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Antigenomes in Sendai virions and Sendai virus-infected cells
VIROLOGY
66, 185-191 (1975)
Antigenomes
in Sendai Virions
DANIEL Dbpartment
KOLAKOFSKY de Biologic moltculaire, Accepted
and Sendai Virus-Infected AND
ANDREE BRUSCHI
Universite March
Cells
de Genbve, 1211, Geneve 4
4, 1975
Several factors which might affect the relative proportions of genomes and antigenomes in Sendai virus have been examined. The relative amount of antigenomes assembled into mature virions was found to be independent of the time of harvest, whereas the amount of cellular RNAs incorporated into virions was found to increase late in infection. Virus strain ard host cell did affect the relative amounts of antigenomes in both mature virions and the infected cell, but in all cases more antigenomes were present in the infected cell than would be expected from their function as intermediates in genome replication.
plus-minus sense of the viral genomes is reversed. Genome-length plus strands or antigenomes have recently been demonstrated in purified Sendai virions (Kolakofsky et al., 1974). Since such antigenomes are thought to function only as intermediates in minus-strand genome synthesis, their presence in purified virions appears to be superfluous. We have, therefore, examined whether the inclusion of antigenomes in mature virions might be due to some physiological anomaly occurring during infection, e.g., packaging of plus nucleocapsids late in infection when cytopathic effects are most severe. We have found that the relative ratio of plus to minus nucleocapsids assembled into mature virions is constant throughout the infection. We have also examined the effect of different viral strains and host cells on the ratio of plus to minus nucleocapsids present both in mature virions and infected cells. Although both the viral strain and the host cell have a small effect on these ratios, in all cases considerably more antigenomes were present than might be expected from their function as intermediates in genome replication.
INTRODUCTION
Sendai virus, a parainfluenza virus type 1, contains an RNA genome which is complementary to the virus-specific mRNAs found in the infected cells (Kingsbury, 1974; Shatkin, 1974). Since viral RNA, which is directly used as template for protein synthesis, is generally referred to as the plus strands, parainfluenza viruses are, therefore, called negative strand viruses to distinguish them from viruses such as QB or polio in which the virion RNA is also mRNA (Baltimore, 1971; Shatkin, 1974). The genome of Sendai virus is found in the virion not as free RNA, but complexed with nucleocapsid protein subunits which are helically arranged to cover the RNA in a manner similar to TMV (Kingsbury and Darlington, 1968; Blair and Robinson, 1970). In our present understanding of the events which occur during Sendai virus infection, viral nucleocapsids containing the virion polymerase enter the cell cytoplasm and transcribe the minus strand genome to produce multiple, monocistronic mRNAs. These virus-specific mRNAs are then used to synthesize virusspecific proteins, some of which are required to replicate the viral genome via a complementary plus strand or antigenome MATERIALS AND METHODS intermediate (Kingsbury, 1974; Shatkin, 1974). The replication of the Sendai viral Purification of Virus genome is analogous to Qfl or polio genome Individual harvests of tissue culture mereplication in this respect, except that the dium were chilled in ice, and clarified of 185
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186
KOLAKOFSKY
cellular debris by centrifuging for 10 min at 5000 g. The virus was then pelleted through a 25ml cushion of 25% glycerol in TNE (25 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA) by centrifuging for 2 hr at 36,000 rpm (7”) in the International A170 rotor. The virus pellet was resuspended in l/100 of the original volume of TNE with the aid of the Dounce homogenizer and banded in a preformed 10%40% potassium tartrate gradient by centrifuging for 2.5 hr at 38,000 rpm (10“) in a Spinco SW 40 rotor. The visible virus band was removed through the side of the tube with a syringe, diluted lo-fold with TNE, and repelleted through a 0.5-ml cushion of 25% glycerol in TNE by centrifuging for 1 hr at 50,000 rpm (7”) in a Spinco SW 56 rotor. The virus pellets were stored at -28” until used. Isolation of Intracellular sids
Viral Nucleocap-
Five confluent cultures of MDBK or CEF cells in lo-cm Falcon tissue culture dishes were infected with 20 EID,,/cell of various strains of parainfluenza virus and incubated with 5 ml/dish of Dulbecco’s modified Eagles Medium containing 0.5% lactalbumin hydrolysate and 10 &i/ml of [SH]uridine (25 Ci/mmole) at 37”. At 24 hr postinfection, when the cytopathic effects of infection were maximal without detachment of cells from the dish for Sendai-infected cells, and 12 hr p.i. for NDVinfected cells, the culture medium was harvested and the virus purified as described above. The cell monolayers were washed once with PBS and then scraped with a rubber policeman into 5 ml of PBS. The cells were then pelleted by centrifuging for 5 min at 5000 g, resuspended in 2.5 ml of 0.15 M NaCl, 0.05 MTris-Cl, pH 8.0, and 0.6% triton-X100, and disrupted by vortexing for 30 set at maximum speed. The resulting suspension was then centrifuged for 10 min at 5000 g, the supematant removed and made 10 mM in EDTA and 0.1% in DOC, and incubated for 5 min at 37”. The supernatant was then layered onto a lo-ml CsCl-sucrose step gradient as described by Compans and Choppin (1967) and centrifuged for 2 hr at 36,000 rpm (10’)
AND BRUSCHI
in the Spinco SW 40 rotor. The visible nucleocapsid band, which was located in the middle of the original 30% CsCl step of the gradient, was removed through the side of the tube with a syringe, diluted 10 times with TNE, and pelleted by centrifuging for 1 hr at 50,000 rpm (7”) in the Spinco SW 56 rotor. Genome-length RNA was then isolated from the intracellular nucleocapsid pellets as described in Fig. 2. Virus Strains
SendailHarris and SendailOhbayashi virus strains were obtained from R. Barry, Cambridge, England, and M. Homma, Sendai, Japan, respectively. NDV strains HP and L were obtained from M. Bratt, Worcester, MA and NDV strains BK and C were obtained from D. Kingsbury, Memphis, TN, and C. Colby, Davis, CA, respectively. To prepare- viral stocks, Sendai viruses were grown in g-day-old embryonated chicken eggs for 3 days at 33” and NDV viruses for l-2 days at 39”. The allantoic fluid was clarified of debris by centrifugation for 10 min at 5000 g and stored at -78”. Viral stocks were titered by end-point dilution in embryonated chicken eggs. RESULTS
The Effect of Time Virion RNA
of Harvest
on the
The genomic RNA of Sendai virus can self-anneal when isolated as free 50 S RNA (Robinson, 1970; Portner and Kingsbury, 1970) and this self-annealing has recently been shown to be due to the presence of both plus and minus nucleocapsids in the virion population (Kolakofsky et al., 1974). Blair and Robinson (1970) have demonstrated that only a small fraction of the viral nucleocapsids which are synthesized in the infected cell are incorporated into mature virions. Since virus is released from the infected cells over an extended period of time (generally for 18 to 72 hr postinfection) we have examined whether the occurrence of relatively large amounts of plus nucleocapsids in purified virions might be due to aberrant packaging of plus nucleocapsids late in infection. At this time
ANTIGENOMES
IN SENDAI
the cytopathic effects of the infection are most pronounced and may interfere with the mechanism by which nucleocapsids are selected for assembly into mature virions. MDBK cells were, therefore, infected with Ohbayashi strain of Sendai virus, [3H]uridine was added to label newly synthesized virus RNA, and virus was harvested over five successive time periods. To determine whether the incorporation of cellular RNAs, such as tRNA (Barry and Bukrinskaya, 1968: Kolakofsky, 1972), into the virions was also dependent on the time of harvest of the virus, the cells were prelabeled with [‘“Cluridine for 24 hr before infection. The virus from each successive harvest was purified as described in Materials and Methods, resuspended in TNE containing 1% SDS and sedimented through a SDS-sucrose gradient (Fig. 1).
FRACTION
NUMBER
FIG. 1. Isolation of 50 S RNA from virus harvested at different times after infection. Eight growing cultures of MDBK cells in 75-cm2 Falcon tissue culture
VIRIONS
187
Gradient fractions containing the 50 S RNA, (indicated by the horizontal bar in each panel of Fig. l), were precipitated with ethanol and the RNA recovered by centrifugation. The 50 S RNAs from virus harvested at different intervals were then allowed to self-anneal for different times and the equilibrium annealing levels reached are shown in the boxes in each panel of Fig. 1. The results demonstrate that the self-annealing levels of the virion 50 S RNA are independent of the time of harvest of the virus. Furthermore, addition of excess unlabeled 50 S RNA from egggrown Sendai virus (which is composed predominantly of minus strands (Portner and Kingsbury, 1970)) to each 3H-labeled 50 S RNA sample before annealing resulted in lowered self-annealing levels of approximately 35% in each case. These reduced self-annealing levels reflect the relative proportion of plus and minus strands in egg-grown Sendai/Ohbayashi virus (Kolakofsky, unpublished results). Virus harvested at different times after infection, flasks were prelabeled with 0.2 &i/ml of [“Cluridine (27 mCi/mmole) in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (15 ml/flask). After 24 hr at 37”, the confluent cultures were infected by adsorption of 20 EID,,/cell of the Ohbayashi strain of Sendai virus for 60 min at 31”. The infecting medium was then removed and replaced with 7 ml of medium containing 0.5% lactalbumin hydrolysate and 10 &i/ml of [3H]uridine (25 Ci/mmole) and the incubation continued at 31”. Medium was successively harvested at 20 hr PI (a), 32 hr PI (b), 41 hr PI (c), 50 hr PI (d), and 66 hr PI (e), and replaced with the above medium containing 2 &i/ml of [“H]uridine. Virus was purified from the individual harvests as described in Materials and Methods and each viral pellet was then dissolved in 0.2 ml of TNE containing 1% SDS, incubated for 5 min at 37”, and centrifuged for 105 min at 50,000 rpm (7”) on 5% to 23% sucrose gradients (50 mM LiCl, 20, mM Tris-Cl, pH 7.5, 4 mM EDTA, 0.1% SDS) in the Spinco SW 56 rotor. After fractionating each gradient, samples of 60 ~1 (a), 10 ~1 (b), 10~1 (c), 15 ~1 (d), and 50 ~1 (e) were spotted on filter paper discs, and the discs were washed and counted as described in Fig. 2. The horizontal bar in each panel indicates those fractions pooled and recovered by ethanol precipitation as 50 S RNA. The enclosed figures in each panel are the self-annealing values of the individual 50 S RNAs determined as described in the legend to Table 1.
188
KOLAKOFSKYANDBRUSCHI
therefore, always contains a majority of minus strands. Since the self-annealing of Sendai 50 S RNA has been shown to be due to a single population of full-length complementary strands, both present in the virions as nucleocapsids (Kolakofsky et al., 1974), we conclude that the relative amounts of plus and minus nucleocapsids assembled into mature virions is independent of the time of harvest. The appearance of other RNA species in purified virions, on the other hand, is quite clearly dependent on the time of harvest of the virus. As shown in Fig. 1, virus harvested within the first 32 hr postinfection contains essentially only 50 S RNA. However, from 32 to 66 hr postinfection, increasing quantities of 4, 18, and 28 S RNA are found in the purified virus preparation and, in contrast to the 50 S RNA, these species are preferentially labeled with [“Cluridine used to prelabel the cellular RNA before infection. The evidence that these 4, 18, and 28 S RNA are located inside the virions and are not merely cellular contaminants on the surface of the virion which have survived the purification procedure is 2fold: (a) their proportions relative to the virion 50 S RNA are not diminished by further purification such as equilibrium sedimentation on sucrose density gradients, and (b) treatment of [3H]uridine-labeled virus with nuclease Sl, which is sufficient to completely degrade an amount of added “C-labeled mouse kidney cellular RNA equal to the virion RNA, did not significantly degrade the virion 4, 18, and 28 S RNAs (data not shown). It would thus appear that virus released from cells late in infection, when the cytopathic effects of the infection are most pronounced, contain significant quantities of cellular RNAs, whereas virus released early after infection does not.
remain associated with the cells. It was, therefore, of interest to examine whether the ratio of plus-to-minus nucleocapsids found in mature virions reflects the ratio found in infected cells, or alternatively, was the result of preferential selection of either plus or minus nucleocapsids during assembly of the virus at the cell surface. Furthermore, since the ratio of plus-tominus nucleocapsids packaged into mature virions is independent of the time of harvest, we have also examined the effect of different viral strains and host cells on this ratio at a single time of harvest. Both CEF and MDBK cells were infected with different strains of parainfluenza virus, [3H]uridine was added immediately after infection to label the virus RNA, and both the culture medium and the cells were harvested when virus production was maximal. Virus was purified from the culture medium and its 50 S RNA isolated as described in Fig. 1. Viral nucleocapsids were isolated from the infected cell by the method of Compans and Choppin (1967) and their 50 S RNA was isolated as described in Fig. 2. The various 50 S RNAs were then self-annealed for different lengths of time and their equilibrium annealing levels determined. The results, reported in Table 1, are summarized as follows: 1. In the four cases in which 50 S RNA from both mature virions and intracellular nucleocapsids were examined in the same infection, 50 S RNA from intracellular nucleocapsids self-annealed to approximately twice the level of virus 50 S RNA in three cases and was only slightly higher in one case. The large amount of plus nucleocapsids found in parainfluenza virions, therefore, reflects an even larger amount of plus nucleocapsids in infected cells. Although there is some bias in selectively packaging minus nucleocapsids, this bias is not very pronounced. Selection of Plus and Minus Nucleocapsids 2. For a given strain of Sendai virus During Virus Assembly grown in a given host, the relative amounts Blair and Robinson (1970) have shown of plus and minus nucleocapsids are fairly that more than 95% of the nucleocapsids constant (*5%). 3. The relative amount of plus nusynthesized during Sendai virus infection are not processed into mature virions but cleocapsids packaged is affected by the
ANTIGENOMES
189
IN SENDAI VIRIONS
viral strain: e.g., the Ohbayashi strain of Sendai virus contains a significantly higher proportion of plus nucleocapsids than the Harris strain of Sendai virus, regardless of whether MDBK cells or CEF have been used as host cells. 4. The host cell influences the ratio of plus to minus nucleocapsids assembled into mature virions. Both the Harris and the Ohbayashi strains of Sendai virus contain a significantly higher proportion of plus nucleocapsids when grown in CEF cells compared to MDBK cells. Selectivity toward which nucleocapsids are assembled into mature virions again appears to be of secondary importance, since both Sendail Harris- and Sendai/Ohbayashi-infected CEF contain a larger proportion of plus nucleocapsids (80% self-annealing) than similarly infected MDBK cells (50% selfannealing). 5. Purified NDV also contains significant, but lower, quantities of plus nucleocapsids than Sendai virus. In the one case where 50 S RNA from both virus and intracellular nucleocapsids were examined in the same infection, intracellular 50 S RNA was found to self-anneal to twice the extent of virus 50 S RNA. DISCUSSION
Sendai and NDV are prototypes of the parainfluenza class of viruses whose ssRNA genome (minus strand) is complementary to the monocistronic mRNA plus strands found in the infected cell (Kingsbury, 1974: Shatkin, 1974). Our present understanding of the events thought to occur during parainfluenza virus replication, like that of the other major class of negative-strand viruses, the rhabdoviruses, does not ascribe any function to full-length plus strands (antigenomes) other than to serve as a template for minus-strand genome synthesis. In this respect, parainfluenza plus strands would appear to serve an analogous function to that determined for minus-strand RNA in the replication of plus-strand viruses such as QB or polio. Antigenome RNA, however, never represents more than l-Z% of the virus-specific RNA found in both QB- and polio-infected
FRACTION
NUMBER
FIG. 2. Isolation of 50 S RNA from intracellular nucleocapsids. Each intracellular nucleocapsid pellet, isolated as described in Materials and Methods, was dissolved in 0.15 ml of TNE containing 1% SDS and centrifuged on a 5% to 23% sucrose gradient as described in Fig. 1. After fractionating the gradient, lo-p1 samples were spotted on filter paper discs, the discs were washed with 6% TCA followed by 95% ethanol, and counted by liquid scintillation to determine radioactivity. The horizontal bar indicates those fractions which were pooled and recovered by ethanol precipitation as intracellular nucleocapsid 50 S RNA. The arrows indicate the positions of the peaks of “C-labeled mouse kidney cellular RNA which was sedimented on a parallel gradient. The above figure represents intracellular nucleocapsid RNA isolated from Sendai/Harris-infected MDBK cells. The second RNA species, which peaks at fraction 13, was found to cosediment with the large ribosomal subunit RNA from uninfected MDBK cells and was not examined further (data not shown).
cells (Weissmann et al., 1968; Oberg and Philipson, 1971). The data presented here demonstrate that, although such factors as virus strain and host cell have a small effect on the relative ratios of plus and minus nucleocapsids present in the parainfluenza virus-infected cell, antigenomes always represent a larger fraction of the intracellular genome-length RNA than would be expected from their function as intermediates in genome replication. Furthermore, genome-length plus strands have recently been shown to represent a similarly large fraction of the genome-length RNA in VSV-infected cells (Schincariol and Howatson, 1972; Sorial et al., 1974). This anomaly, therefore, applies equally to both major classes of negative-strand viruses. Since genome-length plus strands are unlikely to function other than as templates for the synthesis of minus-strand
190
KOLAKOFSKY
AND BRUSCHI
TABLE
1
SELF-ANNEALING OF VIRUS AND INTRACELLULAR 50 S RNA”
Virus strain
Host
Percent self-annealing of 50 S RNA Virion
Intracellular
1 Sendai/Harris 2 Sendai/Harris 3 Sendai/Harris
MDBK MDBK CEF
26.1 + 0.7 27.5 f 0.4 46.4 i 0.7
52.5 zt 1.3 79.0 f 3.9
4 SendailOhbayashi 5 Sendai/Ohbayashi 6 Sendai/Ohbayashi
MDBK MDBK CEF
45.0 * 1.9 47.0 l 1.6 57.2 zt 2.8
51.5 * 1.5 80.5 zt 0.5
CEF CEF CEF CEF MDBK
9.3 f 22.9 f 27.6 * 23.9 * 19.0 f
7 8 9 10 11
NDV/HP NDV/BK NDV/C NDV/L NDV/L
0.2 0.3 0.4 0.9 1.0
-
19.6 * 1.3
0 50 S RNA from purified virus and intracellular nucleocapsids were isolated as described in the legends to Fig. 1 and Fig. 2, respectively. Samples of the different 50 S RNAs containing from 809 to 2000 cpm were annealed in 20 ~1of 2.5 x SSC at 78” in sealed capillaries for 0,30,60,90, and 120 min. The capillaries were then opened, their contents transferred into 0.8 ml of 2 x SSC containing 33 fig of ribonuclease A, and incubated for 30 min at 25”. The remaining RNA was coprecipitated with 30 pg of carrier rRNA by adjusting each solution to 10% in TCA, collected on Millipore filters, and counted by liquid scintillation. Total input radioactivity was determined by the average of one unincubated sample and one sample which was incubated for 120 min at 78’, neither of which received ribonuclease. In all cases, these values did not differ by more than 9%. Annealing equilibrium had been reached in all cases during 60 min at 78”, and the values reported in the table are the average of the 60-, 90-, and 120-min time points, i.e., the equilibrium portion of each annealing curve.
genomes in both parainfluenza virus- and rhabdovirus-infected cells, other explanations for their relative abundance in the infected cell should be considered. Of these, an interesting possibility is that minus-strand viruses simply cannot regulate the relative amounts of full-length plus and minus strands because of the nature of their genome. By comparison with the more extensively studied plus-strand viruses such as Q/3 and polio, minus-strand viruses have properties which might explain their inability to regulate the synthesis of genome and antigenome. For example, Q/3 replicase will preferentially use Q@ minus strands as template in vitro, most probably because Q@replicase can recognize the different structures of the free plus and minus strands in solution (Weissmann et al., 1968). This method of regulation, however, may not apply to parainfluenza and rhabdoviruses since their template for replication is not free RNA but the viral nucleocapsids in which the viral RNA is encapsidated. Since plus and minus nucleocapsids apparently differ only in their
RNA, and this RNA is surrounded by helical arrangement of identical protein subunits (e.g., the RNA in the viral nucleocapsids is insensitive to ribonuclease (Kingsbury and Darlington, 1968)), there may not be sufficient structural differences between plus and minus nucleocapsids that the replicating enzyme can recognize. The fact that the viral transcriptase can distinguish between plus and minus nucleocapsids does not necessarily diminish the above argument since in contrast to transcription, initiation sites for replication must be present on both plus and minus nucleocapsids. Another mechanism which regulates the relative abundance of plus and minus strands in plus-strand virus-infected cells stems from the ability of the viral genome to serve both as a template for replication and protein synthesis. In QB- and polioinfected cells, plus strands are continually being withdrawn from the pool of viral RNA to form polysomes, leaving the pool of RNA available to replicase enriched in minus strands. Such a mechanism is, of
ANTIGENOMES
191
IN SENDAI VIRIONS
course, not applicable to negative-strand viruses. On the contrary, should plus nucleocapsids also be capable of acting as templates for protein synthesis, the pool of nucleocapsids available for replication would be enriched in minus nucleocapsids and an increased synthesis of plus nucleocapsids would result. It is, therefore, possible that minusstrand virus-infected cells contain large amounts of plus nucleocapsids not because they are required in such numbers for viral replication, but rather by default. If this is not the case, it would be worth rexamining other functions for plus nucleocapsids in negative-strand virus replication. Although both rhabdovirus- and parainfluenza virus-infected cells contain large amounts of plus nucleocapsids, only parainfluenza virions contain significant quantities of plus nucleocapsids. From the data presented here we have concluded that parainfluenza virus-infected cells have some bias in selectively packaging minus nucleocapsids, but this bias is very weak. This, however, is not surprising if plus and minus nucleocapsids differ only in their RNA. Rhabdovirus-infected cells, on the other hand, must have a rather stringent mechanism for assembling only minus nucleocapsids into mature virus, which is either nonexistent or poorly functioning in parainfluenza virus-infected cells. Morrison et al. (1975) have recently suggested that the genome length plus strands found in WV-infected cells contain poly A whereas minus strands do not. The presence of poly A tails on plus nucleocapsids and their absence on minus nucleocapsids could serve as a basis for the selection of minus nucleocapsids for assembly into mature virions. Similarly, the absence of poly A tails on both parainfluenza plus and minus nucleocapsids might explain the lack of a stringent mechanism in the packaging of viral nucleocapsids in parainfluenza virus-infected cells. It is so far not known whether parainfluenza plus strands contain terminal poly A sequences and we are presently investigating this matter, ACKNOWLEDGMENTS We thank Pierre-Francois Spahr for generous ad-
vice, encouragement, and support, and Peter Bromley and Julian Davies for helpful discussion. This work was supported by Research Grant 3.331.074 from the Fonds National Suisse de la Recherche Scientifique. REFERENCES BALTIMORE, D. (1971). Expression of animal virus
genomes. Bacterial. Reo. 35, 235-241. BARRY,R. D., and BUKRINSKAYA, A. G. (1968). The nucleic acid of Sendai virus and RNA synthesis in cells infected by Sendai virus. J. Gen. Virol. 2, 71-79. OBERG, B., and PHILIPSON, L. (1971). Replicative structures of polio virus RNA. J. Mol. Biol. 58, 725-737. BLAIR, C. D., and ROBINSON,W. S. (1970). Replication of Sendai virus. II. Steps in virus assembly. J. Viral. 5.639-650. COMPANS, R. W., and CHOPPIN, P. W. (1967). Isolation and properties of the helical nucleocapsid of the parainfluenxa virus SV5. Proc. Nat. Acad. Sci. USA 57.949-956. KINGSBURY, D. W., and DARLMGTON, R. W. (1968). Isolation and properties of NDV nucleocapsids. J. Virol. 2, 248-255. KINGSBURY, D. W. (1974). The molecular biology of paramyxoviruses. Med. Microbial. Immunol. 160,
73-83. KOLAKOFSKY, D., BOY DELATOUR,E., and BRUSCHI, A. (1974). Self-annealing of Sendai virus RNA. J. Virol. 14,33-39. KOLAKOFSKY, D. (1972). tRNA nucleotidyl transferase and tRNA in Sendai virions. J. Viral. 10, 555-559. MORRISON, T. G., STAMPFER, M., LODISH, H. F., and BALTIMORE, D. (1975). In vitro translation of vesicu-
lar stomatitis messenger RNAs and the existence of a 40s plus strand. In “Negative Strand Viruses” (Barry and Mahy, eds.), Pitman Press, Bath. PORTNER, A., and KINGSBURY,D. W. (1970). Complementary RNAs in paramyxovirions and paramyxovirus-infected cells. Nature (London) 228, 11961197. ROBINSON, W. S. (1970). Self-annealing of subgroup 2 myxovirus RNAs. Nature (London) 225.944-945. SCHINCARIOL, A. L., and HOWATSON,A. F. (1972). Replication of vesicular stomatitis virus. Virology 49, 766-783. SHATKIN, A. J. (1974). Animal RNA viruses: Genome structure and function. Annu. Rev. Biochem. 43, 643-665. SORIA, M., LITIXE, S. P., and HUNG, A. S. (1974). Characterization of vesicular stomatitis nucleocapsids. I. Complementary 40 S RNA molecules in nucleocapsids. Virology 61,270-280. WEISSMANN, C., FEIX, G., and SLOR,H. (1968). In vitro synthesis of phage RNA. Cold Spring Harbor Symp. Quant. Biol. 33.83-100.
66, 185-191 (1975)
Antigenomes
in Sendai Virions
DANIEL Dbpartment
KOLAKOFSKY de Biologic moltculaire, Accepted
and Sendai Virus-Infected AND
ANDREE BRUSCHI
Universite March
Cells
de Genbve, 1211, Geneve 4
4, 1975
Several factors which might affect the relative proportions of genomes and antigenomes in Sendai virus have been examined. The relative amount of antigenomes assembled into mature virions was found to be independent of the time of harvest, whereas the amount of cellular RNAs incorporated into virions was found to increase late in infection. Virus strain ard host cell did affect the relative amounts of antigenomes in both mature virions and the infected cell, but in all cases more antigenomes were present in the infected cell than would be expected from their function as intermediates in genome replication.
plus-minus sense of the viral genomes is reversed. Genome-length plus strands or antigenomes have recently been demonstrated in purified Sendai virions (Kolakofsky et al., 1974). Since such antigenomes are thought to function only as intermediates in minus-strand genome synthesis, their presence in purified virions appears to be superfluous. We have, therefore, examined whether the inclusion of antigenomes in mature virions might be due to some physiological anomaly occurring during infection, e.g., packaging of plus nucleocapsids late in infection when cytopathic effects are most severe. We have found that the relative ratio of plus to minus nucleocapsids assembled into mature virions is constant throughout the infection. We have also examined the effect of different viral strains and host cells on the ratio of plus to minus nucleocapsids present both in mature virions and infected cells. Although both the viral strain and the host cell have a small effect on these ratios, in all cases considerably more antigenomes were present than might be expected from their function as intermediates in genome replication.
INTRODUCTION
Sendai virus, a parainfluenza virus type 1, contains an RNA genome which is complementary to the virus-specific mRNAs found in the infected cells (Kingsbury, 1974; Shatkin, 1974). Since viral RNA, which is directly used as template for protein synthesis, is generally referred to as the plus strands, parainfluenza viruses are, therefore, called negative strand viruses to distinguish them from viruses such as QB or polio in which the virion RNA is also mRNA (Baltimore, 1971; Shatkin, 1974). The genome of Sendai virus is found in the virion not as free RNA, but complexed with nucleocapsid protein subunits which are helically arranged to cover the RNA in a manner similar to TMV (Kingsbury and Darlington, 1968; Blair and Robinson, 1970). In our present understanding of the events which occur during Sendai virus infection, viral nucleocapsids containing the virion polymerase enter the cell cytoplasm and transcribe the minus strand genome to produce multiple, monocistronic mRNAs. These virus-specific mRNAs are then used to synthesize virusspecific proteins, some of which are required to replicate the viral genome via a complementary plus strand or antigenome MATERIALS AND METHODS intermediate (Kingsbury, 1974; Shatkin, 1974). The replication of the Sendai viral Purification of Virus genome is analogous to Qfl or polio genome Individual harvests of tissue culture mereplication in this respect, except that the dium were chilled in ice, and clarified of 185
Copyright @ 19’75by Acad-miL
All rights ot ,.
Press, Inc. duction in any form reserved.
186
KOLAKOFSKY
cellular debris by centrifuging for 10 min at 5000 g. The virus was then pelleted through a 25ml cushion of 25% glycerol in TNE (25 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA) by centrifuging for 2 hr at 36,000 rpm (7”) in the International A170 rotor. The virus pellet was resuspended in l/100 of the original volume of TNE with the aid of the Dounce homogenizer and banded in a preformed 10%40% potassium tartrate gradient by centrifuging for 2.5 hr at 38,000 rpm (10“) in a Spinco SW 40 rotor. The visible virus band was removed through the side of the tube with a syringe, diluted lo-fold with TNE, and repelleted through a 0.5-ml cushion of 25% glycerol in TNE by centrifuging for 1 hr at 50,000 rpm (7”) in a Spinco SW 56 rotor. The virus pellets were stored at -28” until used. Isolation of Intracellular sids
Viral Nucleocap-
Five confluent cultures of MDBK or CEF cells in lo-cm Falcon tissue culture dishes were infected with 20 EID,,/cell of various strains of parainfluenza virus and incubated with 5 ml/dish of Dulbecco’s modified Eagles Medium containing 0.5% lactalbumin hydrolysate and 10 &i/ml of [SH]uridine (25 Ci/mmole) at 37”. At 24 hr postinfection, when the cytopathic effects of infection were maximal without detachment of cells from the dish for Sendai-infected cells, and 12 hr p.i. for NDVinfected cells, the culture medium was harvested and the virus purified as described above. The cell monolayers were washed once with PBS and then scraped with a rubber policeman into 5 ml of PBS. The cells were then pelleted by centrifuging for 5 min at 5000 g, resuspended in 2.5 ml of 0.15 M NaCl, 0.05 MTris-Cl, pH 8.0, and 0.6% triton-X100, and disrupted by vortexing for 30 set at maximum speed. The resulting suspension was then centrifuged for 10 min at 5000 g, the supematant removed and made 10 mM in EDTA and 0.1% in DOC, and incubated for 5 min at 37”. The supernatant was then layered onto a lo-ml CsCl-sucrose step gradient as described by Compans and Choppin (1967) and centrifuged for 2 hr at 36,000 rpm (10’)
AND BRUSCHI
in the Spinco SW 40 rotor. The visible nucleocapsid band, which was located in the middle of the original 30% CsCl step of the gradient, was removed through the side of the tube with a syringe, diluted 10 times with TNE, and pelleted by centrifuging for 1 hr at 50,000 rpm (7”) in the Spinco SW 56 rotor. Genome-length RNA was then isolated from the intracellular nucleocapsid pellets as described in Fig. 2. Virus Strains
SendailHarris and SendailOhbayashi virus strains were obtained from R. Barry, Cambridge, England, and M. Homma, Sendai, Japan, respectively. NDV strains HP and L were obtained from M. Bratt, Worcester, MA and NDV strains BK and C were obtained from D. Kingsbury, Memphis, TN, and C. Colby, Davis, CA, respectively. To prepare- viral stocks, Sendai viruses were grown in g-day-old embryonated chicken eggs for 3 days at 33” and NDV viruses for l-2 days at 39”. The allantoic fluid was clarified of debris by centrifugation for 10 min at 5000 g and stored at -78”. Viral stocks were titered by end-point dilution in embryonated chicken eggs. RESULTS
The Effect of Time Virion RNA
of Harvest
on the
The genomic RNA of Sendai virus can self-anneal when isolated as free 50 S RNA (Robinson, 1970; Portner and Kingsbury, 1970) and this self-annealing has recently been shown to be due to the presence of both plus and minus nucleocapsids in the virion population (Kolakofsky et al., 1974). Blair and Robinson (1970) have demonstrated that only a small fraction of the viral nucleocapsids which are synthesized in the infected cell are incorporated into mature virions. Since virus is released from the infected cells over an extended period of time (generally for 18 to 72 hr postinfection) we have examined whether the occurrence of relatively large amounts of plus nucleocapsids in purified virions might be due to aberrant packaging of plus nucleocapsids late in infection. At this time
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the cytopathic effects of the infection are most pronounced and may interfere with the mechanism by which nucleocapsids are selected for assembly into mature virions. MDBK cells were, therefore, infected with Ohbayashi strain of Sendai virus, [3H]uridine was added to label newly synthesized virus RNA, and virus was harvested over five successive time periods. To determine whether the incorporation of cellular RNAs, such as tRNA (Barry and Bukrinskaya, 1968: Kolakofsky, 1972), into the virions was also dependent on the time of harvest of the virus, the cells were prelabeled with [‘“Cluridine for 24 hr before infection. The virus from each successive harvest was purified as described in Materials and Methods, resuspended in TNE containing 1% SDS and sedimented through a SDS-sucrose gradient (Fig. 1).
FRACTION
NUMBER
FIG. 1. Isolation of 50 S RNA from virus harvested at different times after infection. Eight growing cultures of MDBK cells in 75-cm2 Falcon tissue culture
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187
Gradient fractions containing the 50 S RNA, (indicated by the horizontal bar in each panel of Fig. l), were precipitated with ethanol and the RNA recovered by centrifugation. The 50 S RNAs from virus harvested at different intervals were then allowed to self-anneal for different times and the equilibrium annealing levels reached are shown in the boxes in each panel of Fig. 1. The results demonstrate that the self-annealing levels of the virion 50 S RNA are independent of the time of harvest of the virus. Furthermore, addition of excess unlabeled 50 S RNA from egggrown Sendai virus (which is composed predominantly of minus strands (Portner and Kingsbury, 1970)) to each 3H-labeled 50 S RNA sample before annealing resulted in lowered self-annealing levels of approximately 35% in each case. These reduced self-annealing levels reflect the relative proportion of plus and minus strands in egg-grown Sendai/Ohbayashi virus (Kolakofsky, unpublished results). Virus harvested at different times after infection, flasks were prelabeled with 0.2 &i/ml of [“Cluridine (27 mCi/mmole) in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (15 ml/flask). After 24 hr at 37”, the confluent cultures were infected by adsorption of 20 EID,,/cell of the Ohbayashi strain of Sendai virus for 60 min at 31”. The infecting medium was then removed and replaced with 7 ml of medium containing 0.5% lactalbumin hydrolysate and 10 &i/ml of [3H]uridine (25 Ci/mmole) and the incubation continued at 31”. Medium was successively harvested at 20 hr PI (a), 32 hr PI (b), 41 hr PI (c), 50 hr PI (d), and 66 hr PI (e), and replaced with the above medium containing 2 &i/ml of [“H]uridine. Virus was purified from the individual harvests as described in Materials and Methods and each viral pellet was then dissolved in 0.2 ml of TNE containing 1% SDS, incubated for 5 min at 37”, and centrifuged for 105 min at 50,000 rpm (7”) on 5% to 23% sucrose gradients (50 mM LiCl, 20, mM Tris-Cl, pH 7.5, 4 mM EDTA, 0.1% SDS) in the Spinco SW 56 rotor. After fractionating each gradient, samples of 60 ~1 (a), 10 ~1 (b), 10~1 (c), 15 ~1 (d), and 50 ~1 (e) were spotted on filter paper discs, and the discs were washed and counted as described in Fig. 2. The horizontal bar in each panel indicates those fractions pooled and recovered by ethanol precipitation as 50 S RNA. The enclosed figures in each panel are the self-annealing values of the individual 50 S RNAs determined as described in the legend to Table 1.
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KOLAKOFSKYANDBRUSCHI
therefore, always contains a majority of minus strands. Since the self-annealing of Sendai 50 S RNA has been shown to be due to a single population of full-length complementary strands, both present in the virions as nucleocapsids (Kolakofsky et al., 1974), we conclude that the relative amounts of plus and minus nucleocapsids assembled into mature virions is independent of the time of harvest. The appearance of other RNA species in purified virions, on the other hand, is quite clearly dependent on the time of harvest of the virus. As shown in Fig. 1, virus harvested within the first 32 hr postinfection contains essentially only 50 S RNA. However, from 32 to 66 hr postinfection, increasing quantities of 4, 18, and 28 S RNA are found in the purified virus preparation and, in contrast to the 50 S RNA, these species are preferentially labeled with [“Cluridine used to prelabel the cellular RNA before infection. The evidence that these 4, 18, and 28 S RNA are located inside the virions and are not merely cellular contaminants on the surface of the virion which have survived the purification procedure is 2fold: (a) their proportions relative to the virion 50 S RNA are not diminished by further purification such as equilibrium sedimentation on sucrose density gradients, and (b) treatment of [3H]uridine-labeled virus with nuclease Sl, which is sufficient to completely degrade an amount of added “C-labeled mouse kidney cellular RNA equal to the virion RNA, did not significantly degrade the virion 4, 18, and 28 S RNAs (data not shown). It would thus appear that virus released from cells late in infection, when the cytopathic effects of the infection are most pronounced, contain significant quantities of cellular RNAs, whereas virus released early after infection does not.
remain associated with the cells. It was, therefore, of interest to examine whether the ratio of plus-to-minus nucleocapsids found in mature virions reflects the ratio found in infected cells, or alternatively, was the result of preferential selection of either plus or minus nucleocapsids during assembly of the virus at the cell surface. Furthermore, since the ratio of plus-tominus nucleocapsids packaged into mature virions is independent of the time of harvest, we have also examined the effect of different viral strains and host cells on this ratio at a single time of harvest. Both CEF and MDBK cells were infected with different strains of parainfluenza virus, [3H]uridine was added immediately after infection to label the virus RNA, and both the culture medium and the cells were harvested when virus production was maximal. Virus was purified from the culture medium and its 50 S RNA isolated as described in Fig. 1. Viral nucleocapsids were isolated from the infected cell by the method of Compans and Choppin (1967) and their 50 S RNA was isolated as described in Fig. 2. The various 50 S RNAs were then self-annealed for different lengths of time and their equilibrium annealing levels determined. The results, reported in Table 1, are summarized as follows: 1. In the four cases in which 50 S RNA from both mature virions and intracellular nucleocapsids were examined in the same infection, 50 S RNA from intracellular nucleocapsids self-annealed to approximately twice the level of virus 50 S RNA in three cases and was only slightly higher in one case. The large amount of plus nucleocapsids found in parainfluenza virions, therefore, reflects an even larger amount of plus nucleocapsids in infected cells. Although there is some bias in selectively packaging minus nucleocapsids, this bias is not very pronounced. Selection of Plus and Minus Nucleocapsids 2. For a given strain of Sendai virus During Virus Assembly grown in a given host, the relative amounts Blair and Robinson (1970) have shown of plus and minus nucleocapsids are fairly that more than 95% of the nucleocapsids constant (*5%). 3. The relative amount of plus nusynthesized during Sendai virus infection are not processed into mature virions but cleocapsids packaged is affected by the
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viral strain: e.g., the Ohbayashi strain of Sendai virus contains a significantly higher proportion of plus nucleocapsids than the Harris strain of Sendai virus, regardless of whether MDBK cells or CEF have been used as host cells. 4. The host cell influences the ratio of plus to minus nucleocapsids assembled into mature virions. Both the Harris and the Ohbayashi strains of Sendai virus contain a significantly higher proportion of plus nucleocapsids when grown in CEF cells compared to MDBK cells. Selectivity toward which nucleocapsids are assembled into mature virions again appears to be of secondary importance, since both Sendail Harris- and Sendai/Ohbayashi-infected CEF contain a larger proportion of plus nucleocapsids (80% self-annealing) than similarly infected MDBK cells (50% selfannealing). 5. Purified NDV also contains significant, but lower, quantities of plus nucleocapsids than Sendai virus. In the one case where 50 S RNA from both virus and intracellular nucleocapsids were examined in the same infection, intracellular 50 S RNA was found to self-anneal to twice the extent of virus 50 S RNA. DISCUSSION
Sendai and NDV are prototypes of the parainfluenza class of viruses whose ssRNA genome (minus strand) is complementary to the monocistronic mRNA plus strands found in the infected cell (Kingsbury, 1974: Shatkin, 1974). Our present understanding of the events thought to occur during parainfluenza virus replication, like that of the other major class of negative-strand viruses, the rhabdoviruses, does not ascribe any function to full-length plus strands (antigenomes) other than to serve as a template for minus-strand genome synthesis. In this respect, parainfluenza plus strands would appear to serve an analogous function to that determined for minus-strand RNA in the replication of plus-strand viruses such as QB or polio. Antigenome RNA, however, never represents more than l-Z% of the virus-specific RNA found in both QB- and polio-infected
FRACTION
NUMBER
FIG. 2. Isolation of 50 S RNA from intracellular nucleocapsids. Each intracellular nucleocapsid pellet, isolated as described in Materials and Methods, was dissolved in 0.15 ml of TNE containing 1% SDS and centrifuged on a 5% to 23% sucrose gradient as described in Fig. 1. After fractionating the gradient, lo-p1 samples were spotted on filter paper discs, the discs were washed with 6% TCA followed by 95% ethanol, and counted by liquid scintillation to determine radioactivity. The horizontal bar indicates those fractions which were pooled and recovered by ethanol precipitation as intracellular nucleocapsid 50 S RNA. The arrows indicate the positions of the peaks of “C-labeled mouse kidney cellular RNA which was sedimented on a parallel gradient. The above figure represents intracellular nucleocapsid RNA isolated from Sendai/Harris-infected MDBK cells. The second RNA species, which peaks at fraction 13, was found to cosediment with the large ribosomal subunit RNA from uninfected MDBK cells and was not examined further (data not shown).
cells (Weissmann et al., 1968; Oberg and Philipson, 1971). The data presented here demonstrate that, although such factors as virus strain and host cell have a small effect on the relative ratios of plus and minus nucleocapsids present in the parainfluenza virus-infected cell, antigenomes always represent a larger fraction of the intracellular genome-length RNA than would be expected from their function as intermediates in genome replication. Furthermore, genome-length plus strands have recently been shown to represent a similarly large fraction of the genome-length RNA in VSV-infected cells (Schincariol and Howatson, 1972; Sorial et al., 1974). This anomaly, therefore, applies equally to both major classes of negative-strand viruses. Since genome-length plus strands are unlikely to function other than as templates for the synthesis of minus-strand
190
KOLAKOFSKY
AND BRUSCHI
TABLE
1
SELF-ANNEALING OF VIRUS AND INTRACELLULAR 50 S RNA”
Virus strain
Host
Percent self-annealing of 50 S RNA Virion
Intracellular
1 Sendai/Harris 2 Sendai/Harris 3 Sendai/Harris
MDBK MDBK CEF
26.1 + 0.7 27.5 f 0.4 46.4 i 0.7
52.5 zt 1.3 79.0 f 3.9
4 SendailOhbayashi 5 Sendai/Ohbayashi 6 Sendai/Ohbayashi
MDBK MDBK CEF
45.0 * 1.9 47.0 l 1.6 57.2 zt 2.8
51.5 * 1.5 80.5 zt 0.5
CEF CEF CEF CEF MDBK
9.3 f 22.9 f 27.6 * 23.9 * 19.0 f
7 8 9 10 11
NDV/HP NDV/BK NDV/C NDV/L NDV/L
0.2 0.3 0.4 0.9 1.0
-
19.6 * 1.3
0 50 S RNA from purified virus and intracellular nucleocapsids were isolated as described in the legends to Fig. 1 and Fig. 2, respectively. Samples of the different 50 S RNAs containing from 809 to 2000 cpm were annealed in 20 ~1of 2.5 x SSC at 78” in sealed capillaries for 0,30,60,90, and 120 min. The capillaries were then opened, their contents transferred into 0.8 ml of 2 x SSC containing 33 fig of ribonuclease A, and incubated for 30 min at 25”. The remaining RNA was coprecipitated with 30 pg of carrier rRNA by adjusting each solution to 10% in TCA, collected on Millipore filters, and counted by liquid scintillation. Total input radioactivity was determined by the average of one unincubated sample and one sample which was incubated for 120 min at 78’, neither of which received ribonuclease. In all cases, these values did not differ by more than 9%. Annealing equilibrium had been reached in all cases during 60 min at 78”, and the values reported in the table are the average of the 60-, 90-, and 120-min time points, i.e., the equilibrium portion of each annealing curve.
genomes in both parainfluenza virus- and rhabdovirus-infected cells, other explanations for their relative abundance in the infected cell should be considered. Of these, an interesting possibility is that minus-strand viruses simply cannot regulate the relative amounts of full-length plus and minus strands because of the nature of their genome. By comparison with the more extensively studied plus-strand viruses such as Q/3 and polio, minus-strand viruses have properties which might explain their inability to regulate the synthesis of genome and antigenome. For example, Q/3 replicase will preferentially use Q@ minus strands as template in vitro, most probably because Q@replicase can recognize the different structures of the free plus and minus strands in solution (Weissmann et al., 1968). This method of regulation, however, may not apply to parainfluenza and rhabdoviruses since their template for replication is not free RNA but the viral nucleocapsids in which the viral RNA is encapsidated. Since plus and minus nucleocapsids apparently differ only in their
RNA, and this RNA is surrounded by helical arrangement of identical protein subunits (e.g., the RNA in the viral nucleocapsids is insensitive to ribonuclease (Kingsbury and Darlington, 1968)), there may not be sufficient structural differences between plus and minus nucleocapsids that the replicating enzyme can recognize. The fact that the viral transcriptase can distinguish between plus and minus nucleocapsids does not necessarily diminish the above argument since in contrast to transcription, initiation sites for replication must be present on both plus and minus nucleocapsids. Another mechanism which regulates the relative abundance of plus and minus strands in plus-strand virus-infected cells stems from the ability of the viral genome to serve both as a template for replication and protein synthesis. In QB- and polioinfected cells, plus strands are continually being withdrawn from the pool of viral RNA to form polysomes, leaving the pool of RNA available to replicase enriched in minus strands. Such a mechanism is, of
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course, not applicable to negative-strand viruses. On the contrary, should plus nucleocapsids also be capable of acting as templates for protein synthesis, the pool of nucleocapsids available for replication would be enriched in minus nucleocapsids and an increased synthesis of plus nucleocapsids would result. It is, therefore, possible that minusstrand virus-infected cells contain large amounts of plus nucleocapsids not because they are required in such numbers for viral replication, but rather by default. If this is not the case, it would be worth rexamining other functions for plus nucleocapsids in negative-strand virus replication. Although both rhabdovirus- and parainfluenza virus-infected cells contain large amounts of plus nucleocapsids, only parainfluenza virions contain significant quantities of plus nucleocapsids. From the data presented here we have concluded that parainfluenza virus-infected cells have some bias in selectively packaging minus nucleocapsids, but this bias is very weak. This, however, is not surprising if plus and minus nucleocapsids differ only in their RNA. Rhabdovirus-infected cells, on the other hand, must have a rather stringent mechanism for assembling only minus nucleocapsids into mature virus, which is either nonexistent or poorly functioning in parainfluenza virus-infected cells. Morrison et al. (1975) have recently suggested that the genome length plus strands found in WV-infected cells contain poly A whereas minus strands do not. The presence of poly A tails on plus nucleocapsids and their absence on minus nucleocapsids could serve as a basis for the selection of minus nucleocapsids for assembly into mature virions. Similarly, the absence of poly A tails on both parainfluenza plus and minus nucleocapsids might explain the lack of a stringent mechanism in the packaging of viral nucleocapsids in parainfluenza virus-infected cells. It is so far not known whether parainfluenza plus strands contain terminal poly A sequences and we are presently investigating this matter, ACKNOWLEDGMENTS We thank Pierre-Francois Spahr for generous ad-
vice, encouragement, and support, and Peter Bromley and Julian Davies for helpful discussion. This work was supported by Research Grant 3.331.074 from the Fonds National Suisse de la Recherche Scientifique. REFERENCES BALTIMORE, D. (1971). Expression of animal virus
genomes. Bacterial. Reo. 35, 235-241. BARRY,R. D., and BUKRINSKAYA, A. G. (1968). The nucleic acid of Sendai virus and RNA synthesis in cells infected by Sendai virus. J. Gen. Virol. 2, 71-79. OBERG, B., and PHILIPSON, L. (1971). Replicative structures of polio virus RNA. J. Mol. Biol. 58, 725-737. BLAIR, C. D., and ROBINSON,W. S. (1970). Replication of Sendai virus. II. Steps in virus assembly. J. Viral. 5.639-650. COMPANS, R. W., and CHOPPIN, P. W. (1967). Isolation and properties of the helical nucleocapsid of the parainfluenxa virus SV5. Proc. Nat. Acad. Sci. USA 57.949-956. KINGSBURY, D. W., and DARLMGTON, R. W. (1968). Isolation and properties of NDV nucleocapsids. J. Virol. 2, 248-255. KINGSBURY, D. W. (1974). The molecular biology of paramyxoviruses. Med. Microbial. Immunol. 160,
73-83. KOLAKOFSKY, D., BOY DELATOUR,E., and BRUSCHI, A. (1974). Self-annealing of Sendai virus RNA. J. Virol. 14,33-39. KOLAKOFSKY, D. (1972). tRNA nucleotidyl transferase and tRNA in Sendai virions. J. Viral. 10, 555-559. MORRISON, T. G., STAMPFER, M., LODISH, H. F., and BALTIMORE, D. (1975). In vitro translation of vesicu-
lar stomatitis messenger RNAs and the existence of a 40s plus strand. In “Negative Strand Viruses” (Barry and Mahy, eds.), Pitman Press, Bath. PORTNER, A., and KINGSBURY,D. W. (1970). Complementary RNAs in paramyxovirions and paramyxovirus-infected cells. Nature (London) 228, 11961197. ROBINSON, W. S. (1970). Self-annealing of subgroup 2 myxovirus RNAs. Nature (London) 225.944-945. SCHINCARIOL, A. L., and HOWATSON,A. F. (1972). Replication of vesicular stomatitis virus. Virology 49, 766-783. SHATKIN, A. J. (1974). Animal RNA viruses: Genome structure and function. Annu. Rev. Biochem. 43, 643-665. SORIA, M., LITIXE, S. P., and HUNG, A. S. (1974). Characterization of vesicular stomatitis nucleocapsids. I. Complementary 40 S RNA molecules in nucleocapsids. Virology 61,270-280. WEISSMANN, C., FEIX, G., and SLOR,H. (1968). In vitro synthesis of phage RNA. Cold Spring Harbor Symp. Quant. Biol. 33.83-100.