation protocol (unpublished data). Thus,
the “fusion” type DI RNAs seem to be a general property of paramyxoviruses. It remains to be established if the HPIV3 DI RNAs analyzed in this report have conserved the 3’ terminus of the standard genome or if they have a copy-back sequence at the 3’ end. ACKNOWLEDGMENTS This study was supported by the Medical Research Council of Canada, the Natural Science and Engineering Research Council of Canada, and the World Health Organization. K.D. is the recipient of an Ontario Ministry of Health Career Scientist Award. We thank M.-J. C&B and D. G. Storey for preparation of the cDNA clones.
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