Virus RNA and Protein Synthesis in Cells Infected with Different Strains of Newcastle Disease Virus

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1 J. gen. Virol. 097D, ~t3, lll-lzo Printed in Great Britain IIl Virus RNA and Protein Synthesis in Cells Infected with Different Strains of Newcastle Disease Virus By B. LOMNICZI,* A. MAGR'J" AND D. C. BURKt Department of Biochemistry, Marischal College, University of Aberdeen * Veterinary Medical Research Institute of the Hungarian Acadamy of Sciences, Budapest, Hungary "~ Division of Biological Sciences, University of Warwick, Coventry CV4 7AL, Warwickshire (Accepted 3 June I97I ) SUMMARY The virus RNAs and proteins synthesized in chick embryo cells infected with different strains of Newcastle disease virus have been characterized by polyacrylamide gel electrophoresis. By comparison of the rates of electrophoretic mobility of virus RNA with those of RNA molecules of known molecular weight, the molecular weights were estimated to be 4"8 x io 6 for the virus particle RNA and 2"5 x lo 6, 1.1 x IO 6 and 0"4 to 0"7 x lo 6 for the RNA molecules synthesized in infected cells, the latter being heterogeneous. Synthesis of a number of virus particle proteins was readily detected. INTRODUCTION Newcastle disease virus is a member of the paramyxovirus group. It contains a molecule of single-stranded RNA with a sedimentation coefficient of 57s (Duesberg & Robinson, I965) and five or probably six polypeptides, one of which interacts with the RNA to form the virus nucleoprotein, while two others are glycoproteins and are components of the lipoprotein envelope of the virus (vans & Kingsbury, I969; Haslem, Cheyne & White, I969; Caligiri, Klenk & Choppin, I969, Mountcastle, Compans & Choppin, I97I). Infected cells contain a number of virus RNA molecules (Bratt & Robinson, I967), including both the virus particle RNA and a series of RNA molecules of lower sedimentation coefficients 08s, 22s and 35s). These have been partially resolved by sucrose gradient centrifugation and shown by hybridization to be complementary to the virus particle RNA (Kingsbury, I967; Bratt & Robinson, I967). However, almost nothing is known about the role of these RNA molecules in virus replication, although there is evidence that the I8S RNA is associated with cellular polysomes (Bratt & Robinson, ~967; Blair & Robinson, I968). In order to establish the role of these RNA molecules more precisely, it was necessary to separate them by a procedure of higher resolving power than sucrose density gradient centrifugafion and to establish their molecular weights. The synthesis of the virus proteins was also investigated. A large number of strains of Newcastle disease virus have been isolated which differ markedly in their virulence for chickens (Waterson, Pennington & Allan, I967). They can be classed as velogenic (the most virulent), mesogenic (of intermediate virulence) and lentogenic (the least virulent) on the basis of the time taken to cause death in the chick embryo, I-day-old chicks or adult chickens (Hanson & Brandly, I955). A number of 8-2

2 II2 B. LOMNICZI, A. MAGR AND D. C. BURK properties of the different strains have been examined in a search for a property linked with virulence. These include properties of the virus particle, such as morphology, chemical composition, heat stability, cell-fusing ability, serology and enzymology and also properties of the infected cells, such as speed of virus multiplication (Kohn & Fuchs, 1969; Reeve, Rosenblum & Alexander, 197o; Reeve & Waterson, I97O). None of these characteristics showed any correlation with virulence. However, the capacity to produce plaques and syncytia in chick embryo cells was found to be related to virus virulence (Schloer & Hanson, I968; Kohn & Fuchs, 1969; Reeve & Alexander, t97o), and a secondary aim of this work was to compare virus RNA and protein synthesis in cells infected with strains of differing virulence in order to obtain more information about the way in which these strains differed. Six strains have been used in this work- two velogenic (T~XAS and HRTS 33), two mesogenic (H and L) and two lentogenic strains (F and LA SOTA). MTHODS Materials. Actinomycin D was given by Merck, Sharp and Dohme Ltd. Sodium dodecyl sulphate (specially pure grade) was obtained from British Drug Houses, Poole, Dorset. ther and phenol were freshly redistilled before use. [5-3H]uridine (3O'TC/m-mole), [ah]dlvaline (25o mc/mmole); [14C]L-valine (260 mc/m-mole) and asp (orthophosphate in solution containing I mg./ml, phosphate buffer ph 7"o) were obtained from the Radiochemical Centre, Amersham, Buckinghamshire, and acrylamide, NNl-methylenebisacrylamide and NNNiNl-tetramethylethylene diamine from astman Kodak. Acrylamide and NN ~- methylenebisacrylamide were recrystallized from chloroform and acetone, respectively. Media and cells. Chick embryo cells were prepared from 9- or Io-day-old embryos as described by Walters, Burke & Skehel (I967). Virus. The origin, growth and assay of the Newcastle disease virus strains has been previously described (Lomniczi, I97O). Table I gives a summary of their biological properties. Growth curves were done in 8x I3 cm. flasks containing 25 IOG cells. After infection with 15 to 25 p.f.u./cell (for TXAS, HRTS 33, n and ) or I5 to 25 ID5o (egg infecting dose 50 %) for the avirulent strains (F and LA SOTA), the cells were washed, incubated at 39 and small samples removed at intervals for titration. Isotope incorporation experiments were carried out in glass Petri dishes containing IOO x io 6 cells. Purification of radioactive virus and extraction of RNA. Virus, labelled with asp, was prepared as described by Barry & Bukrinskaya 0968), except that the density gradient centrifugation was done on a gradient of potassium tartrate (I5 to 60 %, w/v). When the TXAS strain was purified in this way, the product had an infectivity/haemagglutinin ratio one-tenth of that of the crude virus. The RNA was extracted by the method of Bratt and Robinson 0967) and fractionated on 2.2 % and 2"5 % polyacrylamide gels as described by Peacock & Dingman 0967). Calibration of 2"2 % polyacrylamide gels was done using the method of Loening 0967). Virus protein synthesis in infected cells was measured as described by Joss et al. 0969) Actinomycin (o'5 #g./ml.) was added immediately after infection and the cells pulsed with either [~H]valine (9/~c/culture of infected cells) or [14C]valine (3 #c/culture of uninfected cells) between 4 and 6 hr after infection. The infected and uninfected cells were mixed, nuclei removed by low speed centrifugation, and the proteins in the supernatant solubilized and fractionated by polyacrylamide gel electrophoresis as described by Hay, Skehel & Burke 0968). In some experiments, the isotope was added for t hr either 14 or 24 hr after infection. In this case, actinomycin was added 4 hr before addition of isotope.

3 RNA and proteins in ND V infection 113 Virus RNA synthesis in infected cells was measured as described by Gandhi & Burke (197o). The RNA was precipitated with ethanol at -2o, redissolved and then fractionated on 2.2% or 2"5 % polyacrylamide gels as described by Peacock & Dingman 0967). Preparation of [32p] Hela cell nucleolar RNA. HeLa cells were grown to confluence (72 hr) in Roux bottles in agle's minimal essential medium containing 7 % calf serum and o'5 mc [32P]/bottle. The cells were removed by trypsinization, pelleted by centrifugation (5oo g for 5 min.), and nucleoli were prepared by the method of Penman, Vesco & Penman (1968). Nucleolar RNA was extracted as described by Penman (1966) and Wagner, Katz & Penman (I967). Table i. Biological properties of Newcastle disease virus Strain LASOTA F H L TXAS GB HRTS 33 Cytopathic effect in Plaque size in Mean death time chick embryo chick embryo for embryos* cellst cells:~ (hr) (%) (mm.) ICP IVPll > I20 ~ "O0 O'OO > 120 ~-~ IO -- 0"O0 0"00 48 ~< 5O 3 to 4 O'78 O'42 68 IO0 I O'64 O'48 5O to 6O I00 2 to 3 I"50 > 1"50 50 to 60 ~00 2 to 3 /> I'50 > I'50 * Using the highest dilutions of virus that killed IOO % of the embryos. qr Cytopathic effect was measured after 24 hr at 39. LA SOTA, F and ~ caused rounding of cells, L and HRTS 33 rounding of cells and formation of small syncytia, and TXAS formation of large syncytia. :~ Measured after 48 hr at 39. lntracerebral pathogenicity index in i-day-old chicks (cf. Waterson et al., 1967). [[ Intravenous pathogenicity index in 6-week-old chicks (cf. Waterson, et al., 1967). RSULTS Growth curves of Newcastle disease virus strains in chick embryo cells Measurement of the yield of virus released from infected cells as a function of time showed (Fig. I) that the two velogenic strains grew most rapidly and to highest yield. The two mesogenic strains yielded less extracellular virus although both produced plaques. The L strain caused an extensive cytopathic effect late in infection, but that due to the H strain never exceeded 50 %. The two lentogenic strains (LA SOTA and F) failed to induce plaques or an extensive cytopathic effect, and were therefore titrated in eggs. After a latent period of 4 to 6 hr, titres increased exponentially, reaching maxima I2 to 14 hr after infection. Thus, the kinetics of growth were similar, although the yield of virus released/cell varied between the strains. The relatively poor growth of the I-t strain and also of strains F and LA SOTA was probably due to autointerference (B. Lomniczi, unpublished results). Virus RNA synthesis in infected cells Before investigation of virus RNA synthesis in infected cells by use of polyacrylamide gel electrophoresis, it was necessary to characterize the virus particle RNA. lectrophoresis of the RNA extracted from the TXAS and F strains gave identical patterns; a single peak being obtained (Fig. 2). Similar results were obtained with the I4 strain. lectrophoresis in 2"5 % polyacrylamide gels failed to demonstrate the low molecular weight material reported by Duesberg & Robinson 0965) which was probably a degradation product. Infected ceils were treated with actinomycin to depress the rate of cellular RNA synthesis

4 II 4 B. LOMNICZI, A. MAGR AND D. C. BURK and then pulsed with [SH]uridine before extraction of RNA and fractionation on 2.2 ~o polyacrylamide gels. The results (Fig. 3) showed the presence of multiple species of RNA. These were virus particle RNA (at fraction 7) and RNA species at about fraction 12, fraction I7 and at fraction 25 which probably corresponded to the 35s, 22s and I8s molecules observed by others (Bratt & Robinson, I967; Blair & Robinson, ~968; Bratt, I969). Similar results were obtained using 2"5 % gels, although in this case virus particle RNA I I I f I I I! D t jd-- fla..c] _a 107 i'= x~= 106 4~ 2x ~L ~g m =,7, & o~ 10 s 10 4 I :/ / j~#'-- --~-- --A Q I i i l I I I I Time after infection (hr) Fig. I. Growth curves of Newcastle disease virus strains. Cells were infected as described in Methods and fluids harvested at the times shown and titrated in chick embryo cells (TXAS, HRXS 33, H and L) or fertile eggs (F and LA SOTA). TXAS, ~----[Z; HRTS 33,, H, O----O, L, ~ g, F, ~------~; LA SOTA, A. ( i I I a) 28s 18s (b) I i i 28s 18s o -r e~ Fraction no Fig. 2. Polyacrylamide gel electrophoresis on 2"2 ~o gels of the RNA ( 0) extracted from (a) the TXAS and (b) the F strain of Newcastle disease virus. lectrophoresis was at ~5o v/2 ma tube for I hr. The arrows indicate the position of the ribosomal markers ( in a).

5 RNA and proteins in ND V infection I 15 did not enter the gels. All six strains stimulated the synthesis of the same species of RNA, although, as first observed by Bratt 0969), the virulent strains produced somewhat more I8S RNA than the avirulent. Further investigation showed that both the 22s and I8s RNA were heterodisperse (Fig. 4) I 28 s I 18 s I -- HRTS 33 + ~, _ I - TXAS 28s 1 18s ~/ ~ I P I I I I 28s I 18s I _L ~ + _ I I I I 28s I 18s I z g 100 o "1-400 I 28s I 18sl -~ ~ + - I 28s I 18s I - LA SOTA,~ ~/ 100 I I I Fraction no. Fig. 3. Polyacrylamide gel electrophoresis of virus nucleic acid extracted from cells infected with various strains of Newcastle disease virus. The nucleic acids were fractionated on z-2 % gels. The arrows indicate the position of the ribosomal markers. Note the different scales. Bishop, Claybrook & Spiegelman 0967) have shown that the relative electrophoretic mobility of a series of RNA molecules in polyacrylamide gels was proportional to the log. of the molecular weight. A similar relationship was found (Fig. 4), using the HeLa cell nucleolar RNA (Weinberg & Penman, I97O) and was used to calculate the molecular weights of the virus RNA molecules found in infected cells, which were found to be o. 4

6 II6 B. LOMNICZI, A. MAGR AND D. C. BURK to 0"7 x IO 6 (' 18S'), about I.I x io G ('22s'), 2"5 x lo s (35s) and 4"8 x Io 6 (virus particle RNA). Generally, molecular weights of RNA molecules can be determined from their sedimentation coefficient using the relationship molecular weight = t55 o x s 2"1 (Spirin, ~963) and the molecular weight values of I8S, 22s and 35s as determined by polyacrylamide gel calibration correspond to those calculated from the Spirin formula. However, the virus 28s d 32s d s 100 a. \ I I ~ Fraction no. Fig. 4. The electrophoretic mobility in 2"2 ~o polyacrylamide gels of the HeLa cell 32[P]nucleolar RNA (O---O), HeLa cell [asp]ribosomal RNA ( O), and [ahlvirus nucleic acid (~ O) extracted from cells infected with Newcastle disease virus strain XXAS. The different types of cellular RNA are indicated by the arrows, and the following values were taken for their molecular weights: 45 s, 4I s and 32s nucleolar RNA, 4.i IO 6, 3'I 106, and 2"I x IO g, respectively (Weinberg & Penman, 197o); 28s and I8S ribosomal RNA, 1-65 lo 6 and o'65 x io" (Peterman & Pavlovec, 1966). particle RNA appears to be an exception, for in this case the widely quoted value of 57s corresponds to a molecular weight of 7"5 x io 6. It is interesting that the molecular weight of 45s RNA from Semliki Forest virus particles (4"4 x io 6) as calculated from the Spirin formula is approximately the same as that determined by polyacrylamide gel electrophoresis (A. Meager, unpublished observation).

7 Virus protein synthesis in infected cells RNA and proteins in NDV infection [I7 Virus proteins synthesized in infected cells were extracted and fractionated by polyacrylamide gel electrophoresis, using the double labelling procedure of Hay et al. 0968). The results (Fig. 5) are plotted as the ZH: aac ratios in order to simplify the presentation of the data. The 3H and 14C counts are shown separately for the LA SOTA strain in Fig. 6, and it can be seen that the use of the 3H: 14C ratio removes peaks due to cellular proteins 1 I I I I I ] I I I I I LA SOTA I F I ' ' I ' I I I I I I I ', 1 6 H? L_ 0 L_ u4 o. -l- m 2 FIRTS 33 I. TXAS Fraction no. Fig. 5- Polyacrylamide gel electrophoresis of the proteins extracted from chick embryo cells 6 hr after infection with various strains of Newcastle disease virus. (e.g. at fraction z and z5), and also removes any errors due to unequal gel slices (e.g. at fraction 34). The results showed that all six strains synthesized three virus proteins (at fractions 15, 23 and 3a) which were almost certainly virus particle proteins (vans & Kingsbury, ]969; Haslam et al. I969) and that, in addition, strains L, v and L~, SOTA synthesized a fourth virus protein (at fraction [8), which was not detected in the purified virus by vans & Kingsbury (I969) or Haslem et al. 0969), but may be identical with VP 2 described by Mountcastle et al. (]970. The small peak at fractions 28 to 30 probably

8 II8 B. LOMNICZI, A. MAGR AND D. C. BURK corresponds to the minor virus particle protein found by Mountcastle et al. (I970 in the paramyxoviruses SV 5 and Newcastle disease virus. xamination of virus protein synthesis at later times in infection showed that cells infected with the LA SOTA strain were still synthesizing virus proteins at I4 hr and 24 hr after infection. A similar result was obtained using the r strain. Virus protein synthesis was still just detectable in cells infected with the H strain I4 hr after infection. Cells infected with more virulent strains did not synthesize any protein, either virus or cellular, at I4 hr after infection, and showed an extensive cytopathic effect. Thus, virus protein synthesis continued longer in cells infected with the avirulent strains. I I I I I I ~ 4 -? # x 6._~ -5 = 4 t.) 2 I I I I I I Fraction no. Fig. 6. Polyacrylamide gel eiectrophoresis of the proteins extracted from chick embryo strains 6 hr after infection with the LA SOTA strain. The lower panel shows the ah ( ) and ~4C (O~-----O) radioactive counts separately. DISCUSSION Our figure of 4"8 lo o for the molecular weight of the virus particle RNA is somewhat lower than previous values, which were generally based on the sedimentation coefficient of the RNA. This varied from 4os for Sendai virus and SV 5 RNA (Barry & Bukrinskaya, I968 ; Compans & Choppin, I968) to 57s for Newcastle disease virus (Duesberg & Robinson, I965) depending on the conditions used. Using the Spirin formula (I963) these figures correspond to molecular weights of 5"2 and 7"5 lo6. Using an electromicroscopic method, Compans & Choppin (I968) obtained a figure of 6-8 Io 6 for SV 5, while Nakijima & Obara (t967) obtained a figure of 5"8 Io 6 by a chemical method. However, both these latter estimates involved a number of assumptions, certainly more than those involved in the determination of molecular weights from calibrated gels. The molecular weights of the other virus RNA molecules were 2"5 Io 6, I.I Io 6 and

9 RNA and proteins in NDV infection 0"4 to 0"7 x io 6. It is interesting that these figures and that for the virus particle RNA, fall in the regular series o.6:1.2:2.4:4"8. The significance of this is unknown, although it may reflect a subunit structure of the RNA. The greater resolving power of polyacrylamide gel electrophoresis clearly showed that the 18s RNA was heterogeneous. Kingsbury (197o) has pointed that the hybridization data of Bratt & Robinson (I967) demonstrates that either the 18s RNA is heterogeneous with respect to nucleotide sequences, the total population making up sequences complementary to 5O~o of the genome, or that all the 18s molecules are identical with half the virus genome redundant for their species. The present finding makes the first possibility seem the more likely, and it is interesting that Huang, Baltimore & Stampfer (197o) have found a similar situation in cells infected with vesicular stomatitis virus, a virus that shows a very similar pattern of RNA replication to Newcastle disease virus. These results give, as yet little clue to the molecular basis of virulence. Virus RNA and protein synthesis were broadly similar although, as reported by Bratt (1969), the relative amount of the different RNA molecules made, varied. It is planned to continue the comparison in a more detailed analysis of virus RNA and protein synthesis. We thank Action for the Crippled Child and the Medical Research Council for grants which supported this work and Mr F. Troup for technical assistance. I I9 RFRNCS BARRY, R. D. & BUKmNSKAYA, A. G. (I968). The nucleic acid of Sendai virus and ribonucleic acid synthesis in ceils infected by Sendai virus. Journal of General Virology 2, 7I. BISHOP, D. H. L., CLAYBROOK, J. R. & SP1GLMAN, S. 0967). lectrophoretic separation of viral nucleic acids on polyacrylamide gels. Journal of Molecular Biology 26, 373- BLAre,. D. & ROBINSON, W. S. 0968). Republication of Sendai virus. I. Comparison of the viral RNA and virus-specific RNA synthesis with Newcastle disease virus. Virology 35, 537. BRATT, M. A. (I969). RNA synthesis in chick embryo cells infected with different strains of NDV. Virology 38, 485. BRAXT, M. A. & ROBINSON, W. S. (1967). Ribonucleic acid synthesis in cells infected with Newcastle disease virus. Journal of Molecular Biology 23, I. CALIGUIRI, L. A., KLNK, H. D. & CHOPPIN, P.W. (I969). The proteins of the parainfluenza virus SV5. 1. Separation of virion polypeptides by polyacrylamide gel electrophoresis. Virology 39, 46o. COMPANS, R. W. & CHOPPIN, e. W. (I968). The nucleic acid of the parainfluenza virus SV5. Virology 35, 289. DUSBRG, P. H. & ROBINSON, W. S. (I965). Isolation of the nucleic acid of Newcastle disease virus (NDV). Proceedings of the National Academy of Sciences of the United States of America 54, 794. VANS, M. J. & KINGSBURY, D. W. (I969)- Separation of Newcastle disease virus proteins by polyacrylamide gel electrophoresis. Virology 37, 597. GANOHI, S. S. & BURK, D. C. (I970). Interferon production by myxoviruses in chick embryo cells. Journal of General Virology 6, 95. HANSON, R. P. & BRANOLY, C. A. (I955)- Identification of vaccine strains of Newcastle disease virus. Science, New York I22, I56. HASLAM,. A., CHYN, I. M. & WHIT, D. O. 0969). The structural proteins of Newcastle disease virus. Virology 39, 1 I8. HAY, A. J., SKHL, J. J. & BURK, D. C. (I968). Proteins synthesised in chick cells following infection with Semliki Forest virus. Journal of General Virology 3, I75. HUANG, A. S., BALTIMOR, D. & STAMPFR, M. (I970). Ribonucleic acid synthesis of vesiculars tomatitis virus. III. Multiple complementary messenger RNA molecules. Virology 42, 946. Joss, A., GANDHI, S. S. HAY, A. J. & BURr:, D. C. (I969). Ribonucleic acid and protein synthesis in chick embryo cells infected with fowl plague virus. Journal of Virology 4, 8 ~ 6. KINGSBURY, D. W. (I967). Newcastle disease virus complementary RNA: its relationship to the viral genome and its accumulation in the presence or absence of actinomycin D. Virology 33, 227. KINGSBURV, D. W. (I970). Republication and functions of myxovirus ribonucleic acids. Progress in Medical Virology I2, 49- KOHN, A. & FUCHS, P. (I969)- Cell fusion by various strains of Newcastle Disease virus and their virulence. Journal of Virology 3, 539.

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