Distinct subunits of the ribonucleoprotein of influenza virus

,J. ,Tlol. Biol. (1969) 42, 485499

Distinct Subunits of the Ribonucleoprotein

of Influenza Virus

P. H. DUESBERQ Department of Molecular Biology and Virus Laboratory University of Californiu, Berkeley, Calif. 94720 U.S.A. (Received 28 November 1968, and in revised form 17 February 1969) Influenza virus A was disrupted by deoxycholate, Triton Xl00 or ether extraction into ribonucleoprotein and envelope proteins. The ribonucleoprotein was resolved into a 70 8, a 60 s and a 60 s component by sucrose gradient sedimentation. Three components of viral ribonucleoprotein were also obtained by polyacrylamide gel electrophoresis. The buoyant density of the viral nucleoprotein was 1.266 g/ml. in a sucrose density-gradient. The ribonucleoprotein was relatively resistant to pronase. But the RNA of the ribonucleoproteins of influenza virus was degraded by RNase. Probably the same three ribonucleoprotein components were also detected in virus-infected cells about three hours after infection. Distinct components of the influenza virus nucleoprotein were found to contain distinct viral RNA’s, suggesting that their heterogeneity is original and not an artifact of the isolation procedures used.

1. Introduction There are two groups of myxoviruses

(Waterson,

1962; Robinson

& Duesberg,

1968),

the larger parainfluenza viruses with a diameter of about 150 to 200 rnp and one RNA molecule of about 65 x lo6 daltons (Duesberg & Robinson, 1965; Duesberg, 1968a; Blair & Robinson, 1968; Compans & Choppin, 1968) and the smaller iufluenza viruses with a diameter of about 100 rnp and an estimated RNA content of 2 x lo6 daltons (Frisch-Niggemeyer, 1956). The general structure of myxoviruses is thought to be a helically arranged ribonucleoprotein (Waterson, 1962), also called g-antigen (Schafer & Zillig, 1954) or s-antigen (Schafer, 1963), which is enclosed by a lipoprotein envelope (Cruickshank, 1964). Virus particles can be disintegrated by ether (Hoyle, 1952), a combination of non-ionic detergents and ether (Hosaka, Hosakawa & Fukai, 1959; Rott & Schafer, 1964) or deoxycholate &aver, 1963; Mizutani & Mizutani, 1965; de The & O’Connor, 1966; Styk & Hana, 1968; Blair, 1968) into two main components, hemagglutinin and ribonucleoprotein. There has been considerable uncertainty about the size and structure of the nucleoprotein of influenza virus. Because of many structural and biological similarities between the two groups of myxoviruses (Cruickshank, 1964; Robinson & Duesberg, 1968), the nucleoprotein of influenza virus A was expected to have a relatively large uniform structure, although smaller than the nucleoprotein of the parainfluenza viruses in proportion to the smaller size and lower RNA content of the influenza group. The ribonucleoproteins of several parainfluenza viruses, after isolation by the same method as used for influenza viruses, were shown to consist of large, homogeneous 485

486

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H.

DUESBERG

helices of 1000 rnp length (Compans & Choppin, 1967; Hosaka & Shimizu, 1968) and 20 my in diameter (Waterson, 1962) and to have an S, of about 250 s (Blair, 1968; Compans & Choppin, 1968; Hosaka, 1968; Kingsbury & Darlington, 1968). The first physical analysis of the nucleoprotein of fowl plague (influenza A) virus by Schafer & Zillig (1954) showed that it was present in filaments of variable lengths (15 to 100 rnp) and various sedimentation coefficients (S,,,,) of about 15 to 60 s. This heterogeneity was thought to be the result of various chain-like aggregates of a single subunit with a diameter of 10 to 15 rnp and an S, value of 15 to 20 s. A similar result was obtained with PR8 influenza virus A by Paucker, Birch-Anderson & von Magnus (1959), who suggested that the heterogeneity of the nucleoprotein represented rods fractionated into units of various lengths. Detailed electron micrographic studies by Hoyle, Horne & Waterson (1961) confirmed that the ribonucleoprotein of influenza virus occurred in relatively short pieces of variable lengths with an average length of 60 rnp and a diameter of 10 mp. The failure to isolate the nucleoprotein of influenza in a single large piece stimulated speculation about the actual size of the nucleoprotein in the intact virion. In 1963 Schafer suggested that the small size of the iniluenza virus ribonucleoprotein was probably due to degradation during the isolation from the virus. Hoyle et al. (1961) and Cruickshank (1964) also considered the possibility, which they did not think very likely, that the nucleoprotein might exist in the virus in the form of several distinct relatively short pieces. More recently the RNA of influenza virus A was found to exist in several discrete pieces (Duesberg & Robinson, 1967; Duesberg, 19683; Pons & Hirst, 1968). The present report provides evidence that the puzzling heterogeneity of the nucleoprotein of influenza virus is not an artifact of the isolation procedure but probably reflects its primary structure. It was found that the larger subunits of the nucleoprotein contain the larger and the smaller subunits contain the smaller RNA’s of influenza virus.

2. Materials and Methods (a)

Buffers

Standard buffer contains 0.1 M-NaCl, 0.01 M-Tris-HCl (pH 7.4) and 1 m&f-EDTA; low-salt buffer contains 0.02 an-NaCl, 0.02 M-Tris-HCl (pH 7.4) and 2 m-EDTA; electrophoresis buffer contains 0.02 M-sodium acetate, 0.04 M-Tris-acetate (pH 7.2) and 1 mMEDTA; hypotonic buffer contains 0.026 M-KC& 2 m&r-MgCl,, 5 m&r-CaCl,, and 0.01 MTris-HCl (pH 7.4). Tris-saline contains/l. : 3 g Tris adjusted to pH 7.4 with HCl; 8 g NaCl ; 0.38 g KC1 ; 0.1 g NazHPO,; 1 g dextrose; lo6 units penicillin and 50 mg streptomycin. Dulbecco buffer contains/l.: 8 g NaCl; 0.2 g KCl; O-1 g C&l,; 0.1 g MgCl,, 6HzO; 1.16 g NazHP04, 2 Hz0 and 0.2 g KHzPO,. (b) Growth of radioactive

virus

A WSN strain of influenza virus A obtained from Dr D. W. Kingsbury, St Jude Hospital, Memphis, Tennessee, was used in all experiments. The virus was grown on chick embryo fibroblasts, which were seeded at 1.5 x 10” cells per lo-cm plastic dish and cultured for one day prior to infection (Rubin, 1960). After the medium was removed and the cells were washed with Tris-saline, the virus was added at a multiplicity of infection of about low3 in 3 ml. Tris-saline and incubated for 30 min at room temperature. Then the inoculum was removed and 6 ml. of medium 199 were added. The medium was supplemented by 0.5% lactalbumin hydrolyzate (Difco), 0.1% (w/v) glucose and 0.2 rg Fungazone/ml. (Squibb). After 16 to 18 hr at 41 “C (at this stage usually a few microplaques were already detectable), radioactive precursors were added. [5’-3H]Uridine was added directly to the medium.

NUCLEOPROTEIN

OF

INFLUENZA

48;

VIRUS

\Vhen 32P was used to label the virus, the medium was replaced by phosphate-free medium 199 supplemented as above. When [14C]amino acids were used, the medium was replaced by amino acid-free (except for glutamine) medium 199 which contained 0.1% la&albumin hydrolyzate and was otherwise supplemented as above. In typical experiments, 100 to 200 PC r3H]uridine (20 c/m-mole), 1 mc H3 32P04 or 30 @ [14C]amino acids (0.2 c/m-mole) (reconstituted protein hydrolyzate) were used per lo-cm tissue culture plate. The medium was harvested for virus purification 16 to 24 hr later, when more than 90% of the cells had become rounded and detached from the plate. The hemagglutinin titer of such medium was usually about 160 hemagglutinin units per ml. (0) virus puri,ficution This was as described previously (Duesberg, 196853, the following modification was used. Virus was incubated for 15 min at 20°C with RNase (10 pg/ml.) and 25 units of Pibrio cholerae neuraminidase (CalBiochem) prior to sedimentation to a density interface. Concentration and transfer of purified virus to another buffer was achieved by sedimentation through a 200,6 (w/v) sucrose solution to a cushion of 65% (w/v) sucrose containing the desired buffer. (d) Prepnration of a cytoplasmic extract of ing%uenza virus-infected or uninfected cells Confluent cultures of secondary chick embryo fibroblasts were prepared by seeding 2 x 10” cells on a lo-cm Petri dish (Rubin, 1960). 1 to 2 hr later the cells were washed twice with Tris-saline and infected at a multiplicity of 10 to 100 by the addition of 3 to 4 ml. of stock virus, which was either allantoic fluid of infected IO-day old chick embryos or tissue culture medium of infected cells containing lOa to 10Qinfectious particles/ml. After 30 min at 41°C, 3 ml. of medium 199 supplemented as described above were added. 3 hr after the addition of virus, 26 pg actinomycin D (a gift of Merck, Sharpe & Dohme) were added, and 30 min later the cultures were labeled with 200 pc [5’-3H]uridine for various times. The cells were then trypsinized, pelleted in a clinical centrifuge and resuspended in 0.5 ml. hypotonic buffer containing 0.1 oh Triton Xl00 and homogenized by 6 to 12 strokes in a Dounce homogenizer. After this treatment, more than 95% of the cells were broken, but the nuclei remained intact as indicated by microscopic inspection. The homogenate was centrifuged for 5 min at 600 g to remove the nuclei and the supernatant fraction was used in the experiments described.

(e) Disintegration of the virus Purified influenza in low-salt buffer was mixed at 0°C with an equal volume of 1 ye (w/v) NaDOCt (Sigma) or 1 o/o(v/v) Triton Xl00 in the same buffer and allowed to warm to 20°C over 10 to 15 min. Immediately after addition of the detergents, the opalescence of the virus solution decreased. Alternatively, the virus was disrupted by three ether extractions of a virus solution in low-salt buffer or in Tris-saline in presence or absence of 0*50/ODOC. Disruption of the virus was indicated by a loss or pronounced decrease of infectivity and a considerable reduct,ion of the 8, of radioactive components of the virus (see below).

3. Results (a)

&me

(i) L3edimentation

physical

and chemical properties products

qf the

RNA-contain~ing

viral split

in sucrose gradients

When a mixture of DOC-treated [3H]uridine-labeled influenza virus and of a cytoplasmic extract of 32P-labeled normal chicken cells is analyzed by sucrose gradient sedimentation, a distribution of radioactivity as shown in Figure l(a) is obtained. The 32P-labeled cytoplasmic extract was added to provide 50 s and 30 s ribosomal subunits (Girard, Latham, Penman & Darnell, 1965) as sedimentation markers. As seen in Figure l(a), four 3H-labeled components were discernible. The three fastt Abbreviation

used: DOC, deoxycholate.

488

P. H.

DUESBERG

.i

0

IO

20

30 Fraction

(a)

no.

(b)

FIQ. 1. Sucrose gradient sedimentation of DOC-treated influenza virus. (a) Sedimentation pattern of 100 4. [sH]uridine labeled (-@-a-) influenza virus in low-salt . 1ow-salt buffer for 10 min at 20°C buffer after incubation with an equal vol. of 1% DOG (w / v ) m with 2 pl. of cytoplasmic extract of 3aP-labeled (-A-A-) normal chick embryo fibroblasts (see Materials and Methods). Sedimentation was in a sucrose gradient 15 to 30% (W/V) containing low-salt buffer and 0.06% (w/v) DOC for 100 min at 66,000 rev./n& in a Spinco SW65 rotor at 5°C. 3-drop fractions were collected and counted in 5 ml. Bray’s (1960) scintillation fluid in a Tricarb scintillation oounter. and [3H]uridine labeled (-@-a-) (b) Sedimentation pattern of [14C]amino acid (-A-A-) influenza virus after incubation with DOC as described for (a). Unlabeled virus was added to a total hemagglutinin titer of about 500 hemagglutinin units. Radioactivity was determined by counting 50-4. samples of each fraction as described for (a). The hemagglutinin titer (- x - x -) of 25-4. samples of consecutive fractions was determined after an initial dilution in 200 pl. Dulbecoo’s buffer with 200 c;l. 0.6% (v / v ) ch ic k en red blood cells in Dulbecco’s buffer as described previously (Duesberg & Robinson, 1967).

sedimenting components were estimated by the method of Martin & Ames (1961) to have S, values of about 70, 60 and 50 s, respectively, using the ribosomal subunits as sedimentation markers. The same sedimentation pattern was obtained from virus solutions in low-salt buffer which had been disrupted by the non-ionic detergent Triton Xl00 at a final concentration of 05% (v/v) or by three consecutive extractions with three volumes of ether. However, in the case of ether-disrupted virus, pellets of up to 50% of the total radioactivity applied to the gradient were obtained if the sample contained more than about 1000 HA units in 300 ~1. solution, suggesting incomplete disruption. But disruption was virtually complete if (1000 HA units were disrupted in 300 ~1. solution, i.e. <5% of the applied radioactive virus was pelleted. In contrast, disruption of the virus by detergents was usually complete even if > 1OOOHA units were disrupted in 300 ~1. solution and less than 5% of the radioactivity in the gradient pelleted to the bottom of the tube. Similar sedimentation patterns have previously been described for the purified g-antigen of ether-disrupted fowl plaque virus (influenza A) (Schgfer & Zillig, 1954) and PR8 influenza virus (Paucker et al., 1959).

NUCLEOPROTEIN

OF

INFLUENZA

489

VIRUS

In order to test whether the three fast-sedimenting components of disrupted influenza virus contained protein in addition to RNA, a virus preparation which was labeled with [14C]amino acids and [3H]uridine was disrupted and analyzed as described for Figure l(b). The 14C-labeled viral protein had a less distinct distribution and followed only approximately the three fast-sedimenting components containing the viral [3H]RNA’s. The distribution of the viral hemagglutinin of Figure l(b) suggested that at least part of the fast-sedimenting 14C-labeled viral protein was the viral hemagglutinin. (ii) Electrophoresis in polyacrylamide gels A more obvious congruence between part of the viral 14C-labeled protein and the [3H]RNA containing components of DOGtreated influenza virus was obtained after polyacrylamide gel electrophoresis (as seen in Fig. 2(a)). Again three [3H]RNA components like those described for Figure 1 were resolved, while the distribution of 14C-labeled protein was more complex. Beside the 14C-peaks which coincided with the 3H-peaks, there were 14C-peaks with lower and some with higher electrophoretic mobilities. The 3H/14C ratios of the two major components were very similar (Fig. 2(a) and Fig. 4(a)), whereas the minor component had about a 20% lower 3H/14C ratio. The

+

0

IO

20

30

40

50

60

Distance (a)

IO

20

30

40

50

60

70

moved (mm) (b)

FIG. 2. Polyacrylamide electrophoresis of [sH]uridine (-O-O-) and [i4C]amino acidlabeled (-A--A-) influenza virus after incubation in 0.5% DOC (w/v) as described for Fig. 1, and in 1 y0 sodium dodecyl sulfate (w/v). (a) A mixture of 40 ~1. [3H]uridineand [iW]amino acid-labeled influenza in low-salt buffer (see Materials and Methods) containing about 20% sucrose was incubated with 40 ~1. low-salt buffer containing 1% (w/v) DOC for 10 min at 20%. The solution was then analyzed by electrophoresis in a 2.3% polyacrylamide gel column containing electrophoresis buffer (see Materials and Methods) for 6 hr at 55 v at room temperature as described previously (Duesberg, 19686). Subsequent to electrophoresis, the gels were frozen and sliced with a gel slicer consisting of l-mm spaced razor blades (Diversified Scientific Instruments, Stierlin Road, Mountain View, Calif.). The slices were dissolved in 50 ~1. of 1 M-piperidine and counted after the addition of 0.6 ml. NCS (Nuclear Chicago scintillation fluid) and 10 ml. toluene-based scintillation fluid in a Tricarb scintillation counter. (b) A mixture of 40 ~1. [3H]uridine and [14C]amino acid-labeled influenza virus in low-salt buffer containing about 20% sucrose was incubated with 2 pl. of 20% (w/v) sodium dodecyl sulfate for 5 min at room temperature. The solution was then analyzed by electrophoresis in a 2.30,: polyacrylamide gel column containing electrophoresis buffer and O.lO,/, (w/v) sodium dodecyl sulfate for 2 hr at 55 v and otherwise as described for (a).

490

I’.

H.

UUESUERG

nature of this difference cannot be explained from these expcrimcnts because tl~ proteins have not, been analyzed. Contrary to the effects of DOC and Triton Xl00 on the virus, a short t’reatment with 1% sodium dodecyl sulfate completely dissociated the viral 14C-labeled protein peaks from those of the viral [3H]RNA’e, as evidenced by polyacrylamide gel electrophoresis. As seen in Figure 2(b) the 13H]RNA components of sodium dodecyl sulfate-disrupted virus showed the same electrophoretic pattern as the purified viral RNA’s (Fig. 6; Duesberg, 19686), and the viral proteins were resolved into two major components, both with higher mobilities than the RNA’s. Two major protein components were also previously obtained after polyacrylamide electrophoresis of influenza virus protein in 8 M-urea at pH 4.5 (Duesberg & Robinson, 1967). In accord with a dissociation of the ribonucleoproteins into RNA and protein, the electrophoretic mobilities of both proteins and RNA’s became significantly higher after the addition of sodium dodecyl sulfate. The viral RNA’s, for example, moved about three times faster after incubation of the virus with sodium dodecyl sulfate than after incubation with DOC (compare Fig. 2(a), 6 hr at 55 v, and (b), 2 hr at 55 v). (iii) Equilibrium

density-gradient centrifugation

The buoyant density of the RNA containing components of influenza virus released by DOC was l-265 g/ml. in a sucrose density-gradient, as shown in Figure 3(a). It was higher than that of intact virus under the same conditions (1.22 to 1.23 g/ml.) (Duesberg & Robinson, 1967). There was also a peak of 14C-labeled viral protein with a density of 1.265 g/ml. (Fig. 3(a) ) which coincided with the peak of the RNA-containing components. The rest of the viral 14C-labeled protein had lower densities or possibly much lower S, values under these conditions. Part of the hemagglutinin had also the same density as the RNA-containing components. To decide whether the RNA-containing components and the hemagglutinin are associated or just happen to coincide under the conditions used, the hemagglutinin was separated from the nucleoprotein components by a modification of the method described by Schiifer & Zillig (1954). In this experiment, the same amount of 14Clabeled protein and [3H]RNA-labeled virus was used as in the previous experiment (Fig. 3(a)). The virus was disrupted with ether (Fig. 3(b)) and then incubated with chicken erythrocytes to adsorb the viral hemagglutinin. After pelleting the erythrocytes, the supernatant fraction was analyzed by sucrose density-gradient centrifugation. It can be seen in Figure 3(b) that the yield of radioactive nucleoprotein with a density of l-265 g/ml. as obtained by this method was about 20% of that described for Figure 3(a), while the recovery of viral hemagglutinin was less than 1% of that described in Figure 3(a). This experiment suggested that at least 20% of the nucleoprotein was separated from the hemagglutinin by ether and could be freed from the hemagglutinin by adsorption of the hemagglutinin to erythrocytes. Possibly the rest of the nucleoprotein remained associated with hemagglutinin and was removed by adsorption to erythrocytes or was degraded to non-sedimentable material (see below). The 3H-peak on top of the gradient in Figure 3(b) is thought to be viral RNA which was degraded or dissociated from the nucleoprotein during the incubation with ether and erythrocytes and did not sediment to equilibrium density under the conditions of the experiment. The density of the viral nucleoprotein of 1.265 g/ml. is similar to those reported previously for Tween-ether-treated influenza virus in potassium citrate (1.24 to l-26

NUCLEOPROTEIN

OF

1NFLUENZA

VIRUS

\

0

0

~

2 4 6 8 IO 12 14 16 Fraction

(a)

no.

6

\ 4, 8IO 12 14 16

-

(b)

FIG. 3. Equilibrium sucrose density-gradient centrifugation of DOC-disrupted influenza virus (a) ; and influenza virus after disruption with ether and incubation with chicken red blood cells to remove the viral hemagglutinin (b). (a) A mixture of [‘%]amino acid- (-A-A-) and [3H]uridine-labeled (-e-e--) influenza virus in 200 ~1. low-salt buffer containing 200 pg sRNA (to inhibit RNase, see text) was divided in two equal parts (A) and (B). (A) was incubated with DOC as described for Fig. 1 and then directly layered on a preformed sucrose density-gradient made of equal volumes of 20% (w/v) sucrose in low-salt buffer and 70% (w/v) sucrose in Da0 containing low-salt buffer. Centrifugation was for 16 hr at 45,000 rev./min in a Spinco SW65 rotor at 4°C. About 300-~1. fractions were collected. The solution densities (-n--n-) were determined by weighing lOO-~1. samples. The radioactivit,y was determined by precipitating lOO-~1. samples with 200 pg yeast RNA carrier in 5% trichloroacetic acid at 0% and washing the precipitates on Millipore filters. After drying at 60°C, the filter6 were counted in toluene-based scintillation fluid in a Tricarb scintillation counter. The hemagglutinin units (- x - x -) were determined after an initial dilution of 50-p]. samples of each fraction in 200 ~1. Dulbecco’s buffer as described for Fig. l(b). (b) Sample B was mixed with 1.2 ml. Tris-saline (see Materials and Methods) and then extract’ed three times with 2 vol. of ether at 6 to 10%. After complete removal of the ether by pipetting and then by reduced pressure, 60 4. of a 60% (v / v ) so 1u t’Ion of red cells in Tris-saline were added and the mixture was incubated with oocasional shaking for 5 min at 5 to 10°C. The red cells were then pelleted by centrifugation in a clinical centrifuge and the supernatant fraction was incubated twice more with 50 pl. of fresh red cells, which were no further agglutinated after the last incubation. ah The final supernatant fraction was layered on a preformed sucrose gradient and centrifuged described for sample (A).

g/ml.) (de ThB & O’Connor, 1966) and in CsC1 (1.28 g/ml.) (Rosenbaum, McCollum & Brandon, 1967). However, a direct comparison of these results with the present, experiments is difficult, because the density distribution of the viral RNA was not reported. But it was mentioned by Rosenbaum et al. (1967) that the hemagglutinin of Tween-ether-treated virus had a density of 1.28 g/ml. in CsCl. A density of l-27 g/ml. in sucrose density-gradients has previously been obtained for the helical tobacco mosaic virus (Schachman & Lauffer, 1949), which contains about 5% RNA, as well as for the helical nucleo-capsid of Sendai virus (Blair, 1968). and for the icosahedral bushy stunt virus (Cheng, 1953) which contains about 16”{, RNA. Hence the density of 1.265 g/ml. of the ribonucleoprotein of influenza virus is

492

P. H.

DUESBERG

consistent with its composition of 15% RNA and 85% protein, as determined by Schafer & Zillig (1954) and Zillig, Schafer & Ullmann, (1955). The above results indicate that about 90% of the RNA of influenza virus can be released from the virus by DOC and at least 20% by ether in the form of three distinct components in electrophoresis or sedimentation analysis as shown in Figures 1,2 and 3, all coinciding with viral protein. Their S, values are much higher than those of the free viral RNA’s, and correspondingly their electrophoretic mobilities are much lower than those of the free viral RNA’s. In addition, the RNA-protein components have a buoyant density (l-265 g/ml.) which is higher than that of the intact virus (1.23 g/ml.) but lower than that of RNA (> I.3 g/ml.). It therefore seems likely that they are identical with the g-antigen (Schafer & Zillig, 1954; Paucker et al., 1959) or ribonucleoprotein (Hoyle et al., 1961; Waterson, 1962) of influenza virus, although this has not directly been demonstrated. (b) Effects of enzymes on the ribonucleoproteins of inJluenu;c virus After a 15- to 30-minute incubation of DOC-treated influenza virus with pronase, the sedimentation pattern of the viral 14C-labeled protein components was considerably changed, whereas the sedimentation pattern of the three [3H]RNA-containing components was unchanged (Fig. 4(a)). Only the 14C-labeled protein which cosedimented with the three ribonucleoproteins retained its high S,. The rest of the protein remained on top of the gradient. This experiment indicated that there are fast-sedimenting viral proteins other than the nucleoproteins in a preparation of DOC-treated influenza virions and that these proteins are susceptible to digestion with pronase. Although pronase does not cause any detectable alteration of the nucleoprotein under the conditions described (Fig. 4(a)) (20°C for 10 min), prolonged incubation under the same conditions or at elevated temperatures caused degradation of the nucleoprotein to more slowly sedimenting heterogeneous products. Most of the viral hemagglutinin sedimented faster than the nucleoprotein before the pronase treatment (Fig. l(b)), which agrees with the observation of Schafer & Zillig (1954) that the hemagglutinin has an Srp~sO value of about 100 s. After pronase digestion, however, most of the hemagglutinin was rendered nonsedimentable under the conditions of the experiment (Fig. 4(a)). The 8, values of the RNA’s of the ribonucleoproteins of influenza were found to be sensitive to RNase. Incubation with RNase at 5 pg per 100 ~1, for five minutes at 5°C in low-salt buffer containing from O-02 to 0.2 M-NaCl completely converted the S, of the RNA of the nucleoproteins from 70, 60 and 50 s to about 2 to 6 s (Fig. 4(b)). There was no difference in RNase-sensitivity between the nucleoproteins prepared by DOC, Triton Xl00 or ether extraction of the virus. The finding that even the S,values of the RNA of ether-released nucleoproteins were changed by RNase suggests that the RNase-sensitivity was not the result of an alteration of the original ribonucleoprotein by the detergent treatment. This observation is consistent with the results of Schafer & Wecker (1958), who found that the RNA of the nucleoprotein of fowl plague (influenza A) virus could be digested by RNase although at least some antigenicity was retained. The RNase-sensitivity of the nucleoprotein may also explain the previously noted tenfold reduction of the complement fixation titer of the gantigen of fowl plague virus, which was thought to be the result of a complex formation between RNase and ribonucleoprotein rather than enzymic degradation (Zillig et al. 1955).

i’OO0 IL NUCLEOPROTEIN

OF

Pronase

40

INFLUENZA

Rriu:,r

4000

20

1

2000

1

493

VIRUS

-----I 1 RNose , Pronose

- 3oc

-?OO

v) .; II) .L? 5 '; w ;il 3:

-103

:“ -0 Fraction

((1)

no. Cd

FIQ. 4. Sucrose gradient sedimentation of DOC-treated influenza virus after incubation with pronese (a), RNase (b) and pronase following RNase (c). A mixture of [“C]amino acid (-A-A-) and [3H]uridine-labeled (-O-e-) influenza, virus was divided into 3 equal 160-g. parts A, B and C, after the addition of DOG aa described for Fig. 1. (E) Sample A was incubated for 20 min at 20°C with 20 pg pronase, (b) B was incubated for 20 min at 20°C with 20 pg RNase, (c) C was incubated for 10 min with 20 pg RN&se and subsequently for 10 min with 20 pg pronase. Each sample was then analyzed by sucrose gradient sedimentation as described for Fig. 1. Hemagglutinin units ( x - x ) were determined as described for Fig. 1.

The sedimentation pattern of the proteins of DOC-treated virus, however, was not significantly changed after incubation with RNase (Fig. 4(b)), and resembled closely that of the virus treated only with DOC (Fig. l(b)). In contrast, treatment of DOC-disrupted influenza virus with RNase followed by pronase completely degraded all fast-sedimenting viral RNA and proteins (Fig. 4(c)). These experiments suggest that the protein of the viral nucleoproteins was protected against proteolytic degradation by virtue of its association with the viral RNA, and was rendered pronase-sensitive after digestion of the RNA. The question whether t,he fast-sedimenting protein after RNase digestion of DOC-disrupted influenza virus (Fig. 4(b)) is an RNA-free protein “core” of the viral nucleoprotein or a mixture of it, with other viral proteins, cannot be answered from the experiments described here, because the viral proteins have not been analyzed. It seems likely, however, that protein of the viral nucleoproteins retains a high S, value after RNase digestion, because approximately the same amount of l*C-labeled protein remains in the sucrose gradient in the position of the viral nucleoproteins after RNase digestion (Fig. 4(b)) as after pronase digestion (Fig. 4(a)), or after direct sedimentation of DOGtreated virus (not shown).

I’.

404

H.

(c) Intracellular (i) Comparison

DUESBERG

ribonucleoprolein

with the n~ucleoprotein of mature virus

It has been shown that the g-antigen of the influenza virus appears as early as two to three hours after infection (Schilfer, 1963), about two to three hours before the appearance of mature virus in the medium. Similarly, the rate of synthesis of intracellular viral single-stranded and double-stranded RNA was found to be a maximum at about three hours (Schiifer, 1963; Duesberg, 1968b). An attempt was, therefore, made to isolate nucleoprotein of influenza virus from the infected cell prior to its processing into mature virus, in order to find out whether the heterogeneity of the nucleoprotein of mature virus was primary or a secondary product of a late intraFor this experiment, freshly seeded chick cellular or extracellular degradation. fibroblasts were infected one hour after plating at a multiplicity of 10 to 100, and three to four hours later actinomycin D and 25 minutes later [3H]uridine were added, and the culture was incubated for one hour (Materials and Methods). It was previously shown that only viral RNA is labeled under these conditions (Duesberg & Robinson, 1967). A cytoplasmic extract of these cells was then prepared (see Materials and Methods) and mixed with DOC-disrupted 32P-labeled virus and analyzed The distribution of the viral intracellular by sucrose-gradient centrifugation. of mature virus [3H]uridine-labeled components and the 32P-labeled nucleoproteins were found to be very similar (E’ig. 5). The fast-sedimenting material of the cell showed the same heterogeneity as the nucleoproteins of mature virus; however, t’he intracellular material appeared to sediment slightly slower than the 32P-labeled components of mature virus. Despite this minor difference in the S, values, isolation and analysis of the RNA of the pooled fast-sedimenting intracellular 3H-labeled ribonucleoproteins (fractions 8 to 20, Fig. 5) showed the typical patterns for influenza virus RNA’s in sucrose gradients or polyacrylamide gels. This experiment, suggests that the relatively small size and heterogeneity of the influenza virus nucleoprotein already exist during the eclipse period in the infected cell. (ii) Kinetics

of the formation

of the nucleoprotein

The rate of incorporation of the newly synthesized viral RNA’s into the components of the nucleoprotein was studied by pulse-labeling of infected cells three hours after infection. It was found that after pulses as short as five minutes, the sedimentation patterns of the cytoplasmic extracts were almost identical to that shown in Figure 5, except for a smaller amount of incorporated [3H]uridine, and no faster-sedimenting possible precursor nucleoproteins were seen after similar and shorter periods of centrifugation. Shorter pulses were not practical, because too little [3H]uridine was incorporated for analysis of the products. This result indicates that the newly synthesized viral RNA is very rapidly incorporated into the viral nucleoproteins and makes the existence of a large precursor nucleoprotein unlikely. (d) Distribution

of the in$uenza

virus RNA’s in the three compon,ents of thP viral nucleoprotein

The following experiment was carried out to decide whether the hydrodynamic and electrophoretic heterogeneity of the three viral nucleoproteins is the result of various types of aggregation of a common minor subunit which might contain all of the viral RNA’s, or of an artificial degradation of one large fragile nucleoprotein as suggested by

NUCLEOPROTEIN

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INFLUENZA

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Fraction no FIG. 5. Cosedimentation of intracellular influenza virus-specific [3H]uridine-labeled components 32P-labeled influenza virus (--A-A-). (-a-@-) and DOG-released nucleoproteins of mature A cytoplasmic extract of influenza virus-infected chick embryo fibroblasts which had been labeled with [3H]uridine in the presence of actinomycin D from 3.5 to 4.5 hr after infection was prepared (see Materials and Methods). After the addition of EDTA to 0.05 M, 300 ~1. of the cytoplasmic extract containing 15,000 cts/min of trichloroacetic acid-precipitable 3H-labeled material were mixed with 20 pl. of a solution of about 80,000 cts/min of 32P-labeled influenza virus in low-salt buffer containing 0.5% (w/v) DOC. The solution was then layered on a 17-ml. sucrose for Fig. 1 and centrifuged for 18 hr at 25,000 rev./min in gradient (15 to 3096, w/v) as described an SW25.3 Spinco rotor at 4%. After centrifugation, S-drop fractions were collected. After determination of the absorbancy at 260 rnp (-O-c-), the trichloroacotic acid-precipitable radioactive material of each fraction was determined as described in Fig. 3.

Hoyle et al. (1961), Schgfer (1963) and Cruickshank (1964) which might contain one la,rge hypothetical RNA, or whether they are due to distinct pre-existing nucleoproteins containing each one distinct component of the recently described distinct viral RNA’s (Duesberg, 1968b). The nucleoprotein of 32P-labeled influenza virus was fractionated by sucrose gradient sedimentation as described for Figure 1. Fractions comprising the 70 s component and fractions comprising about half of t,he 60 s and all of the 50 s component of the viral nucleoprotein were pooled and their RNA extracted by the phenol-sodium dodecyl sulfate method (Duesberg, 19686). The [32P]RNA of each pool was then mixed with [3H]RNA of unfractionated virus and analyzed by polyacrylamide gel elect’rophoresis. The results are shown in Figure 6. It can be seen that the fast-sedimenting (70 s) nucleoprotein contained mainly th(l largest viral RNA component (i.e. with the lowest electrophoretic mobility). Some of the intermediate viral RNA’s were also present at a relatively lower ratio than in intact virus. They might originate from contamination of the 70 s component with the 60 s component or from breakage of t*he RNA of the 70 s component. The more slowly sedimenting (60 and 50 s) components of the nucleoprotein contained the small viral RNA’s with the higher electrophoretic mobilities. This indicates t’hat the RNA’s of the nucleoprotein components are probably not degra’ded, or at least not 33

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more degraded, than RNA’s directly isolated from infectious virus. This result furthe suggests that the hydrodynamic and electrophoretic differences of the DOC-released viral ribonucleoproteins reflect the differences of the sizes of the viral RNA’s which they contain.

Distance

moved (mm) lb)

(a)

FIG. 6. Co-electrophoresis of [32P]RNA (-A--A--) of the 70 s (a) and the 80 and 50 s (b) components of the influenza virus nucleoprotein with [3H]RNA (-a-@-) of unfractionated virus. (a) Sucrose gradient fractions containing the 70 s component of DOC-treated aZP-labeled influenza virus were pooled (see Fig. 1) and the RNA was isolated by the phenol-sodium dodecyl sulfate method (Duesberg, 19685) in the presence of 30 pg sRNA carrier. After 2 alcohol precipitations, the RNA was dissolved in 30 ~1. electrophoresis sample buffer (Duesberg, 1968b) containing 0.2% sodium dodecyl sulfate and 10% glycerol and mixed with 20 pl. of [3H]uridine-labeled influenza virus which had been digested for 15 min at 37% in the same buffer containing 0.2% sodium dodecyl sulfate and 1 mg pronase per ml. Electrophoresis was for 2 hr at 60 v at room temperature in a 2.2% polyaorylamide gel column containing electrophoresis buffer and 0.1% sodium dodecyl sulfate (w/v) as described previously (Duesberg, 19683). Gel slicing and determination of radioactivity were as described for Fig. 2. (b) Co-electrophoresis of the [3aP]RNA of part of the 60 s and all of the 50 s component of DOG-treated 3aP-labeled influenza virus with [aH]RNA of unfractionated influenza virus as described for (a).

4. Discussion (a) Form of the nucleoprotein

Ribonucleoprotein of influenza virus A exists in several distinct subunits. This finding is compatible with the notion that influenza virus RNA also occurs in several distinct pieces (Duesberg & Robinson, 1967; Pons & Hirst, 1968; Duesberg. 19683). Also it may explain the failure to detect in influenza virus a single large nucleoprotein such as those detected in various parainfluenza viruses, and it may explain the antigenic heterogeneity of DOC-treated influenza virus in the Ouchterlony diffusion test (Styk & Hana, 1968). The possibility that the heterogeneity of the nucleoprotein is due to aggregation of a single minor subunit or degradation of a single large unit is unlikely for the following reasons: (a) the same number and relative proportions of nucleoprotein components are reproducibly obtained from the virus after various methods of isolation and from the infected cell during the eclipse period ; (b) B’-pulselabeled intracellular nucleoprotein shows the same heterogeneity as that of mature virus, which makes the existence of a single large nucleoprotein precursor unlikely;

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(c) the S, values of the nucleoprotein components are unaffected by detergents like Triton Xl00 and DOC, by ether and by EDTA; (d) distinct components of the nucleoprotein contain distinct viral RNA’s, which are indistinguishable from components of the viral RNA obtained directly by the phenol-sodium dodecyl sulfate method from infectious virus. In addition, if distinct nucleoproteins were the result of specific breakage of a single large RNA of a large hypothetical nucleoprotein, the same distinct pieces of RNA would not also be expected to be present in infectious virus and in virus-infected cells (Duesberg, 1968b). Although it is conceivable that shear forces might break the RNA of a large nucleoprotein, it is unlikely that these breakage points would be the same as the putative breakage points of the viral RNA obtained by phenol extraction of the virus or the virus-infected cell. The possibility that distinct nucleoproteins of influenza virus might originate from a mixture of mutually dependent viruses present in roughly equal proportions is also not probable, for the following reasons. (a) A linear dependence between plaque count and virus dilution has been described for influenza virus (Choppin, 1962). This is evidence that a single particle is sufficient to initiate infection. (b) The ultraviolet inactivation of plaque-purified influenza virus was found to follow first-order kinetics up to a 103-fold inactivation (Tumova & Pereira, 1965), which is compatible with a single particle being the infectious unit. At higher degrees of ultraviolet inactivation, however, a lower rate of inactivation was observed, which is presumably the result of cross-reactivation of several partially inactivated virus particles infecting the same cell. Accordingly, the y-ray inactivation of influenza virus was shown to follow a single-hit inactivation curve over the first three orders of magnitude (Yoshishita, 1959). Thus it is likely that the distinct nucleoproteins stem from single virus particles, although it cannot be excluded that the virus populations used are heterogeneous with respect to the number and type of ribonucleoproteins enclosed in each envelope (e.g. containing von Magnus-type influenza virus). It appears, therefore, that the nucleoproteins of the two types of myxoviruses exist in two different forms, i.e. the nucleoproteins of the parainfluenza viruses consist of single large units, whereas the nucleoprotein of influenza virus exists in several distinct small units. The question of the exact number of distinct nucleoproteins in an infectious virion is comparable to that of the number of the viral RNA’s. The present results suggest that there are at least three nucleoproteins and there are probably five RNA’s in influenza virus (Duesberg, 19683). There could be more than t.hree, possibly also five nucleoproteins in influenza virus, each containing a distinct viral RNA which was not) resolved by the methods used in the present study. This, however, is difficult to decide, because only the largest and the smallest of the viral RNA’s are completely resolved by polyacrylamide electrophoresis, whereas the RNA’s of intermediate size are incompletely resolved and can, therefore, not be used for identification of unresolved nucleoproteins. At least two RNA’s and two nucleoproteins are required in order to explain t,he high recombination frequency of influenza virus (Burnet, 1956; Hirst, 1962; Simpson & Hirst, 1968). But since all stocks of influenza virus have a high ratio of physical particles to infectious particles, and neither the viral RNA’s nor the nucleoproteins have been shown to be biologically active, it is not possible to draw firm conclusions about the structure of those particles which are infectious, from biochemical studies on the total virus population. Nevertheless, despite these uncertainties, it seems very

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likely that each infectious particle contains more than one ribonucleoprotein and that the high recombination frequencies observed in influenza virus result from this. The occurrence of the influenza virus nucleoprotein in distinct subunits olfcrs a possibly unique mechanism for influenza virus recombination. It may bc that recombination does not represent (only) the exchange of distinct viral RNA’s in the replicating pool of the cell, but may occur by exchange of complete subunits of the viral nucleoprotein. (b) Structure of the nucleoprotein Distinct properties of the nucleoprotein of influenza virus indicate that its structure differs from that of nucleoproteins of parainfluenza viruses. For example, the RNasesensitivity of the influenza virus ribonucleoproteins is in contrast to t’he complete RNase-resistance of the nucleoproteins isolated from parainfluenza viruses (Compans & Choppin, 1968; Blair, 1968; Hosaka, 1968). The RNase-sensitivity of the nucleoprotein of influenza virus suggests that the RNA is an outer structural component of it which can function in protecting the protein core against proteolytic enzymes. Likewise, it was found that the RNA in the influenza virus particle is accessible to whereas the RNA of parainflaenza hydroxylamine like the RNA of poliovirus, viruses (Newcastle disease, mumps), like that of tobacco mosaic virus, is not accessible to hydroxylamine (Schafer, 1963). In addition, the diameter of the nucleoprotein of influenza virus is about 10 rnp whereas the diameter of the nucleoproteins of parainfluenza viruses is about 20 111~ (Watcrson, 1962). Further, a helical structure like that of tobacco mosaic virus has been clearly demonstrated for the nucleocapsid of several parainfluenza viruses (Cruickshank, 1964), while the structural symmetry of the ribonucleoprotein of influenza virus is still a matter of controversy (Shafer & Zillig, 1954; Hoyle et al., 1961; de The & O’Connor, 1966). Because of these ambiguities about the structure of the nucleoprotein of influenza virus, and because of its sensitivity to RNase, the term ribonucleoprotein instead of nucleocapsid was used in this report, since the nucleocapsid of viruses has been defined as being “synonymous with symmetry” and was postulated to function in protecting the viral nucleic acid (Lwoff, Horne & Tournier, 1962). I thank Drs W. M. Stanley and H. Rubin for encouragement and support, and Drs M. Halpern and G. S. Martin for review of the manuscript. This investigation was supported by Public Health Service research grant AI01267 from the National Institute of Allergy and Infectious Diseases; and grants CA04774, CA11426, CA05619, and training grant CA05028 from the National Cancer Institute, U.S. Public Health Service. Note added in proof: obtained

Results by Dr D. W. Kingsbury

similar to those reported (personal communication).

in this

paper

were

REFERENCES Blair, C. D. (1968). Ph.D. Thesis, University of California, Berkeley. Blair, C. D. & Robinson, W. S. (1968). Virology, 35, 537. Bray, S. A. (1960). Analyt. Biochem. 1, 279. Burnet, F. M. (1956). Xcience, 123, 1101. Cheng, P. Y. (1953). Ph.D. Thesis, University of California, Berkeley, Calif. Choppin, P. W. (1962). V+oZogy, 18, 332. Compans, R. W. & Choppin, P. W. (1967). Proc. Nat. Acad. Sci., Wash. 57, 949. Compans, R. W. & Choppin, P. W. (1968). Virology, 35, 289.

recently

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Cruickshank, F. G. (1964). In Cellular Biology of Myxovirus Infections, ed. by G. E. LT. Wolstenholme & J. Knight, p. 5. Boston: Little, Brown & Co. Duesberg, P. H. (1968a). Proc. Nut. Acad. Sci., Wash. 60, 1511. Duesberg, P. H. (19686). Proc. Nut. Acad. Sci., Wash. 59, 930. Duesberg, P. H. & Robinson, W. S. (1965). Proc. Nat. Acad. Sci., WCZ-?~.54, 794. Duesberg, P. H. &. Robinson, W. S. (1967). J. Mol. Biol. 25, 383. Frisch-Niggemeyer, W. (1956). Nature, 178, 307. Girard, M., Latham, H., Penman, S. & Darnell, J. E. (1965). J. &1oZ. Biol. 11, 187. Hirst, G. K. (1962). Cold Spr. Harb. Symp. Quay&t. Biol. 27, 303. Hosaka, Y. (1968). Virology, 35, 445. Hosuka, Y., Hosakawa, Y. 8: Fukai, K. (1959). B&en’s J. 2, 367. Hosaka, Y. & Shimizu, K. (1968). J. Mol. Biol. 35, 369. Hoyle, L. (1952). J. Hyg. 50, 229. Hoyle, L., Horne. R. W. & Waterson, A. P. (1961). Virology, 13, 448. Kingsbury, D. W. & Darlington, R. W. (1968). J. Virology, 2, 248. Laver, W. S. (1963). Virology, 20, 251. Lwoff, A., Horne, R. & Tournier, P. (1962). Cold Spr. Hurb. Symp. Quant. BioZ. 27, 51. Martin, R. G. & Ames, B. N. (1961). J. BioZ. Chem. 236, 1372. Mizutani, H. S: Mizutani, H. (1965). Virology, 26, 761. Paucker, K., Birch-Anderson, A. & von Magnus, P. (1959). Virology, 8, 1. Pons, M. W. It. Hirst, G. K. (1968). Virology, 34, 385. Robinson, \V. S. S: Duesberg, P. H. (1968). In Molecular Basis of Virology, ed. by H. Fraenkel-Conrat, p. 255. New York: Reinhold Book Corp. Rosenbaum, M., McCollum, C. & Brandon, F. B. (1967). Bact. Proc. 104, 151. Rott, R. & Schafer, W. (1964). In Cellular Biology of Myxovirus Infections, ed. by G. E. W. Wolstenholme & J. Knight, p. 27. Boston: Little, Brown & Co. Rubin, H. (1960). Proc. Nat. Acad. Sci., Wash. 46, 1105. Schachman, H. K. & Lauffer, M. A. (1949). J. Amer. Chem. Sot. 71, 536. Schafer, W. (1963). Bact. Rev. 27, 1. Schafer, W. &. Wecker, E. (1958). Arch. Ezp. Vet. Med. 12, 418. Schafer, W. & Zillig, W. (1954). 2. Nuturf. 96, 779. Simpson, R. & Hirst, G. (1968). Virology, 35, 41. Styk, B. & Hana, L. (1968). Acta Viral. 12, 203. de The, G. & O’Connor, T. E. (1966). Virology, 28, 713. Tumova, B. &; Pereira, H. G. (1965). Virology, 27, 253. VVatorson, A. I?. (1962). Nature, 193, 1163. Yoshishita, T. (1959). B&en’s J. 1, 151. Zillig, W., Schafer, W. & Ullmann, S. (1955). 2. Naturf. 106, 199.