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Studies on the pleomorphism of HVJ virions
VIROLOGY
29,
205-221 (1966)
Studies
on the Pleomorphism
YASUHIRO L)epal’tment
HOSAKA
ASD
of HVJ’ Virions
HIDEKO
KITANO
of Preventive Medicine, Research Znslitute for ~Jioobial Osaka University, Osaka, Japan AND
I’he Radiation
SAKIKO
Diseases,
IKEGUCHI
Center of Osaka Prefecture, Accepted February
Osaka, Japan
23, 1966
Partially purified preparations of HVJ were fractionated by zone centrifugation in sucrose gradient,s, and several properties of the separated classes of virions were compared. The most slowly sedimenting band of HVJ consists of the smallest virions, which contain the shortest nucleocapsids, enclosed by the smallest envelopes. The lengths of the nucleocapsids distribute regularly. The minimum length associated with infectivity is 1.0 ,,L This nucleocapsid is designated the mononucleocapsid: its multiples are called polynucleocapsids. The maximum length of polynucleocapsids was more than 20 +. Usually the unfractionated viral population contains in the highest proportion the virions with mononucleocapsids. The separated classes of virions are all infectious. Their progenies are again heterogeneous. Experiments on inactivation of HVJ virions with polynucleocapsids by Co@ show that they contain multiply infectious units. The pleomorphism of HVJ virions can be explained in terms of heterogeneity in length and flexible conformation of nucleocapsids, enclosed by flexible envelopes. MATERIALS
INTRODUCTION
AND METHODS
The morphology of large myxoviruses Virus. HVJ, strain 2, was used. Dilutions has been extensively studied. They consist of 10m2of infected chorioallantoic fluids were of an outer envelope and an inner heIica1 inoculated into the chorioallantoic cavity of nucleocapsid 180 A wide (Horne and Waterlo-day-old embryonated eggs; after 72 son, 1960; Horne et al., 1960; Hosaka et al., hours’ incubation at 35” the infected chorio1961; Waterson et al., 1961; Rott and SchSi- allantoic fluids were harvested. fer, 1961; Hermodsson and Westman, 1962; The infected fluids were centrifuged at Choppin and Stoeckenius, 1964; Cruick2500 rpm for 20 minutes, and then the shank, 1964). Little is known about the supernatants were centrifuged at 20,000 capsid length or the cause of pleomorphism. rpm for 30 minutes. The resulting pellets We studied the pleomorphism, employing were resuspended by pipetting in 0.15 M HVJ, a member of the parainfluenza 1 group. NaCl + 0.015 M sodium citrate (SSC) and We fractionated HVJ virions by sucrose th en centrifuged at 2000 rpm for 15 minutes. gradient centrifugation and compared the Th e resulting superna,tant was used as puriproperties of the fractions obtained. The fied HVJ results showed that the separated classes of Fractionation of HVJ by sucrose gradient virions differ in their construction. centrifugation. Sucrose was dissolved in SSC to give 15, lS, 22, 26, and 30 % (w/v) * Hemagglutinating virus of Japan, synonym: solutions, respectively. Volumes of 5 ml of Serldai virlls. 205
206
HOSAKA,
KITANO
each solution were overlayered in order from the bottom of the tube. The purified HVJ was layered on the sucrose gradient, and the tubes were centrifuged at 15,000 rpm for 50 minutes in a SW 25 rotor. The main band was removed carefully with a l-ml syringe; it was designated band 1 (Bl). The diffuse band below the main band was also removed and called band B2” (B2,); the pellets were called band 4 (B4). Bl and B2, were diluted and centrifuged at 25,000 rpm for 30 minutes. The resulting pelletIs and B4 were resuspended in a small volume of SSC and centrifuged at 2000 rpm for 10 minutes. The supernatants were further centrifuged in sucrose gradients. All bands were purified by a total of three cycles of zone centrifugation in 15-30% sucrose. B20 was further divided into two bands in the second zone centrifugation; the upper and the lower bands were called B2 and B3, respectively. These bands were purified once more by zone centrifugation. Pig. 1 shows the bands of fractions Bl, B2, and B3 purified in this way. Infectivity assay. Aliquots of 0.2 ml of tenfold serial dilutions of HVJ in broth were inoculated into the chorioallantoic cavity of four lo-day-old embryonated eggs. After 3 days of incubation, the chorioallantoic fluids were harvested and tested for hemagglutinin production. The infectivity of HV.J was expressed as the 50 % egg infectivity dose (EIDSO). Hwmgglutinatzon titration. Salk’s pattern method (Salk, 1944) was employed. Aliquots of 0.5 ml of twofold serial dilu-
FIG. 1. Bands of the virions of fraction Bl (left), B2 (middle), and B3 (right) on centrifugation in 15-30’30 sucrose gradients at 15,000 rpm for 50 minlltes in an SW 25 rotor. Fraction Bl sediments slowly, B3 rapidly, and B2 intermediatel~~. Fraction H4 sediments to the bottom.
AND
IKEGUCHI
tions in PBS (phosphate-buffered saline, pH 7.2) were mixed with 0.5 ml of 0.5% fowl red cells in PBS. The hemagglutination titer is expressed as the reciprocal of the final dilution which agglutinated the red cells after 2 hours’ incubation in hhe cold. Assay of hemolytic activity. One milliliter of HVJ was mixed with 2 ml of 2 % fowl red cells in PBS. The mixtures were incubated at 37” for 60 minutes and then centrifuged at 2000 rpm for 5 minutes. The hemoglobin liberated into the supernatant was measured spectrophotometrically at 540 mp and expressed as the optical density. Assay of fusion activity. Ehrlich’s ascites tumor cells (ETC) were washed twice with glucose-free Hanks’ solution by centrifugation at 500 rpm for 3 minutes and suspended in Hanks’ solution at a concentration of 5 volume %. Aliquots of 0.75 ml of 5% ETC were mixed with 0.25 ml of HVJ; the mixturcs were shaken at 37” for 60 minutes. After incubation, the number of cells in each mixture was measured. The fusion activity of HVJ was det’ermined by the decrease in the cell number by fusion and expressed as the fusion index (FI) (Okada and Tadokoro, 1962). Chemical analysis. Protein content was measured by the method of Lowry et al. (1951), using bovine serum albumin as a standard. RNA was extracted by Ogur and Rosen’s (1950) method and estimated by t.he orcinol reaction, using yeast, RNA as a standard. Phospholipid in the acid-insoluble material of HVJ was extracted twice with ether-ethanol (1:3) at 50”, and the phosphorus content of the combined extracts was determined. Electron microscopy. HVJ suspended in SSC was centrifuged at 20,000 rpm for 30 minutes and the pellet was resuspended by gently shaking in a small volume of distilled water. The virus suspension was mixed with an equal volume of 2 % phosphotungstic acid (pH 7.2) (Brenner and Horne, 1959). Then the mixt,ure was placed 011 collodioncoated microgrids and examined in a Hitachi HUl IA t’ype clcctron microscope. E’stimation of the length of the nucleocapsid of HVJ. The length of the nucleocapsid within a virion was estimated as t,he total length of nuclcocapsid meaurable wit,hin the
PLEOMORPHISM
virion negatively stained. The total lengths of the nucleocapsids in relatively large virions into which phosphot’ungstic acid penetrates diffusely are measured easily.
OF HVJ
VIRIONS
207
However, usually little phosphotungstic acid penetrates into small HVJ virions. But when the virions are suspended in distilled water containing a small volume of SSC
FIG. 2. Morphology of negatively stained virions in fract,ion Ul. The phosphotungstic acid penetrates inta) the hollow tubes of the helical nucleocapsids within the virions, forming winding black belts, when HJ\- virions are suspended iu distilled water containing a small volume of SSC and then mixed with 2% phosphotungstic acid. The fine belts of the nucleocapsids are of a relatively uniform and continuous length. Most of the virions with a relatively circular outline are between 180 and 250 mr in diameter.
208
HOSAKA, KITANC)
and then mixed with phosphotungstic acid, the acid often penetrates into the hollow tube of the helical nucleocspsid, forming :L winding black belt, as shown in Fig. 2. The length of t’he nucleocapsid within the virion was measured by a map meter on photographs at, magnifications of 160,000 or 240,000. There was the possibilit’y t#hat only part of the nucleocapsid was visible and measurable within negatively stained virions. In order to examine whether the lengths of the nucleocapsids measured within negatively stained virions represent the real lengths of the nucleocapsidx, t#he measured lengths were compared with those of free nucleocapsids liberated from the virions. Ether-emasol treatment of HVJ (Hosaka et al., 1959) disrupts the HVJ virions and liberates the inner nucleocapsids, but most
FIG. 3. Negatively stained nucleocnpsids capsids have a uniform size of 1 .O p.
liberated
ANI) IKEGUXI
of the nucleocapsids are shortscrIed by this treatment. Therefore another method was employed. It was found t’hat, Athough HVJ virions retain their infectivity around pH 10 in K:LHCO:~-~ Ka2(:03 (0.02 Al), their envelopes become fragile. When HVJ virions are left suspended in the alkali for a few days, a small number of nucleocupsids are liberated (Fig. 3). The distribution of the lengths of the free nucleocapsids t,hus liberated is shown in Fig. 4. The peak at 1.0 p agrees with the main peak at I .O fi in t’he distribut,ion (Fig. 12) of the lengths of t’he nucleocapsids measured within unfractionated virions ncgat8ively stained. Therefore the lengths of the nucxleocnpsids measured within negatively st’ained virions are reliable. y-Ray irradiation. HVJ suspended in SSC was diluted ten times with broth and
from HVJ virions
in alkali
solution.
hIany ~~uc:leo-
PLEOMORPHISM Number
20.
~‘JG. 4. 1)istribution of the lengths of free notclee bcapsids liber:rt,ed from HV.J virions suspended in :rlkali. There is a sharp peak at 1.0 p.
irradiated with CoGo. -At int’ervals during irr:ldiation, aliyuots of HVJ were removed ant 1 their infect,ivity was measured. RESGLTS
Xi;es of Fraction.ated I’irions Sedimentation patfern. Fraction Bl has only a single sharp peak with a sedimentation constant of 1000 S. Fraction B2 has a main peak of 1600 S and two accessory peaks of 1100 S and 2200 S. Fraction B3 has a diffuse peak of 2000 S and a small peak with a lower sedimentation constant. When fralations 132 and B3 were further centrifuged in sucrose gradients and removed from finer zones of the bands, the accessory peaks decreased greatly, but the recoveries were also smaller. Icraction B4 does not show a peak, but only a diffusely sedimenting boundary of about 4000 S with a shaded area, possibly due to large particles of HVJ virions, shown in (Llectron microscopy (Fig. 7). Electron microscopy. Figures 2, 5, 6, and 7 are photographs of fraction Bl, B2, B3, and B4 respectively. Almost all the virions in fract’ion B4 but few of the virions in fraction Bl have been penetrated diffusely with phwphotungstic acid. The number of virions penetrated diffusely with phosphotungstic acid decreases in order from fract’ion B4 to Bl. The sizes of the HVJ virions increase from fraction Bl to B4. Since there are various shapes of virions, it is dificult to express exact distributions of sizes of the virions in the fractions. Therefore the cliamet)ers of the
OF 1IV.J WRIONS
209
virions showing more or less circular outlines in each fraction were roughly compared. The diameters of most of the virions in fraction Bl are 160-250 mp. In fraction B2, many virions have diameters of 250-340 rnp, but t,here are also virions with smaller diameters of 200-230 mp. In fraction 133 many virions have diameters of 360-480 111,~~ but a considerable number of virions are smaller (diameters of 200-340 nij~). The diamct,ers of the virions in fraction B4 have a wide range from 200 to 800 nip; virions with diameters of 400-600 nip are predominant. The distribution pattern of each fraction seemed to be roughly compatible with t,he distribution pattern (Fig. 8) in t.hc nucleocapsid length, respectively. l+ope~~ties of Nucleocapsids of Fractionated 17irions Lengths of nucleocapsids within the visions. The lengths of t,he nucleocapsids within negatively stained, fractionated virions were estimated using more than 100 virions from each fraction. E’igure 8 shows their distributions. Fraction Bl has a peak around 1.0 ,.L. This broad peak was formed partly because data were obtained from different observations on different samples and partly because of variations in t’he measurements, parts of the nucleocapsids within some virions being obscured. The peak probably represent’s the presence of many nucleocapsids of a uniform length of 1.0 p. This interpretation agrees with t,he presence of a sharp peak at, 1.0 p in the distribution of the lengths of free nucleocapsids (Fig. 3) and also with the occurrence of a single, sharp peak in the sedimentation pattern of fraction Bl. The comparison of t’he nucleocapsid length wit’h the sedimentation pattern of fraction Bl leads to a conclusion that 1000 S virions of HVJ contain nucleocapsids with a uniform length of 1.0 p. Frwtion B2 has a main peak around 2.0 p and two smaller peaks around 1.0 and 3.0 ~1. Fract’ion B3 has a main broad peak around 3.0 p and two peaks around 2.0 and 1.0 ~1 These peaks are much broader than that in fraction Bl. The width of the peak in the distribution is t’hought to be more when the nucleocapsid is longer, for the reasons given above.
210
FIG. 5. Morphology outlines are between some virions.
HOSAKA,
KITANO
of negatively stained virions 200 and 340 rnp in diameter.
From comparisons of t’hese distributions with their sedimentation patterns, it is concluded that 1600 S and 2000 S virions contain nucleocapsids of uniform lengths of 2.0 and 3.0 p, respectively. The diffuseness of the peaks of 1600 S and 2000 S is thought to be due to variation in the shape of virions with uniform constitutions or due to slightly different sizes of envelopes enclosing nucleocapsids of uniform lengths.
AN11 IKEG
1JC 11I
of fraction B2. Many virions with relat,ively circular Phosphotungst,ic acid has penetrated diffusely into
It is difficult to remove completely the accessory peaks of fractions B2 and B3 in the length distribution under the present experimental conditions. The factors contributing to the occurrence of these accessory peaks may be (1) simple contamination with virions of other fractions during isolation of these fractions by syringe from bands in the sucrose gradients, (2) the presence of aggregates of smaller virions, (3) the pos-
FIG. 6. Morphology of negat’ively stctilled vii-ions of fr:tci,ioll lar outlines are between 400 and 600 mp in size. Phosphotlmgstic siderable Ilumber of virions.
sible degradation of virions with long nucleocapsids into virions with nucleocapsids of shorter lengt’h during resuspension and during the preparation of negatively stained specimens for electron microscopy. Such degradation readily occurs during sonication of virions, as will be reported in a forthcoming paper. Fraction B4 has no peak in the distribution of nucleocapsid lengths and presents a wide distribution from 1 p to more than 10 p. There is a tendency to show periodic
B3. Man)- virions with relatively circulacid has pcuetrated diEusely into a con-
distributions at intervals of 1.0 ~1, within the range of shorter nucleocapsids. These findings indicat’e that t.he lengths of the nucleocapsids vary on the basis of units of 1.0 p lengt,h. It remains to be determined whether there are a few virions with nucleocapsids which do not have int’egral unit lengths. It is thought that the 1.0 ,.Llength of the nucleocapsids is t#he unit which endows the HVJ virion with infectivity, because under the present experimental condit’ions (1) it
HOSAKA,
FIG. 7. Morphology of llegutively from 200 to 800 rnp. Phosphotungstic
KITANO
AND
IKEGUCHI
stained virions of fractiwl 134. The virions vary acid has penetrated diffusely iuto most of them.
represents a sharp peak of minimum nucleocapsid length within virions, associated with infectivity; (2) it represent’s the first value of serial integral units of peaks observed in the distribut,ion of nucleocapsid lengths; (3) the inactivat’ion curve of the virions containing the nucleocapsids of 1.0 P length, in fraction Bl, by Co60 suggests that, they contain one infectious lmit (Fig. 11); (4) it is found in the majority of unfractJionat8ed HVJ virions and usually has t,he highest I : H ratio; (5) virions of shorter nucleocapsid
widely
in size
lengt#h can be produced artificially by sonication but have a low 1:H ratio. The nucleocapsid of 1.0 IL length could be called the mononucleocapsid of HVJ, and its multiples the polynuoleocapsids. The maximum length of polynucleocapsids observed was more than 20 ,.L. Virions with mono-, di-, tri-, tet’ra-, possibly octa- and possibly dodecanucleocapsids are shown in Fig. 9a, b, c, d, c, and f, respectively. Generally the larger virion contains the longer nucleocapsid.
PLEOMORPHISM
OF HVJ
VIRIONS
213
Nymber
2o I
B2
FIG. 8. Distribution of the lengt,hs of nucleocapsids within virions negat,ively stained, in fractions Bl, B2, B3, and B4. Fraction Bl has a sharp peak around 1.0 p. Fraction B2 has a main peak around 2.0~ and accessory peaks around 1.0 and 3.0 p. Fraction B3 has a diffuse peak around 3.0 p and accessory peaks around 1.0 and 2.0 1. Fraction B4 has no peak and a wide distribution.
The polynucleocapsids within virions a.ppear to be continuous. Relatively few free ends of nucleocapsids have been observed within virions with polynucleocapsids. The maximum length of free nucleocapsids ever seen by us is 2.1 p, approximately corresponding to a dinucleocapsid. We have not seen a definitely circular conformat’ion of a nucleocapsid within a virion. RNA content of visions with polynucleocapsids. If one hema,gglutinating unit’ is due to a constant number of virions, irrespective of the sise of the virion, the RNA content per hemagglutinating unit should be proportional to the length of the nucleocapsid. This is the case (Table 1). The value RNA: 104 HA of fraction B2 is nearly twice that of fraction Bl, and the value of fraction B3 is 2 to 3 times larger. These ratios are
higher than those expected from the nucleo capsid lengths, shown in Fig. 8, if one assumes that the nucleocapsids are all in individual free virions with hemagglutinating activity. This discrepancy can be explained if one supposes that the factors described in the preceding section, which possibly contribute to the occurrence of the accessory peaks in the distributions of nucleocapsid lengths in fraction 52 and B3 (except the first factor) do not contribute to the formation of the accessory fra,ction in the present determination. The rat,ios of bhe protein cont’ent of each fraction per 10“ HA u&s are also shown in Table 1. The relative rat.ios for the fraction? are similar t.o those for RKA. Protein is contained in the envelope as well as in
214
HOSAKA,
KITANO
AND
IKfSC;UC:HI
FI[G. 0. a, b, c, d, c, and f represent virions with mono-, di-, t,ri-, tetrs-, possibly octn-, and possibly respectively. The virion illcreascs in size with the length of the nucleocapsid. dode canucleocapsids,
the nucleocapsid. The result!s suggest that’ POlYmucleocapsids contain not only multiple am0‘unts of (sapsid protein but also are cm close2d wit,h mrrcspondingly larger envelopes.
These considerations are based on the assumpt.ion that one hemagglutirtat,ion unit is due t.o a constant number of visions, irrespwtive of t)hc fraction.
PLEOiVO11PHIti~IOF H\‘J Wl:IONS
FIG.
Prop ties of Envelopes of Fmcfionated It that
with
i?iOTLS ‘as suggested in the previous section le longer nucleocapsids are enclosed Ile la,rger envelopes. To examine this
(,
interpretation the sizes of the enve:lope:s of fractionated virions were compared. Hemol.ylzc and cell fusing act& Ities. In Fig. 10s a.nrl b the hemolytic and ce!I1fu lsing activities, associated with the envel or=, are
1:raction Expt.
-~
No.
~-
~-~ __
1
I:BA: Ibtio
~~-
__~
WHA to Bl
..~~~~
(&
Proteill: 1O’IIA llatio to Bl
(pg)
I31 ~~~~~
B2
-~
R3-
B4
1.5 I.0
7.9 1.8
12.0 2.7
350 1 .o
090 1.9
900 2.6
s). -0 1.0
9.5 1.9
13.0 2.6
22.6 1.5
331 1.0
660 2.0
820 2.5
1370 4.1
15-M 4.4 --~ .~ --~~~-
~__ 2
RNA: Ratio
1O’lIA to Bl
(&
Protein: 10”HA Ratio to Bl
OD
HL
(~g)
FI
TABLE
FS PHOSPHOLIPID-P
2
CONTENTS
OP
E.xpt. No.
Bl,
FIIACTIONR
B2 + 3, and B4 PEH HEM.~(;QLU.L’INSTION
TITEIL
Phospholipid-P per lOa HA (pg) _ ~~__. 114 1~1 B2 + 3
_-___
1 2
9.0 8.1
3
7.3
u Value for fraction FIG. 10. Ilemolytic activities (HL) and fusing act,ivities (FS) of the virions in fractions Bl, B2, B3, and B4 versus the hemagglutination titer. Both activities, associated with the envelope, illcrease in order t,hrough fraction Bl to B4. --a-fraction Bl; -Ofraction B2; ---a--fraction B3; ---O--- fract,ion B4.
Expt. NO.
-I--l
plotted against hemagglutinating titer. The hemolytic and cell fusing activities of each fraction vary in parallel. Both activities are highest in fraction B4. Phospholipid-phosphoms content. Table 2 shows that the content of phospholipid-I? is also highest in fraction B4 and is lower in fraction B2 + 3 and Bl in that order. These results (Fig. 10 and Table 2) lead to the conclusion that the virions with the longer nucleocapsids are enclosed in larger envelopes, assuming that OIK hemagglutination unit is due to a constant number of virions, irrespective of the fractions.
B2.
TABLE R.ATIO OF INFECTIVITY: OF FRACTIONS
2
30 25
20 15 12.0"
3
HEM AGGLUTINATION TITER Bl, B2, B3, AND B4
I:raction Parameter
HA/ml I EIUJml I:11 ’ HA/ml EIUjo/ml 1:H
Infectivity
Bl
B2
B3
15,360 1011.2
2,560 10'0.0
2,500 109.7
7.1
6.0
G.3
II160,000 1011.8 G.6
12,000 lci,OOO 1(“J’O.8 1()10.3 6.3 , 0.1
B4 2,560 109.5
6.1 1,200 108.9
5.8
of Fractionated Visions
Infectivity and hemagglutinating titer as well as the ratio of the two in each fraction were determined and are shown in Table 3. The 1:H ratio ranges from 5.8 to 7.1. The 1:H ratio is highest in fraction Bl and lowest in B4. The infectivity of fraction B4
PLEOMORPHISM
is the most unstable on storage. Thus the question arises whether only Bl is infectious or dso the others. Another question is whether, if the others are infectious, they are multiply infectious. The experiment on the inactivation of infectivities with Co60 indicates that the latter is the case. Figure 11 shows t’he inactivation curves of t’he infectivities of t,hree fract,ions. Infect,ivity in fraction Bl is inactivated according t’o single-hit kinetics; decrease in infectivity in fractions B2 + 3 and B4 follows curves parallel to Bl with a shoulder, although B4 Curve with a larger shoulder.
OF IIVJ
VIRIONS
217
These findings suggest that fraction Bl is homogeneous and consists of virions with one infectious unit and that fractions B2 + 3 and B4 are heterogeneous and consist of virions with one or multiple infectious units. Progeny of Fractionated Vbions The progeny produced by limiting dilutions of each fraction has a heterogeneous distribution similar to t,hat of the original HVJ, both in sucrose gradient’s and by electron microscopy. Limiting dilutions of each band of t’he progeny again produced heterogeneous virions. Propodion oj’ I’iGms with Polynucleocapsids an Unfi~actionated Viral Populations
Figure 12 shows the distribution of the lengths of t,he nucloocapsids within about one hurldred virions in an unfractionated but partially purified population negatively stained. There is a peak around 1.0 p which tails off toward longer nucleocapsids. In the figure, 23 of the 105 virions had nucleocapsids which \vcre more than 1.8 p in length. I’hosphotungstic acid did not penetrat.e into a few of the small virions so that their lengths could not he measured. Therefore, about 20% of total HVJ virions seem to have the long nucleocapsids. This value varies somewhat from preparation to preparat’ion. Virions in unpurified allantoic fluids infected with HVJ were also observed in elecE’IG. 11. Inactivation of the infectivities of the tron microscopy. The fluids were dialyzed virions in fractions Bl, B2 + 3, and B4 with Coca. The presence of shoulders in the survival curves for 12 hours sgainst distilled water conof fractions B2 + 3 and B4 indicates that these taining SSC in a volume of 1: 10 and mixed fractions contain virions with multiple infectious wit,h phosphotungstil: acid, then observed. unit.s. -@- fraction Bl; -O- fraction B2 -+ 3; The proportion of virions with polynucleocapsids is approximately 30%, which is --:3- fract.ion B4.
N.105
10 1.0
2.0
.-%-n-I3.0
4.0
FIG. 12. Distribution of lengths of nucleocapsids within unfractionated IIVJ viriorrs, negatively stained. The distribut,ion of the nucleoeapsids has a peak around 1.0p with a t.sil toward longer nucleocapsids.
215
TTOSAK.4, KITANO
ANI)
similar t,o that in t,he purified unlfract,ion:lted sample. After furt,her banding, pelleting, and resuspending of all classes of fractionated virions, the occurrence of virions with polynucleocapsids in higher proport ion than before was never observed. These firrdiiigs indicat,e that HVJ virions with polynucleocapsids are not artifacts formed on pelleting or resuspending the virions. In negatively st,ained preparations, aggregates of individual virions arc easily det’ectcd. On pelleting and resuspending fraction HI we have never observed the appearance of 1600 S and 2000 S components. This (‘onfirms that t,hese components are not artifacts. Pleonzorphisna of HVJ Vikons Pleomorphism of HVJ has been reported by lcukumi and Nishikawa (1954), Fukai and Suzuki (lg.%), Horne et al. (1960), Hosaka et al. (1961). Naturally the heterogeneity of the lengths of the nuclcocapsids within HVJ virions contributes to the pleomorphism of t’hc virions. However even the virions cont,aining nucleocapsids of a uniform length vary in shape, as seen in
FIG. 13. Typically rllclll OLlS.
pleomorphic
forms of II\‘J
IKIS(;lTCIII
Fig. 2. Further, I:ig. 2 shows t’hat t’here are various types of winding configurations of the nucleocapsids, conforming to the shape of the virion, which reflects the shape of the enclosing envelope. Therefore the nucleocapsid must bc flexible, and the envelope is probably also flexible. In Fig. 13, typical virions (doughnuts, filamcntous, tadpole, and crescent forms) are shown. The flexible configuratjion of the nucleoaapsids is illustrated sc~hcmatically in l;ig. 14.
The present study showed that the 1000 S, 1600 S, and 2000 S virions of HVJ cont’ain nucleocapsids of 1.0 p, 2.0 p, and 3.0 p in length, respectively. Cunha et al. (1947) reported the presence of I,WO peaks of 1200 S and 1800 S in the sedimentation pattern of Newcastle disease virus (NDV), which is a large myxovirus, but t,hcy did not describe their biological significance. Schafer and Iiott (1959) found a strandform structure of 2 p length in NDV after &her 1rcatmem. Cruickshank (1964) esti-
viriolm:
:I, tloughr~ut;
b, crescent;
C, t:rclpolr;
tl, fila-
PLEOMOl:PIIIShI
OF IIVJ
219
\.II:IOSS
b
d e FIG. 14. Models of typically mclltous; c, spherical forms.
shaped viriolls
of IIYJ:
mated t)hat, the to&l length of the nucleoprotein of KDV was between 3.5 and 5.0 p. Choppin and Stoeckenius (1964) observed segments over 2 p long as internal components of SV5, another large myxovirus. If the length of the nucleooapsid of a large myxovirus is uniform, it is possible to regard the maximum length observed as approximately its exact length. However, if the length of the nucleocapsid is not uniform, the virus must be fractionated into fractions of uniform lengths to measure the exact length of t’he nucleocapsid. This has b(sen done in the present study. The polynuclcocapsids within t’he virion actually seem t,o be joined end-to-end. In tobacco mosaic virus (TMV), end-t’o-end aggregation of the nucleocapsids of freshly extracted sap of infected leaves has been report,ed (l+ancki, 1962). The present st’udy on the nucleocapsids of HV,J indicates that, some of these aggregates of TMV may be
a, clough~~ut ; k), CI‘PSC’~II~; c:, ttrclpolr;
(1, fil:~-
produced intrucellularly. In sections of Tl\ZV crystals, some of the virus rods appear to be connected linearly (Kolehmainen et al., 1965). In t’he case of HVJ, if the nuclcocapsids of HVJ arc connected linearly, then the polynucleocapsids will be enclosed in a correspondingly larger-sized envelope. Usually one nucleocapsid of a virus forms one virion. In HVJ, polynucleocapsids can form one virion. Inactivation of influenza virus with Co6” showed that, some incomplete virus of t)he van &gnus type contains multiinfectious unit’s (Yoshiehita, 1959). His data suggested that influenza virus can produce virions with polynucleocspsids. The present interpretation on the pleomorphism of HVJ virions SCCI~ t’o apply also to other pleomorphic myxoviruses. From a genetical point of view, the virion with polynucleocapsids can be regarded as a virion with homonuclcocapsids. Viriorls with heteronucleoaapsids may be produced in
220
IIOSAKA,
KITANO
ASI1
IKEGUCHI
mixed infection with closely related myxoCUNH.\, I:., WEII,, Al. L., BEMD, I)., TAYLOR, A. I(., SH:\ttt’, I). (:., and BE.\RD, J. W. (1947). viruses. We interpret, the het’erozygote of Petrification altd charact,eristirs of the NewKDV, reported by Granoff (1959, 1962) castle disease virlts (California strainj. J. Inin this way. Granoff showed that hctrroxymuno/ 55, G!)-89. gotes were produced with a high frequency FILANCIX, 1:. 1. B. (19@2). Infectivity of tobacco in mixed infections with two st,rains of iUDV mosaic virtls from tobacco leaves treat,ed with and suggested that all virions of NDV had 2-t~hiouracil. I~i~ology 17, {l-21. two genomcs and hct’crozygotes of XDV were Frl~.\r, K., nild Suzoltr, T. (1955). On t.he characproduced by random pairing of the two tcristics of a newly-isolated hemagglutinat,ing kinds of genomcs in mat’uring parl,icles. This virrls from mice. Med. J. Osaka c?niv. 6, l-15. FIX~JMI. II., and NISHJJQ\VA, F. (1954). A pneltidea was supported by the nlorphological motropic virtts from mice cattsing hemagglllobservation that the nucleocapsid of a myxotinatiott. Jupan. .I. Med. Sci. Hiol. 7, 345-353. virus appears to consist of double heliccs (Horne and Waterson, 1960; HoPaka et al., ~IL.\NOFF, A. (1959). St,ttdies OII mixed infection wit.11 Newcastle disease virtts. II. The occttr1901). However thcrc is recent morphologiretrce of iYewcastle disease virus heteroxygotes cal evidence that the nucleocapsid of HVJ aud strldy of phcllot ypic mixing involving seroconsists of :L single helix (in preparat’ion), t,ype and thcrtnal stability. l-i&jgy 9, 649%670. and Choppin and St’oeokenius (1964) rc- GILLVOFF, A. (1962). Heteroxygosis and phcnoported t,hat ihe nucleocapsid of the 8V.5 t)ypic mixitlg wit,11 Newcastle disease virrts. c’olcl Spring Harhr Sjymp. Quant. Bid. 27, 31% virus consists of a single helix. Icm%her, the 326. inactivat,ion of infectivit,y in fraction 131 $., :rl~tl WES,~M.%K, J. (1962). Illtrawith Co60 suggests that there is one genomc HIXMODSSON, strtlctttre of parainflttf~ttza 3 virtta. ,/. I:ltmstruc?. in every virion in the fraction. Thus, t.he Kes. 6, 499-510. produ&on of het’crozygotes with high frcIlons~, It. W., atld W.\.L.ERSON, A. I’. (l!XiO). B quency can be explained more reasonably helical st)rrtctrtre ilt mumps, Newcastle disease on the basis of the presenw of virionr with alld Scndai virtues. .J. Mol. Niol. 2, i5-77. heteronucleoc:Lpsids : if :L Iargc myxovirus HOME, 1:. W., W.\.I.EHSON, ,4. I’., WJLIJY, I’., :t~d usually contains virions with homonualcoE’.I~~NH.IJ~, A. E. (1960). ‘I% st,rllctllre and romcapsids to the extent of 20 YS,ad 011 mixed position of the myxovirltscs. I. Electron microscope stttdirs of l.he strttctttre of mpxovirus parinfect,ion the two kinds of nucleocapsids ticles by negative st aillirlg techniqrtes. Virolog!/ pair at random, then virions wit,h hd.ero11, T-M. nucleocapsids will bc produced with the Hos.\K.\, I-., IIf~so~c.~~v.~, y., arid FUIC.ZJ, K. high frequcwy of 10 ‘35. ACKNOWLEIXMENTS The attthors thank Professor K. Fttkai for his continnotts slipport and advice, I)r. Y. Okads for his helpfld discrtssioll, l)r. &I. Nakai for allowing us to use the electron microscope, rnodcl HUllA, and I)r. Kausde for his help in work wit,h t,hc Spine0 I< centrifuge. REFERENCES S., and HOJ~NE, It. W. (1959). il negative staining method for high resollttion electron microscopy of viruses. Hiochinl. Riophys. .IcYa 34, 103-110. CHOPPJN, P. W., and S~TOEC’I
BRENNEIL,
Cellular
Chttrchill.
Biology
London.
of
Myxovi~trs
Injections,
p.
5.
(1959). A thew device for preparing srtbrtnits of myxovirrls. Hiken’s J. 2, 36i-370. IIOS.\KA, \i., -\jI,H,, \i., and F~JLII, K. (1961). The strttctltre of II\-J. II. The fine strrtctttre of t,he subunits. Biken’s J. 4, 243-254. KOLEHM.UNEN, L., ZECH, H., and VON WETTSTEJN, 11. (1965). The st,rrtctnre of cells during tobacco mosaic virlts rcprodttction. Mesophyll cells containitlg virlts crystals. J. CR/I, Hio/. 25, (2) 77-79. LO\VJ
Chenb. 193, 2GS2i5. OGT-IL, hf.,
and and 262. Ori~\o.\, giant II\-.J
:rlld I
IT. Quantitative analysis of gi:tllt polyn~~clcar cell formatioll. Esptl. Ce(I Kes. 26, 108-118. l:o~‘r, I<., and SCHAFER, W. (19Cil). Fine strlwtlwc of sllblmits isolated from Newckle disease virus (ND]‘). Vi,ology 14, 298-299. 8.11,x, J. E. (1944). A simplified procedure for titrating hemnggl~~timtting capacit,y of iuflllenxa virrls and the corresponding ant ibody. -1. Ttw ,t,unol. 49, 8i-98. $( tl:iFEI<, W, :LII~ J:oT~‘, I(. (1959). T.Jllterheiten
des Newrastle IIisease- rllkd hlumps vinls. %. .\‘clt~c/fo?xc/l. 14h, Ci29-f31. WATERSON, A. I’., JENSEK, K. 15., TYNELL, I). A. J., 2nd HONNE, 1:. W. (1961). The fillc struttllre of pnr:titlflrIenza 3 virlis. T’i,ology 14, X74378. YostrmHIT.\. T. il959j. C~:tmma ray illact iwttiotl :trialysis of ilkfective particles ill van AI:tgn~~s type il~complete preparation of ilklrlrlua virlls. Hidc’n ‘S J. 1, 15lLl(i:x
29,
205-221 (1966)
Studies
on the Pleomorphism
YASUHIRO L)epal’tment
HOSAKA
ASD
of HVJ’ Virions
HIDEKO
KITANO
of Preventive Medicine, Research Znslitute for ~Jioobial Osaka University, Osaka, Japan AND
I’he Radiation
SAKIKO
Diseases,
IKEGUCHI
Center of Osaka Prefecture, Accepted February
Osaka, Japan
23, 1966
Partially purified preparations of HVJ were fractionated by zone centrifugation in sucrose gradient,s, and several properties of the separated classes of virions were compared. The most slowly sedimenting band of HVJ consists of the smallest virions, which contain the shortest nucleocapsids, enclosed by the smallest envelopes. The lengths of the nucleocapsids distribute regularly. The minimum length associated with infectivity is 1.0 ,,L This nucleocapsid is designated the mononucleocapsid: its multiples are called polynucleocapsids. The maximum length of polynucleocapsids was more than 20 +. Usually the unfractionated viral population contains in the highest proportion the virions with mononucleocapsids. The separated classes of virions are all infectious. Their progenies are again heterogeneous. Experiments on inactivation of HVJ virions with polynucleocapsids by Co@ show that they contain multiply infectious units. The pleomorphism of HVJ virions can be explained in terms of heterogeneity in length and flexible conformation of nucleocapsids, enclosed by flexible envelopes. MATERIALS
INTRODUCTION
AND METHODS
The morphology of large myxoviruses Virus. HVJ, strain 2, was used. Dilutions has been extensively studied. They consist of 10m2of infected chorioallantoic fluids were of an outer envelope and an inner heIica1 inoculated into the chorioallantoic cavity of nucleocapsid 180 A wide (Horne and Waterlo-day-old embryonated eggs; after 72 son, 1960; Horne et al., 1960; Hosaka et al., hours’ incubation at 35” the infected chorio1961; Waterson et al., 1961; Rott and SchSi- allantoic fluids were harvested. fer, 1961; Hermodsson and Westman, 1962; The infected fluids were centrifuged at Choppin and Stoeckenius, 1964; Cruick2500 rpm for 20 minutes, and then the shank, 1964). Little is known about the supernatants were centrifuged at 20,000 capsid length or the cause of pleomorphism. rpm for 30 minutes. The resulting pellets We studied the pleomorphism, employing were resuspended by pipetting in 0.15 M HVJ, a member of the parainfluenza 1 group. NaCl + 0.015 M sodium citrate (SSC) and We fractionated HVJ virions by sucrose th en centrifuged at 2000 rpm for 15 minutes. gradient centrifugation and compared the Th e resulting superna,tant was used as puriproperties of the fractions obtained. The fied HVJ results showed that the separated classes of Fractionation of HVJ by sucrose gradient virions differ in their construction. centrifugation. Sucrose was dissolved in SSC to give 15, lS, 22, 26, and 30 % (w/v) * Hemagglutinating virus of Japan, synonym: solutions, respectively. Volumes of 5 ml of Serldai virlls. 205
206
HOSAKA,
KITANO
each solution were overlayered in order from the bottom of the tube. The purified HVJ was layered on the sucrose gradient, and the tubes were centrifuged at 15,000 rpm for 50 minutes in a SW 25 rotor. The main band was removed carefully with a l-ml syringe; it was designated band 1 (Bl). The diffuse band below the main band was also removed and called band B2” (B2,); the pellets were called band 4 (B4). Bl and B2, were diluted and centrifuged at 25,000 rpm for 30 minutes. The resulting pelletIs and B4 were resuspended in a small volume of SSC and centrifuged at 2000 rpm for 10 minutes. The supernatants were further centrifuged in sucrose gradients. All bands were purified by a total of three cycles of zone centrifugation in 15-30% sucrose. B20 was further divided into two bands in the second zone centrifugation; the upper and the lower bands were called B2 and B3, respectively. These bands were purified once more by zone centrifugation. Pig. 1 shows the bands of fractions Bl, B2, and B3 purified in this way. Infectivity assay. Aliquots of 0.2 ml of tenfold serial dilutions of HVJ in broth were inoculated into the chorioallantoic cavity of four lo-day-old embryonated eggs. After 3 days of incubation, the chorioallantoic fluids were harvested and tested for hemagglutinin production. The infectivity of HV.J was expressed as the 50 % egg infectivity dose (EIDSO). Hwmgglutinatzon titration. Salk’s pattern method (Salk, 1944) was employed. Aliquots of 0.5 ml of twofold serial dilu-
FIG. 1. Bands of the virions of fraction Bl (left), B2 (middle), and B3 (right) on centrifugation in 15-30’30 sucrose gradients at 15,000 rpm for 50 minlltes in an SW 25 rotor. Fraction Bl sediments slowly, B3 rapidly, and B2 intermediatel~~. Fraction H4 sediments to the bottom.
AND
IKEGUCHI
tions in PBS (phosphate-buffered saline, pH 7.2) were mixed with 0.5 ml of 0.5% fowl red cells in PBS. The hemagglutination titer is expressed as the reciprocal of the final dilution which agglutinated the red cells after 2 hours’ incubation in hhe cold. Assay of hemolytic activity. One milliliter of HVJ was mixed with 2 ml of 2 % fowl red cells in PBS. The mixtures were incubated at 37” for 60 minutes and then centrifuged at 2000 rpm for 5 minutes. The hemoglobin liberated into the supernatant was measured spectrophotometrically at 540 mp and expressed as the optical density. Assay of fusion activity. Ehrlich’s ascites tumor cells (ETC) were washed twice with glucose-free Hanks’ solution by centrifugation at 500 rpm for 3 minutes and suspended in Hanks’ solution at a concentration of 5 volume %. Aliquots of 0.75 ml of 5% ETC were mixed with 0.25 ml of HVJ; the mixturcs were shaken at 37” for 60 minutes. After incubation, the number of cells in each mixture was measured. The fusion activity of HVJ was det’ermined by the decrease in the cell number by fusion and expressed as the fusion index (FI) (Okada and Tadokoro, 1962). Chemical analysis. Protein content was measured by the method of Lowry et al. (1951), using bovine serum albumin as a standard. RNA was extracted by Ogur and Rosen’s (1950) method and estimated by t.he orcinol reaction, using yeast, RNA as a standard. Phospholipid in the acid-insoluble material of HVJ was extracted twice with ether-ethanol (1:3) at 50”, and the phosphorus content of the combined extracts was determined. Electron microscopy. HVJ suspended in SSC was centrifuged at 20,000 rpm for 30 minutes and the pellet was resuspended by gently shaking in a small volume of distilled water. The virus suspension was mixed with an equal volume of 2 % phosphotungstic acid (pH 7.2) (Brenner and Horne, 1959). Then the mixt,ure was placed 011 collodioncoated microgrids and examined in a Hitachi HUl IA t’ype clcctron microscope. E’stimation of the length of the nucleocapsid of HVJ. The length of the nucleocapsid within a virion was estimated as t,he total length of nuclcocapsid meaurable wit,hin the
PLEOMORPHISM
virion negatively stained. The total lengths of the nucleocapsids in relatively large virions into which phosphot’ungstic acid penetrates diffusely are measured easily.
OF HVJ
VIRIONS
207
However, usually little phosphotungstic acid penetrates into small HVJ virions. But when the virions are suspended in distilled water containing a small volume of SSC
FIG. 2. Morphology of negatively stained virions in fract,ion Ul. The phosphotungstic acid penetrates inta) the hollow tubes of the helical nucleocapsids within the virions, forming winding black belts, when HJ\- virions are suspended iu distilled water containing a small volume of SSC and then mixed with 2% phosphotungstic acid. The fine belts of the nucleocapsids are of a relatively uniform and continuous length. Most of the virions with a relatively circular outline are between 180 and 250 mr in diameter.
208
HOSAKA, KITANC)
and then mixed with phosphotungstic acid, the acid often penetrates into the hollow tube of the helical nucleocspsid, forming :L winding black belt, as shown in Fig. 2. The length of t’he nucleocapsid within the virion was measured by a map meter on photographs at, magnifications of 160,000 or 240,000. There was the possibilit’y t#hat only part of the nucleocapsid was visible and measurable within negatively stained virions. In order to examine whether the lengths of the nucleocapsids measured within negatively stained virions represent the real lengths of the nucleocapsidx, t#he measured lengths were compared with those of free nucleocapsids liberated from the virions. Ether-emasol treatment of HVJ (Hosaka et al., 1959) disrupts the HVJ virions and liberates the inner nucleocapsids, but most
FIG. 3. Negatively stained nucleocnpsids capsids have a uniform size of 1 .O p.
liberated
ANI) IKEGUXI
of the nucleocapsids are shortscrIed by this treatment. Therefore another method was employed. It was found t’hat, Athough HVJ virions retain their infectivity around pH 10 in K:LHCO:~-~ Ka2(:03 (0.02 Al), their envelopes become fragile. When HVJ virions are left suspended in the alkali for a few days, a small number of nucleocupsids are liberated (Fig. 3). The distribution of the lengths of the free nucleocapsids t,hus liberated is shown in Fig. 4. The peak at 1.0 p agrees with the main peak at I .O fi in t’he distribut,ion (Fig. 12) of the lengths of t’he nucleocapsids measured within unfractionated virions ncgat8ively stained. Therefore the lengths of the nucxleocnpsids measured within negatively st’ained virions are reliable. y-Ray irradiation. HVJ suspended in SSC was diluted ten times with broth and
from HVJ virions
in alkali
solution.
hIany ~~uc:leo-
PLEOMORPHISM Number
20.
~‘JG. 4. 1)istribution of the lengths of free notclee bcapsids liber:rt,ed from HV.J virions suspended in :rlkali. There is a sharp peak at 1.0 p.
irradiated with CoGo. -At int’ervals during irr:ldiation, aliyuots of HVJ were removed ant 1 their infect,ivity was measured. RESGLTS
Xi;es of Fraction.ated I’irions Sedimentation patfern. Fraction Bl has only a single sharp peak with a sedimentation constant of 1000 S. Fraction B2 has a main peak of 1600 S and two accessory peaks of 1100 S and 2200 S. Fraction B3 has a diffuse peak of 2000 S and a small peak with a lower sedimentation constant. When fralations 132 and B3 were further centrifuged in sucrose gradients and removed from finer zones of the bands, the accessory peaks decreased greatly, but the recoveries were also smaller. Icraction B4 does not show a peak, but only a diffusely sedimenting boundary of about 4000 S with a shaded area, possibly due to large particles of HVJ virions, shown in (Llectron microscopy (Fig. 7). Electron microscopy. Figures 2, 5, 6, and 7 are photographs of fraction Bl, B2, B3, and B4 respectively. Almost all the virions in fract’ion B4 but few of the virions in fraction Bl have been penetrated diffusely with phwphotungstic acid. The number of virions penetrated diffusely with phosphotungstic acid decreases in order from fract’ion B4 to Bl. The sizes of the HVJ virions increase from fraction Bl to B4. Since there are various shapes of virions, it is dificult to express exact distributions of sizes of the virions in the fractions. Therefore the cliamet)ers of the
OF 1IV.J WRIONS
209
virions showing more or less circular outlines in each fraction were roughly compared. The diameters of most of the virions in fraction Bl are 160-250 mp. In fraction B2, many virions have diameters of 250-340 rnp, but t,here are also virions with smaller diameters of 200-230 mp. In fraction 133 many virions have diameters of 360-480 111,~~ but a considerable number of virions are smaller (diameters of 200-340 nij~). The diamct,ers of the virions in fraction B4 have a wide range from 200 to 800 nip; virions with diameters of 400-600 nip are predominant. The distribution pattern of each fraction seemed to be roughly compatible with t,he distribution pattern (Fig. 8) in t.hc nucleocapsid length, respectively. l+ope~~ties of Nucleocapsids of Fractionated 17irions Lengths of nucleocapsids within the visions. The lengths of t,he nucleocapsids within negatively stained, fractionated virions were estimated using more than 100 virions from each fraction. E’igure 8 shows their distributions. Fraction Bl has a peak around 1.0 ,.L. This broad peak was formed partly because data were obtained from different observations on different samples and partly because of variations in t’he measurements, parts of the nucleocapsids within some virions being obscured. The peak probably represent’s the presence of many nucleocapsids of a uniform length of 1.0 p. This interpretation agrees with t,he presence of a sharp peak at, 1.0 p in the distribution of the lengths of free nucleocapsids (Fig. 3) and also with the occurrence of a single, sharp peak in the sedimentation pattern of fraction Bl. The comparison of t’he nucleocapsid length wit’h the sedimentation pattern of fraction Bl leads to a conclusion that 1000 S virions of HVJ contain nucleocapsids with a uniform length of 1.0 p. Frwtion B2 has a main peak around 2.0 p and two smaller peaks around 1.0 and 3.0 ~1. Fract’ion B3 has a main broad peak around 3.0 p and two peaks around 2.0 and 1.0 ~1 These peaks are much broader than that in fraction Bl. The width of the peak in the distribution is t’hought to be more when the nucleocapsid is longer, for the reasons given above.
210
FIG. 5. Morphology outlines are between some virions.
HOSAKA,
KITANO
of negatively stained virions 200 and 340 rnp in diameter.
From comparisons of t’hese distributions with their sedimentation patterns, it is concluded that 1600 S and 2000 S virions contain nucleocapsids of uniform lengths of 2.0 and 3.0 p, respectively. The diffuseness of the peaks of 1600 S and 2000 S is thought to be due to variation in the shape of virions with uniform constitutions or due to slightly different sizes of envelopes enclosing nucleocapsids of uniform lengths.
AN11 IKEG
1JC 11I
of fraction B2. Many virions with relat,ively circular Phosphotungst,ic acid has penetrated diffusely into
It is difficult to remove completely the accessory peaks of fractions B2 and B3 in the length distribution under the present experimental conditions. The factors contributing to the occurrence of these accessory peaks may be (1) simple contamination with virions of other fractions during isolation of these fractions by syringe from bands in the sucrose gradients, (2) the presence of aggregates of smaller virions, (3) the pos-
FIG. 6. Morphology of negat’ively stctilled vii-ions of fr:tci,ioll lar outlines are between 400 and 600 mp in size. Phosphotlmgstic siderable Ilumber of virions.
sible degradation of virions with long nucleocapsids into virions with nucleocapsids of shorter lengt’h during resuspension and during the preparation of negatively stained specimens for electron microscopy. Such degradation readily occurs during sonication of virions, as will be reported in a forthcoming paper. Fraction B4 has no peak in the distribution of nucleocapsid lengths and presents a wide distribution from 1 p to more than 10 p. There is a tendency to show periodic
B3. Man)- virions with relatively circulacid has pcuetrated diEusely into a con-
distributions at intervals of 1.0 ~1, within the range of shorter nucleocapsids. These findings indicat’e that t.he lengths of the nucleocapsids vary on the basis of units of 1.0 p lengt,h. It remains to be determined whether there are a few virions with nucleocapsids which do not have int’egral unit lengths. It is thought that the 1.0 ,.Llength of the nucleocapsids is t#he unit which endows the HVJ virion with infectivity, because under the present experimental condit’ions (1) it
HOSAKA,
FIG. 7. Morphology of llegutively from 200 to 800 rnp. Phosphotungstic
KITANO
AND
IKEGUCHI
stained virions of fractiwl 134. The virions vary acid has penetrated diffusely iuto most of them.
represents a sharp peak of minimum nucleocapsid length within virions, associated with infectivity; (2) it represent’s the first value of serial integral units of peaks observed in the distribut,ion of nucleocapsid lengths; (3) the inactivat’ion curve of the virions containing the nucleocapsids of 1.0 P length, in fraction Bl, by Co60 suggests that, they contain one infectious lmit (Fig. 11); (4) it is found in the majority of unfractJionat8ed HVJ virions and usually has t,he highest I : H ratio; (5) virions of shorter nucleocapsid
widely
in size
lengt#h can be produced artificially by sonication but have a low 1:H ratio. The nucleocapsid of 1.0 IL length could be called the mononucleocapsid of HVJ, and its multiples the polynuoleocapsids. The maximum length of polynucleocapsids observed was more than 20 ,.L. Virions with mono-, di-, tri-, tet’ra-, possibly octa- and possibly dodecanucleocapsids are shown in Fig. 9a, b, c, d, c, and f, respectively. Generally the larger virion contains the longer nucleocapsid.
PLEOMORPHISM
OF HVJ
VIRIONS
213
Nymber
2o I
B2
FIG. 8. Distribution of the lengt,hs of nucleocapsids within virions negat,ively stained, in fractions Bl, B2, B3, and B4. Fraction Bl has a sharp peak around 1.0 p. Fraction B2 has a main peak around 2.0~ and accessory peaks around 1.0 and 3.0 p. Fraction B3 has a diffuse peak around 3.0 p and accessory peaks around 1.0 and 2.0 1. Fraction B4 has no peak and a wide distribution.
The polynucleocapsids within virions a.ppear to be continuous. Relatively few free ends of nucleocapsids have been observed within virions with polynucleocapsids. The maximum length of free nucleocapsids ever seen by us is 2.1 p, approximately corresponding to a dinucleocapsid. We have not seen a definitely circular conformat’ion of a nucleocapsid within a virion. RNA content of visions with polynucleocapsids. If one hema,gglutinating unit’ is due to a constant number of virions, irrespective of the sise of the virion, the RNA content per hemagglutinating unit should be proportional to the length of the nucleocapsid. This is the case (Table 1). The value RNA: 104 HA of fraction B2 is nearly twice that of fraction Bl, and the value of fraction B3 is 2 to 3 times larger. These ratios are
higher than those expected from the nucleo capsid lengths, shown in Fig. 8, if one assumes that the nucleocapsids are all in individual free virions with hemagglutinating activity. This discrepancy can be explained if one supposes that the factors described in the preceding section, which possibly contribute to the occurrence of the accessory peaks in the distributions of nucleocapsid lengths in fraction 52 and B3 (except the first factor) do not contribute to the formation of the accessory fra,ction in the present determination. The rat,ios of bhe protein cont’ent of each fraction per 10“ HA u&s are also shown in Table 1. The relative rat.ios for the fraction? are similar t.o those for RKA. Protein is contained in the envelope as well as in
214
HOSAKA,
KITANO
AND
IKfSC;UC:HI
FI[G. 0. a, b, c, d, c, and f represent virions with mono-, di-, t,ri-, tetrs-, possibly octn-, and possibly respectively. The virion illcreascs in size with the length of the nucleocapsid. dode canucleocapsids,
the nucleocapsid. The result!s suggest that’ POlYmucleocapsids contain not only multiple am0‘unts of (sapsid protein but also are cm close2d wit,h mrrcspondingly larger envelopes.
These considerations are based on the assumpt.ion that one hemagglutirtat,ion unit is due t.o a constant number of visions, irrespwtive of t)hc fraction.
PLEOiVO11PHIti~IOF H\‘J Wl:IONS
FIG.
Prop ties of Envelopes of Fmcfionated It that
with
i?iOTLS ‘as suggested in the previous section le longer nucleocapsids are enclosed Ile la,rger envelopes. To examine this
(,
interpretation the sizes of the enve:lope:s of fractionated virions were compared. Hemol.ylzc and cell fusing act& Ities. In Fig. 10s a.nrl b the hemolytic and ce!I1fu lsing activities, associated with the envel or=, are
1:raction Expt.
-~
No.
~-
~-~ __
1
I:BA: Ibtio
~~-
__~
WHA to Bl
..~~~~
(&
Proteill: 1O’IIA llatio to Bl
(pg)
I31 ~~~~~
B2
-~
R3-
B4
1.5 I.0
7.9 1.8
12.0 2.7
350 1 .o
090 1.9
900 2.6
s). -0 1.0
9.5 1.9
13.0 2.6
22.6 1.5
331 1.0
660 2.0
820 2.5
1370 4.1
15-M 4.4 --~ .~ --~~~-
~__ 2
RNA: Ratio
1O’lIA to Bl
(&
Protein: 10”HA Ratio to Bl
OD
HL
(~g)
FI
TABLE
FS PHOSPHOLIPID-P
2
CONTENTS
OP
E.xpt. No.
Bl,
FIIACTIONR
B2 + 3, and B4 PEH HEM.~(;QLU.L’INSTION
TITEIL
Phospholipid-P per lOa HA (pg) _ ~~__. 114 1~1 B2 + 3
_-___
1 2
9.0 8.1
3
7.3
u Value for fraction FIG. 10. Ilemolytic activities (HL) and fusing act,ivities (FS) of the virions in fractions Bl, B2, B3, and B4 versus the hemagglutination titer. Both activities, associated with the envelope, illcrease in order t,hrough fraction Bl to B4. --a-fraction Bl; -Ofraction B2; ---a--fraction B3; ---O--- fract,ion B4.
Expt. NO.
-I--l
plotted against hemagglutinating titer. The hemolytic and cell fusing activities of each fraction vary in parallel. Both activities are highest in fraction B4. Phospholipid-phosphoms content. Table 2 shows that the content of phospholipid-I? is also highest in fraction B4 and is lower in fraction B2 + 3 and Bl in that order. These results (Fig. 10 and Table 2) lead to the conclusion that the virions with the longer nucleocapsids are enclosed in larger envelopes, assuming that OIK hemagglutination unit is due to a constant number of virions, irrespective of the fractions.
B2.
TABLE R.ATIO OF INFECTIVITY: OF FRACTIONS
2
30 25
20 15 12.0"
3
HEM AGGLUTINATION TITER Bl, B2, B3, AND B4
I:raction Parameter
HA/ml I EIUJml I:11 ’ HA/ml EIUjo/ml 1:H
Infectivity
Bl
B2
B3
15,360 1011.2
2,560 10'0.0
2,500 109.7
7.1
6.0
G.3
II160,000 1011.8 G.6
12,000 lci,OOO 1(“J’O.8 1()10.3 6.3 , 0.1
B4 2,560 109.5
6.1 1,200 108.9
5.8
of Fractionated Visions
Infectivity and hemagglutinating titer as well as the ratio of the two in each fraction were determined and are shown in Table 3. The 1:H ratio ranges from 5.8 to 7.1. The 1:H ratio is highest in fraction Bl and lowest in B4. The infectivity of fraction B4
PLEOMORPHISM
is the most unstable on storage. Thus the question arises whether only Bl is infectious or dso the others. Another question is whether, if the others are infectious, they are multiply infectious. The experiment on the inactivation of infectivities with Co60 indicates that the latter is the case. Figure 11 shows t’he inactivation curves of t’he infectivities of t,hree fract,ions. Infect,ivity in fraction Bl is inactivated according t’o single-hit kinetics; decrease in infectivity in fractions B2 + 3 and B4 follows curves parallel to Bl with a shoulder, although B4 Curve with a larger shoulder.
OF IIVJ
VIRIONS
217
These findings suggest that fraction Bl is homogeneous and consists of virions with one infectious unit and that fractions B2 + 3 and B4 are heterogeneous and consist of virions with one or multiple infectious units. Progeny of Fractionated Vbions The progeny produced by limiting dilutions of each fraction has a heterogeneous distribution similar to t,hat of the original HVJ, both in sucrose gradient’s and by electron microscopy. Limiting dilutions of each band of t’he progeny again produced heterogeneous virions. Propodion oj’ I’iGms with Polynucleocapsids an Unfi~actionated Viral Populations
Figure 12 shows the distribution of the lengths of t,he nucloocapsids within about one hurldred virions in an unfractionated but partially purified population negatively stained. There is a peak around 1.0 p which tails off toward longer nucleocapsids. In the figure, 23 of the 105 virions had nucleocapsids which \vcre more than 1.8 p in length. I’hosphotungstic acid did not penetrat.e into a few of the small virions so that their lengths could not he measured. Therefore, about 20% of total HVJ virions seem to have the long nucleocapsids. This value varies somewhat from preparation to preparat’ion. Virions in unpurified allantoic fluids infected with HVJ were also observed in elecE’IG. 11. Inactivation of the infectivities of the tron microscopy. The fluids were dialyzed virions in fractions Bl, B2 + 3, and B4 with Coca. The presence of shoulders in the survival curves for 12 hours sgainst distilled water conof fractions B2 + 3 and B4 indicates that these taining SSC in a volume of 1: 10 and mixed fractions contain virions with multiple infectious wit,h phosphotungstil: acid, then observed. unit.s. -@- fraction Bl; -O- fraction B2 -+ 3; The proportion of virions with polynucleocapsids is approximately 30%, which is --:3- fract.ion B4.
N.105
10 1.0
2.0
.-%-n-I3.0
4.0
FIG. 12. Distribution of lengths of nucleocapsids within unfractionated IIVJ viriorrs, negatively stained. The distribut,ion of the nucleoeapsids has a peak around 1.0p with a t.sil toward longer nucleocapsids.
215
TTOSAK.4, KITANO
ANI)
similar t,o that in t,he purified unlfract,ion:lted sample. After furt,her banding, pelleting, and resuspending of all classes of fractionated virions, the occurrence of virions with polynucleocapsids in higher proport ion than before was never observed. These firrdiiigs indicat,e that HVJ virions with polynucleocapsids are not artifacts formed on pelleting or resuspending the virions. In negatively st,ained preparations, aggregates of individual virions arc easily det’ectcd. On pelleting and resuspending fraction HI we have never observed the appearance of 1600 S and 2000 S components. This (‘onfirms that t,hese components are not artifacts. Pleonzorphisna of HVJ Vikons Pleomorphism of HVJ has been reported by lcukumi and Nishikawa (1954), Fukai and Suzuki (lg.%), Horne et al. (1960), Hosaka et al. (1961). Naturally the heterogeneity of the lengths of the nuclcocapsids within HVJ virions contributes to the pleomorphism of t’hc virions. However even the virions cont,aining nucleocapsids of a uniform length vary in shape, as seen in
FIG. 13. Typically rllclll OLlS.
pleomorphic
forms of II\‘J
IKIS(;lTCIII
Fig. 2. Further, I:ig. 2 shows t’hat t’here are various types of winding configurations of the nucleocapsids, conforming to the shape of the virion, which reflects the shape of the enclosing envelope. Therefore the nucleocapsid must bc flexible, and the envelope is probably also flexible. In Fig. 13, typical virions (doughnuts, filamcntous, tadpole, and crescent forms) are shown. The flexible configuratjion of the nucleoaapsids is illustrated sc~hcmatically in l;ig. 14.
The present study showed that the 1000 S, 1600 S, and 2000 S virions of HVJ cont’ain nucleocapsids of 1.0 p, 2.0 p, and 3.0 p in length, respectively. Cunha et al. (1947) reported the presence of I,WO peaks of 1200 S and 1800 S in the sedimentation pattern of Newcastle disease virus (NDV), which is a large myxovirus, but t,hcy did not describe their biological significance. Schafer and Iiott (1959) found a strandform structure of 2 p length in NDV after &her 1rcatmem. Cruickshank (1964) esti-
viriolm:
:I, tloughr~ut;
b, crescent;
C, t:rclpolr;
tl, fila-
PLEOMOl:PIIIShI
OF IIVJ
219
\.II:IOSS
b
d e FIG. 14. Models of typically mclltous; c, spherical forms.
shaped viriolls
of IIYJ:
mated t)hat, the to&l length of the nucleoprotein of KDV was between 3.5 and 5.0 p. Choppin and Stoeckenius (1964) observed segments over 2 p long as internal components of SV5, another large myxovirus. If the length of the nucleooapsid of a large myxovirus is uniform, it is possible to regard the maximum length observed as approximately its exact length. However, if the length of the nucleocapsid is not uniform, the virus must be fractionated into fractions of uniform lengths to measure the exact length of t’he nucleocapsid. This has b(sen done in the present study. The polynuclcocapsids within t’he virion actually seem t,o be joined end-to-end. In tobacco mosaic virus (TMV), end-t’o-end aggregation of the nucleocapsids of freshly extracted sap of infected leaves has been report,ed (l+ancki, 1962). The present st’udy on the nucleocapsids of HV,J indicates that, some of these aggregates of TMV may be
a, clough~~ut ; k), CI‘PSC’~II~; c:, ttrclpolr;
(1, fil:~-
produced intrucellularly. In sections of Tl\ZV crystals, some of the virus rods appear to be connected linearly (Kolehmainen et al., 1965). In t’he case of HVJ, if the nuclcocapsids of HVJ arc connected linearly, then the polynucleocapsids will be enclosed in a correspondingly larger-sized envelope. Usually one nucleocapsid of a virus forms one virion. In HVJ, polynucleocapsids can form one virion. Inactivation of influenza virus with Co6” showed that, some incomplete virus of t)he van &gnus type contains multiinfectious unit’s (Yoshiehita, 1959). His data suggested that influenza virus can produce virions with polynucleocspsids. The present interpretation on the pleomorphism of HVJ virions SCCI~ t’o apply also to other pleomorphic myxoviruses. From a genetical point of view, the virion with polynucleocapsids can be regarded as a virion with homonuclcocapsids. Viriorls with heteronucleoaapsids may be produced in
220
IIOSAKA,
KITANO
ASI1
IKEGUCHI
mixed infection with closely related myxoCUNH.\, I:., WEII,, Al. L., BEMD, I)., TAYLOR, A. I(., SH:\ttt’, I). (:., and BE.\RD, J. W. (1947). viruses. We interpret, the het’erozygote of Petrification altd charact,eristirs of the NewKDV, reported by Granoff (1959, 1962) castle disease virlts (California strainj. J. Inin this way. Granoff showed that hctrroxymuno/ 55, G!)-89. gotes were produced with a high frequency FILANCIX, 1:. 1. B. (19@2). Infectivity of tobacco in mixed infections with two st,rains of iUDV mosaic virtls from tobacco leaves treat,ed with and suggested that all virions of NDV had 2-t~hiouracil. I~i~ology 17, {l-21. two genomcs and hct’crozygotes of XDV were Frl~.\r, K., nild Suzoltr, T. (1955). On t.he characproduced by random pairing of the two tcristics of a newly-isolated hemagglutinat,ing kinds of genomcs in mat’uring parl,icles. This virrls from mice. Med. J. Osaka c?niv. 6, l-15. FIX~JMI. II., and NISHJJQ\VA, F. (1954). A pneltidea was supported by the nlorphological motropic virtts from mice cattsing hemagglllobservation that the nucleocapsid of a myxotinatiott. Jupan. .I. Med. Sci. Hiol. 7, 345-353. virus appears to consist of double heliccs (Horne and Waterson, 1960; HoPaka et al., ~IL.\NOFF, A. (1959). St,ttdies OII mixed infection wit.11 Newcastle disease virtts. II. The occttr1901). However thcrc is recent morphologiretrce of iYewcastle disease virus heteroxygotes cal evidence that the nucleocapsid of HVJ aud strldy of phcllot ypic mixing involving seroconsists of :L single helix (in preparat’ion), t,ype and thcrtnal stability. l-i&jgy 9, 649%670. and Choppin and St’oeokenius (1964) rc- GILLVOFF, A. (1962). Heteroxygosis and phcnoported t,hat ihe nucleocapsid of the 8V.5 t)ypic mixitlg wit,11 Newcastle disease virrts. c’olcl Spring Harhr Sjymp. Quant. Bid. 27, 31% virus consists of a single helix. Icm%her, the 326. inactivat,ion of infectivit,y in fraction 131 $., :rl~tl WES,~M.%K, J. (1962). Illtrawith Co60 suggests that there is one genomc HIXMODSSON, strtlctttre of parainflttf~ttza 3 virtta. ,/. I:ltmstruc?. in every virion in the fraction. Thus, t.he Kes. 6, 499-510. produ&on of het’crozygotes with high frcIlons~, It. W., atld W.\.L.ERSON, A. I’. (l!XiO). B quency can be explained more reasonably helical st)rrtctrtre ilt mumps, Newcastle disease on the basis of the presenw of virionr with alld Scndai virtues. .J. Mol. Niol. 2, i5-77. heteronucleoc:Lpsids : if :L Iargc myxovirus HOME, 1:. W., W.\.I.EHSON, ,4. I’., WJLIJY, I’., :t~d usually contains virions with homonualcoE’.I~~NH.IJ~, A. E. (1960). ‘I% st,rllctllre and romcapsids to the extent of 20 YS,ad 011 mixed position of the myxovirltscs. I. Electron microscope stttdirs of l.he strttctttre of mpxovirus parinfect,ion the two kinds of nucleocapsids ticles by negative st aillirlg techniqrtes. Virolog!/ pair at random, then virions wit,h hd.ero11, T-M. nucleocapsids will bc produced with the Hos.\K.\, I-., IIf~so~c.~~v.~, y., arid FUIC.ZJ, K. high frequcwy of 10 ‘35. ACKNOWLEIXMENTS The attthors thank Professor K. Fttkai for his continnotts slipport and advice, I)r. Y. Okads for his helpfld discrtssioll, l)r. &I. Nakai for allowing us to use the electron microscope, rnodcl HUllA, and I)r. Kausde for his help in work wit,h t,hc Spine0 I< centrifuge. REFERENCES S., and HOJ~NE, It. W. (1959). il negative staining method for high resollttion electron microscopy of viruses. Hiochinl. Riophys. .IcYa 34, 103-110. CHOPPJN, P. W., and S~TOEC’I
BRENNEIL,
Cellular
Chttrchill.
Biology
London.
of
Myxovi~trs
Injections,
p.
5.
(1959). A thew device for preparing srtbrtnits of myxovirrls. Hiken’s J. 2, 36i-370. IIOS.\KA, \i., -\jI,H,, \i., and F~JLII, K. (1961). The strttctltre of II\-J. II. The fine strrtctttre of t,he subunits. Biken’s J. 4, 243-254. KOLEHM.UNEN, L., ZECH, H., and VON WETTSTEJN, 11. (1965). The st,rrtctnre of cells during tobacco mosaic virlts rcprodttction. Mesophyll cells containitlg virlts crystals. J. CR/I, Hio/. 25, (2) 77-79. LO\VJ
Chenb. 193, 2GS2i5. OGT-IL, hf.,
and and 262. Ori~\o.\, giant II\-.J
:rlld I
IT. Quantitative analysis of gi:tllt polyn~~clcar cell formatioll. Esptl. Ce(I Kes. 26, 108-118. l:o~‘r, I<., and SCHAFER, W. (19Cil). Fine strlwtlwc of sllblmits isolated from Newckle disease virus (ND]‘). Vi,ology 14, 298-299. 8.11,x, J. E. (1944). A simplified procedure for titrating hemnggl~~timtting capacit,y of iuflllenxa virrls and the corresponding ant ibody. -1. Ttw ,t,unol. 49, 8i-98. $( tl:iFEI<, W, :LII~ J:oT~‘, I(. (1959). T.Jllterheiten
des Newrastle IIisease- rllkd hlumps vinls. %. .\‘clt~c/fo?xc/l. 14h, Ci29-f31. WATERSON, A. I’., JENSEK, K. 15., TYNELL, I). A. J., 2nd HONNE, 1:. W. (1961). The fillc struttllre of pnr:titlflrIenza 3 virlis. T’i,ology 14, X74378. YostrmHIT.\. T. il959j. C~:tmma ray illact iwttiotl :trialysis of ilkfective particles ill van AI:tgn~~s type il~complete preparation of ilklrlrlua virlls. Hidc’n ‘S J. 1, 15lLl(i:x