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Mosaics of capsid components produced by cocultivation of certain human adenoviruses in vitro
VIROLOGY
44, 383-3%
Mosaics
(1977)
of Capsid Certain ERLING
Department
of Virology,
Components Human
by Cocultivation
Adenoviruses
NORRBY Karolinska
Produced
YLVA
AND
Institufet,
of
in Vitro’ GOLLMAR
School of Medicine,
Stockholm,
Sweden
Accepted December 31, 19YO Products of cocultivation of seven human adenovirus serotypes in different pairwise combinations were characterized. Capsids composed of fibers (or more likely pentons) and hexons of different serotypes were identified by use of different techniques. One evidence for this was the demonstration by electron microscopy of the occurrence of fibers of different lengths, i.e., specific for different serotypes, in single virions. Capsid mosaics also could involve hexons of different types. The frequency of occurrence of mixed capsids was related to the degree of biological relatedness between the serotypes used for mixed infections. Cocultivation products also included mixed polymeric soluble components. Dodecons composed of pentons of two different serotypes or of core component(s) of one type and pentons mainly of another type were identified. Pentons of serotypes, which during single infections do not produce dodecons, e.g., types 6 and 16, were found to be capable of participating in the formation of structures of this kind. Monomeric soluble capsid components were characterized by reference to theil anion exchange chromatography behavior. Cocultivat,ion of types 3 and 11 caused t,he appearance of four different populations of hexons. Two of these corresponded to hexons of types 3 and 11 present in the control mixture. The two other populations of hexons contained type-specific determinants of both serotypes in varying proportions. It is proposed that these hexons represent hybrid soluble components containing polypeptides derived from the virus of two different serotypes. No evidence for amixingbetweenstructural proteins of penton components of different serotypes was found. INTRODUCTIOX
B considerable structural and functional diversity of capsid components of human adenoviruses has been demonstrated (cf. Norrby, 1969a). Various components belonging to individual serotypes can be readi identified by different biological tests. Thus it is possible to test to what extent mixed capsid products may occur after cocult’ivation of different types of adenoviruses. In the present, study a phenotypic characterization of products of cocultivation 1 Supported by grants from the Swedish Medical Research Council (projects Nos. B71-16X-54807C, B71-16X-744-06), and t,he Swedish Cancer Society (Project No. 171-B70-04X) and Lotten Bohmans fund.
of different human adenoviruses was attempted. The progeny was not genetJically analyzed at this stage of the investigat,ion. Various serot’ypes representing the three subgroups of human adenoviruses (Rosen, 1960) mere employed. Product,s specific for two different serotypes were demonstrated to occur together (a) in the capsid of complete and incomplete virus particles and (b) in soluble components, bot,h in their oligomeric forms and as regards hexons also in their monomeric form. The frequency of occurrence of mosaics of capsid components varied depending upon the degree of biological relationship between the k-o types used for mixed infections. 383
384
NORRBY MATERIALS
AND
AND
METHODS
Virus and cell cultures. An iso1at.e of t(ype 3 and prototype strains of human adenovirus types 4, 6, 9, 11, 15, and 16 were used. The serological specificity of strains was controlled in neutralization and hemagglutination-inhibition tests. The different strains were propagated in heteroploid cell lines, MAS-A cells, under conditions described previously (Norrby, 1966a) and KB cells. The latter cells were kindly provided by L. Philipson, Department of Microbiology, Uppsala, Sweden. Eagle’s minimal essential medium supplied with 2% calf serum was used for their maintenance. At an advanced stage of cytopathic degeneration in infected cultures, cells were scraped off with a “rubber policeman” into the medium and then sedimented by low speed centrifugation (200 y). The pellet of cells from one Roux bottle (about lo* cells) was resuspended in 5 ml of phosphate-buffered, 0.067 M, pH 7.2-7.4, physiological saline (PBS) (Dulbecco and Vogt), 1954). The cell-associated virus products were released either by treatment with a nonionic detergent (Nonidet P40, Shell Co.) in a final concentration of 0.5% for 15 minutes on an ice bath, or by 3-5 cycles of freezing and thawing. The material was then centrifuged at, low speed (200 g) in order to remove cell debris. Technique for cocultivation of adenoviruses of differen.t serotypes. Purified virions were used as inocula. These were prepared by centrifugation of detergent extracted tissue culture materials in discont’inuous CsCl gradients as described previously (Norrby et al., 1969a). The purified virions were dialyzed against PBS to remove CsCl immediately before being used for inoculation of cultures. Input multiplicities of ten 50 % tissue culture infectious doses or higher were used. Since the eclipse period for multiplication of members of subgroup III is somewhat longer than that for members of subgroup I (Ginsberg, 1958), the former (as well as members of subgroup II) were inoculat’ed 4 hours before the latter. Two serotypes belonging to the same subgroup were inoculated simultaneously. Cell monolayers were infect.ed with purified virus in 10 ml complete main-
GOLLMAR
tenance medium per Roux bottle. Usually some different concentrations of virions of one type were propagated together with a constant amount of the second type. After incubation for 2 hours at 37”, medium was added to a final volume of 150 ml per bottle. The infected cell monolayer was harvested after incubation of cultures for 4048 hours at 37”. Virus products were released from cells as described above. On the basis of different biological tests, harvests of virus material containing about equal quantities of the two virus types grown together were selected for further experimentation. An,tisera. Techniques for purification of soluble components of different serotypes of adenovirus and the procedure used for immunization of rabbits with these preparations were presented in previous publications (Norrby, 1969b; Norrby and Wadell, 1969; Wade11 and Norrby, 1969). The specificity of hyperimmune sera was controlled as described in the same publications. Biological tests. Techniques for infectivity tit,rations, hemagglutination, hemagglutination-enhancement (HE), group-specific complement fixation (CF) tests were already described (Norrby, 1969b). In addition to these tests a variant’ of t,he hemagglutination inhibition antibody consumption (HIC) test (Norrby and Skaaret, 1967) was used. This test, was designed not to demonstrate fiber structures but to indicate the typespecific antigenic component of free hexons. This hexon HIC test was based upon the finding that antihexon antisera free of antifiber antibodies are capable of causing a type-specific inhibition of the hemagglutinin (HA) activity of komotypic virions, presumably due to an aggregation of these (Norrby, 196913;Norrby and Wadell, 1969). Serial 2-fold dilutions (volume 0.05 ml) of the material to be tested, which has to be devoid of all complete HA activity, were mixed with 0.025 ml antiserum against honzotypic hexons dilut,ed to contain four ant.ivirion HI units. Aft,er incubation for 1 hour at room temperat)ure 0.025 ml containing 4 HA units of purified freshly prepared homotypic virions was added. Erythrocytes were added after incubat.ion for another hour at room temperature. The last
tubal, which, after sediment,ation of cells at 37”C, displayed $1 complete or a clear-cut partial agglutination was considered to contain one HIC unit (HICU) of typespecific hexon ant)igenic components. Separation techJrigues. The techniques previously described for adsorption on and elut.ion from red cells of the complete HAS of serobypes 3 (Norrby, 1966a) and 9 (Yorrby et al., 1967) were employed. Zonal cent,rifugation, discontinuous CsCl gradient centrifugat ion and anion exchange chromatography 1echniyues have also been presented in earlier publications (Norrby, 1966a, b; Korrby and Skaaret, 1967; Xorrby FLal., 1969a). Electron microscopy. Preparations t,o be examined in the tGctron microscope were added dropwise to n carbon-coated copper grid. Aft,er lo-20 set, the grid was rinsed carefully with a 1 ?;, ammonium acetate solution and one drop of a 4 % solution of sodium tungstosilicate was added to the grid. Excess fluid was removed with filter paper after incubation for another minute and the grid was allowed to dr>. in the air. The preparations were examined in a l’hilips Ii:11 -200 electron microscope at :L primnrv magnification of about 46,000. The magnification of t,he microscope was calibrated in terms of a grating spacing (Ladd Research Indusiries, Inc., Burlington, Vermont j.
Chamctekhx ft/’ Capsids Produced by Cocultivatio,,, of Two Aclenovims Xerotypes The possible simultaneous occurrence of $bws (or pen.to?l,s)of two different, serotypes of adenovirus in individual capsids was studied by the use of three different techniques. Virions were purified by centrifugaCsCl gradient tion in a discontinuous (Korrby et al., 1969:~) and then either (a) precipitated with excess amounts of specific ant isrra against fibers of the two serotypes, (b) adsorbed on and eluted from red cells in cases when t,his \vas possible, or (c) analyzed in the electron microscope. Thr precipitation technique (a) was used for annlysis of products of cocultivat’ion of
virus of serotypes agglutinating t w different’ kinds of erythrocytes, e.g., monkey and rat cells. hntisera against fibers of one type gave :I significant (4.fold or higher) reduct,ion in the amount of virion-:LssociBtCd HA of both kinds in certain cases. This concerned products from cocultivat ion of biologically relatively closely related adenovirus serotypes, e.g., 3 and 4, 4 and l(i (Norrbp and Wadell, 1969), or 9 and 15. The adsorption-rlution procedure (b) WL:: used for analysis of cocultivation product :: including either type 3 or type 9 (or both) and in addil ion a acrotgpe agglut inat ilkg rrd cells of :t different kind. Eluates from red cells were analyzed for a possible occurrcnc~~ of agglutinins for two kinds of wd cells. Results obtained in experiments of this kind are exemplified in Tables 1 and 2. I~Xuates of virions from monkey erythrocytest\-pe :i (Simon, 3962; Xorrby, 1966a--\vrre iountl to carry fibtw of types 4 (Table 1) and also in other experiments of the biologically relatively distantly related wrot ype 6, :w demonstrated by the presence of r:tt c~xll HA. It was furthermore found that virions produced in cocultivation of types 13:md !I included particles cluting from monkt~y ~11~ or from human 0 cells b\- trcatmrnt \vith cholera filtrate (Korr’by it al., 19A7j, \vhic+ were capable of agglut&ting rat ant1 morj key erythrocyt es, respectivel\- (TabIt, 2 1. The length of fibers varies from ontl stw)type of adenovirus to another, in :t \~a> correlated with their biological subgrouping (Yorrbl., 1969a). Individual virions cwq+ng fiber:: of distinctly diffrrent fibw 1(,11gt hs were identified by electron microscopy- (c J in a number of different expwimwt:: ~1s exemplified in Icigs. 1 and 2. The i’rc~lntwc!~ of occurrence of mixed particles of this kirltl varied, :M should be expected, ill proport ion to the rclat ive \-ieltl of hcmugglut iniw representing thr t n-o wrotypes inclutlt~cl itI t,he exprriment , but in :tddition :dso 1o tllr) biological relatednew of the t\vo wrotylws concerned. Thus particles carrying fibws of both serotype 1 (17-1s nm long; \\‘:rclcll et al., 1967), which is :\ member of Howl’s subgroup 111 displaying a numbtbr of biological characteristics relating it IO wbgroul) I members (cf. Sorrb>-, 196S:t: Nowh~~ :rntl
386
NORRBY TABLE
AND
GOLLMAR TABLE
1
ADSORPTION TO AND ELUTION ERYTHROCYTES OF PURIFIED MIXED AND SINGLE INFECTIONS VIRUS TYPES 3 AND 4
FROM MONKEY VIRIONS FROM WITH ADENO-
2
ADSORPTION ON AND ELUTION FROM MONKEY AND HUMAN 0 RED CELLS OF PURIFIED VIRIONS FROM MIXED AND SINGLE INFECTIONS WITH ADENOVIRUS TYPES 3 AND 9
HA activity (units per 0.05 ml) in tests with Preparation
of virions
HA activity (units per 0.05 ml) in tests with Preparation
tionkey Rat cells, cells, type 3) (we 4) :
4
Cocultivation products
Control mixture from single infections
I
of virions Monkey , Rat cells cells Ihe 3) (:tse 9) .__-
-
exe&-,
Starting material Supernatant after adsorptiona Eluate from red cellsa Starting material Supernatant aft,er adsorptiona Eluate from red cellsa
320
<8
80
I
80
320
20
640
160
<8
160
320
Cocultivation products
~ <4
Q The samples were absorbed at 37” for 1 hour with packed monkey erythrocytes in a final concentration of 10%. Elution from monkey red cells was brought about by incubation at 4’ for 2 hours (Norrby, 1966a).
Wadell, 1969; Wade11 and Norrby, 1969), and of members of subgroup I, types 3, 11, and 16 (10 nm long; Norrby, 1966b, 1968b) were readily demonstrated. Cocultivation of other representatives for different subgroups (e.g., types 3 and 6,6 and 15,9 and 4) caused the appearance of virions carrying fibers of two different lengths at a frequency of about 5 % or less of all virions in preparations containing good yields of HA of both serotypes. In an attempt to determine whether hexons originating from two different serotypes can appear together in the same capsid, mixtures of virions and specific antisera against hexons were examined in the electron microscope. In previous studies it was found t,hat antibodies against hexons of members of subgroups I (t,ype 3; Norrby et al., 196913) and II (types 9 and 15; Norrby, 1969c) and of a single member of subgroup 111 (type 4;
Control mixture from single infections
Starting material Supernatant after adsorption with monkey cells Eluate from monkey cells” Supernatant after adsorption with human 0 cells Eluate from human 0 cellsa Starting material Supernatant after adsorption with monkey cells Eluate from monkey cellsa Supernat,ant after adsorption with human 0 cells Eluate from human 0 cellsa
32
256 128
<4
8
16
-
8
<4
8
128
128
256
<4
128
64
<4
128
<4
<4
256
-
a Elution from monkey cells (final concentration 5’%) was brought about as described in the legend of Table 1 and from human 0 cells (final concentration 1070) by treatment with a standard reagent cholera filtrate product (N. V. PhilipsRoxane. The Netherlands) diluted 1:8 (Norrbv et al., 1967; Norrby, 1968cj.
PRODUCTS
FROM
COCULTIVATION
OF AI1EXOVIKUK
SEROTYI’ES
i$Si
FIG. 1. Ultrastructure of virions purified from cocultivation products of adenovirus t,ypes 3 and -1. Note the appearance of short (10 nm; short, arrows) and long (li-18 run; long arrows) fibers in almost all pttrfirlrs. Negnrivc contrasting with STS. X 400,000. The hnr represents 30 nm.
388
NORRBY
AND
GOLLMAR
FIG. 2. Ultrastructure of virions isolated from cocultivation products of adenovirus types 3 and 4 (a) and types 3 and 6 (b). In (a) four 10 nm (type 3) and two 17-18 nm (type 4) long fibers can be seen, whereas in (b) two fibers are 10 nm (type 3) and four fibers are 26-28 nm (type 6). Negative contrasting with STS. X 400,000. Bars represent 30 nm.
Norrby and Wadell, unpublished) were capable of coating the surface of homotypic, but not of heterotypic virions. The interaction between virions from cocultivation experiments including various pairwise combinations of type 4 and/or selected members of subgroups I and II and specific antihexon antibodies were examined in the electron microscope. Antihexon antibodies were employed in quantities giving a complete coating of control Cons at a particle concentration corresponding to that of the test sample. In some experiments virion preparations absorbed wit,h red cells of one type or another were studied. The degree of ant’ibody coating of the surface of virions as well as the extent of aggregation of virions was determined. Analyses of the degree of antibody coating revealed that particles in a free-lying form or more frequent’ly in aggregates could display what was interpreted to represent only a partial coating of their surface (Figs. 3a and b). Part.icles of this kind occurred not only in the center of aggregates, in which case they might have represented trapped particles, but also in their periphery (arrows, Fig. 3). Under comparable conditions antibody-coat,ed particles were partially more frequently encountered in studies of virions produced in cocultivation of relatively closely related serotypes of virus, e.g., types 3 and 4, than in studies of other types such as 3 and 9 or 13. The extent of aggregation of virions by
different antihexon sera also indicated a somewhat higher tendency for serotypes belonging to the same subgroup to give hexon mosaics, than for serotypes of two different subgroups. In experiments with cocultivation products of adenovirus types 3 and 4 in about equal yields, antisera against hexons of both types aggregated the majority of all virions. A somewhat smaller percentage of all virions from cocultivation experiments with type 3 and types 9 or 15 were aggregated under the corresponding conditions. Table 3 illustrates findings in an experiment with adenovirus serotypes 3 and 15 in which the contribution to cocultivation product’s is somewhat higher for t,ype 3 than for type 15. After absorption with red cells of one kind, remaining virions were found to associate preferentially with antihexon sera against the serotype agglutinating red cells of another kind. Thus virions (rat HA) remaining after absorpt,ion with monkey red cells were coated and aggregated predominantly by an ant.iadenovirus type 15 antihexon serum (Table 3). This finding suggests that virions containing fibers or probably pentons of only one type tend to contain hexons of the same t,ype. Polymeric Forms of Soluble Components Derived from Mixed Infections with Two Di$erent Serotypes
Polymeric forms of soluble components, e.g., dodecons and dimers of pentons or
PROINCTS
FROM
COCULTIVATION
OF Al~ENOVII:UH
HEROTYI’JW
339
FIG. 3. Electron microscopy of the association between specific antihrxon antibodies and virions plkfic,d from a mixed infection with adenovirus types 3 atld 4 (a and hj and a single infection with type 1 (c). The control (c) displays a complete coating by homotypic antihexon antibodies. Al?tisercml against type 3 hrxolls gave neither antibody coating nor aggregation of the same virions. The reverse> sit elation w:w t YIN for the second control of the experiInerrt, type 3 viriolls (Irot included in the figrlrc%. Cocultivatiou products in (a) and (b) were incubated with sera against hexons of types 3 at~d 1. respcsclively, Imder condition (particle to antibody ratio) identical with t,hose of the c~)ntrols. Note the :ip pearancr of somr poorly antibody-coat,ed particles (arrows) itI (a) and the small nrunber of ant itrod!. moles-rdcs visihlc ill (b). Xegat,ive contrasting with STS. X 130,O~)O.
fibers, occur in characteristic fashion among different serotyprs of human adenoviruses (cf. Sorrby, 196%). In order t,o characterize solublr> components among products from
material barcocult ivat ion experiments, vested from the low density region of discont,inuous CsCl gradients MW dialyzed then ~ubjrctetl to ZOTM~ against I’?%3 and
390
NORRBY
AND
GOLLMAR
TABLE
3
INTERACTIONS BETWEEN PURIFIED VIRIONS FROM A COCULTIVATION EXPERIMENT M~ITH TYPES 3 AND 15 AND FROM SINGLE INFECTIONS WITH EACH SEROTYPE AND ANTIHEXON SERA AGAINST BOTH SEROTYPES Preparation virions
of
Cocultivation products”
Absorption with red cells from-
Addition of antihexon serum against type
-b
3
-
15
Monkey
3
Relative amount of virions in aggregates Almost
all
5(t7570 of all particles Few
Extent of coating of surface of virions with IgG antibodies Some virions only partly Only partial
in aggregates coated coating of
virions in aggregates Only sparse coating
of virions
in aggregates Monkey Rat Rat
15 3 15
All All Few
Partial t’o complete Complete Only partial coating
of vi-
rions in aggregates Type
3 (control)
-
Type
15 (control)
-
3 15 3 15
All No No All
Complete None None Complete
a Absorptions were repeated until all HA for a certain kind of red cells had been removed. b No absorption. c Hemagglutination and virion HIC tests with soluble components from the harvest of cocultivation products examined revealed the presence of about four times more type-specific components of type 3 ihan of type 15.
centrifugation at a preselected time and speed. Cocultivation of serotypes 3 and 11 caused the appearance of dodecons agglutinating monkey erythrocytes at +4” (type 11 HA) which sedimented with a rate (SO-SO S) similar to that of type 3 dodecons (Fig. 4). In the control mixture, type 11 dodecons sedimented with a rate of 100-120 S, as was previously described (Norrby, 1968b). Spontaneously occurring soluble complete HAS of types 4 and 16 predominantly are represented by dodecons (Wade11 et al., 1967) and fiber dimers (Norrby and Skaaret, 1968), respectively. However after cocultivation of these two serotypes it was found that the monkey HA, i.e., type 16 oligomers, sedimented together with type 4 dodecons (Fig. 5). A similar effect was encountered when type 16 was propagated together with type 15, which by itself yields dodecons and fiber dimers (Norrby, 196%). Additional examples of a similar phenomenon were encountered in studies of other serotypes. The possible occurrence of dodecons including pentons of two serotypes was ana-
lyzed by erythroeyte absorption or adsorption-elution experiments and by electron microscopy. Absorption of dodecons from mixed infections, purified by zonal centrifugation, with one kind of red cells in a number of experiments caused a significant (4-fold or greater) reduction in the amount of HA for another kind of red cells. Furthermore under conditions which allowed adsorption-elution, e.g., analyses of cocultivation products of serotypes 3 and 4, it was found that the monkey cell eluate contained rat cell HA as described above for virions. The occurrence of dodecons containing two kinds of pentons, in some cases mainly pentons of a serotype which normally is not capable of forming this kind of polymeric structure (e.g., type 6) was confirmed by electron microscopy (Fig. 6). Properties of Monomeric Soluble Components Produced by Cocullivation of Two Serotypes of Adenovirus Physical characteristics of monomeric soluble components from single and mixed infections, were compared by anion exchange
PRO1 )UCTS
FROM
COCULTIVATION
HAU per 0.05 ml
256 -
B I
12864 32 ;/JJ~a,,,a,L -*-..+-. -- --x__._ <2 5 0 10 FRACTION
15
20
NUMBER
FIG. 4. Zonal centrifugation of soluble complete HBs from a mixed infection with adenovirus types 3 and 11 (A), and as a control the corresponding components from single infections with the same serotypes (B). HA activity at f4” (X----X ; type 11) and at 37” (O--O; types 3 + 11) have been recorded. The samples were centrifuged in a linear 5-20% sucrose gradient at 25,000 rpm for 6 hours (4”) in rotor SW 25 (Spinco). The bottom of the tube is to the left.
chromatography fractionation. In order to allow an analysis of monomeric soluble components, virions, and soluble complete HA were removed by consecutive centrifugations of material in discontinuous CsCl gradients and linear sucrose gradient,s, respectively, as described above. An analysis of hexons was performed with harvests from cult,ures mixedly infected with types 3 and 11, bot’h members of subgroup I, or with types 11 and 15, representing two different subgroups. The reason for choosing these combinations was a pract’ical one. Among different, serotypes characterized in this laborat,ory (cf. Norrby, 1969a) the position of hexons of t)ypes 3 or 15 and 11 in the elution diagram obtained by anion exchange chromatography are dist,inctly different. The results of an experiment with cocultivation products of types 3 and 11 are presented in Fig. 7. Hexons were monitored in CF test,s
OF ADENOVIRUS
SEROTYPES
391
with antiserum against type 2. Furt,hermore the hexon HIC test described under Materials and Methods was used to indicate the Dype-specific part of hexons of both types. After separation of cocultivation products. 4 peaks (NOS. I through IV) of hexon activities could be distinguished. The position of peaks I and IV corresponded to those of type 3 and type 11 hexons in the cont,rol mixture as confirmed by conductomet,ric measurements. The hexon HIC tests revealed the serotype identity of the t,wo peaks in both materials. Hexon peaks II and IT1 were found in cocultivation products, but not in control materials. Peak II, eluting immediately after t,he peak of type 3 hexons, contained mainly type 3 hexon HIC test positive material, but, also some of type 11 origin. The reverse sit)uation was true for hexons in peak III, which eluted closest to type 11 hexons. Fract’ionation and characterization of products of cocultivat’ion of t)ypes 11 and 15 also caused t’he appearance of :I barely detectable fraction of hexons eluting in a posiHAU per 0.05 ml 32~
16. 8. 4: <2
FRACTION NUMBER FIG. 5. Zonal centrifugat,ion of soluble complete HAS from a mixed infection with adenovirus types 4 and 16 (A) and as a control the corresponding components from single infections wit.h the same serotypes (B). The distribution of rat (X----X; type 4) and monkey cell HAS (O-------O; type 16) is illustrated. Conditions of cerrtrifugation as given in the legend of Fig. 1.
AND
GOLLMAR
terials from mixed and single infections could be demonstrated. DISCUSSION
FIG. 6. Ultrastructure of dodecons isolated by aonal centrifugation from cocultivation products ‘of adenovirus types 4 and 16 (a), types 3 and 6 (b), and types 6 and 11 (c). Note the appearance of fibers of varying lengths in individual dodecons as well as the participation of type 16 pentons (10 nm long fibers) and type 6 pentons (26-28 nm long fibers) in the formation of dodecon structures. The latter two serotypes under conditions of single infections produce dimers of pentons, but no dodecons. Negative contrasting with STS. X250,000. Bars represent 30 nm.
tion intermediate between those of “parental” hexons. The fractionated materials in Fig. 7 were also analyzed for the presence of types 3 and 11 pentons. Unfortunately, the latter pentons, as previously experienced (Norrby, 1968d), are labile under the conditions of fractionation and no satisfactory recovery could be obtained. Type 3 pentons, which were readily identified, displayed exactly the same position and distribution in the elution diagram obtained after fractionation of cocultivation products and of control material. A further analysis of the effect of cocultivation on charact’eristics of pentons was performed with products of mixed infections with types 3 and 15. In this system pentons are readily recovered and appear at distinctly different positions. No difference between the distribution of pentons in ma-
A mixed infection with two different adenoviruses provides an opportunity for peptides of these two serotypes to assemble into hybrid capsomers. The present findings demonstrate that cocultivation products of types 3 and 11 included two populations of hexons (peaks II and III in Fig. 7), which were not present in control materials. These hexons carried type-specific antigenic components of both types and eluted at a position intermediate between that of hexons of the two “parental” types. It could be argued t.hat peaks II and III contain aggregates of hexons of types 3 and 11. However, this explanation seems less likely since (a) a similar appearance of hexons in the elution diagram was never encountered in fractionation of control materials and (b) there was a clear-cut preference for hexons occurring only among cocultivation products to appear in two readily distinguishable peaks of intermediate position. This preference may be of some significance in relationship to the structure of hexons. One might speculate that hexons of peak II are composed of two adenovirus type 3 subunits and one type 11 subunit, of the size described by Xaizel (1968) and that for hexons of peak III the relative occurrence of subunits might be the converse. Structural hexon proteins of adenovirus types 11 and 15, which represent two different subgroups, also seemed to have some capacity to form hybrid hexons although in a much lower frequency than those of types 3 and 4. This is of interest since, on the basis of the well pronounced immunological dissimilarities between hexons of types 11 and 15 (Norrby and Wadell, 1969), one would anticipate the occurrence of corresponding distinct differences between the polypeptides of their hexons. Monomeric soluble components of mixed polypeptide origin theoretically might also occur in cocultivation products such as fibers, vertex capsomers or the joint structure, pentons. No evidence for the occurrence of this was found. Unfortunately no
PKOl>UCTS
FROM
CFU per 0.025ml
COCULTIVATION
OF rll)ENOVIlZUS
SlSI~OTYl’I~:S
393
,
Hexon
HICU IV
I ::
32.
FIG. 7. Anion exchange chromatography separation of soluble components (excluding rnost~ dodefrom a mixed infection wit.h adenovirus types 3 and 11 (upper part of diagram) aJJd a corltrol mixture of the corresponding soluble components of the same two serotypes (bottom part of diagram). A linear gradient of 0.1 to 0.3 M N&l in 0.04 M Tris-HCl buffer, pH 8.4, was used for elut’ion of components from t,he column. The following activities were determined: group-specific hexon CF antigen (0 ---O) identified with an ant,iadenovirus type 2 hexon serum and type-specific adenovirus 3 (U---W) and 11 (A-----A) hexon antigens demonstrated in hexon HIC tests (see Materials and Methods). P denotes the position of type 3 pentons and D that of trace amounts of type 3 dodecons remaining in the samples. The position of different peaks in the two fractionations were monitored by conductometric measurements, whereafter the distribution of fractions m-ere adjusted to comparable scales. cons)
mixed infectlion lvith two serotypes belonging to the same subgroup suitable for an analysis of the effect, on both kinds OF pentons was available. However, in case a mixing between vert,ex capsomers and fibers of, for example, types 3 and 11 into penton hybrids could occur one would have anticipated to see some modification of the distribut,ion of penton incomplete HAS in the anion exchange chromatography experiment illustrat’ed in Fig. 7, since type 11 pentons (in cases when this component is detected) elute at rela-
tively low molarity of S&l. This was not found. lcurthermore, experiments employing serotypes representing two different subgroups in no case suggested the occurrence of modified populations of pentons. It is possible that t,he degree of fitting bet,ween polypeptides of different serotypes, i.e., t,he possibilitjy of occurrence of hybrid components, is much lower in pentons than in hexons, since the former represent a more specialiaed structure (cf. Sorrby, 1970). Pentons and hexons of two serotypc+ were
394
NORRBY
AND
found capable of assembling into mixed products of both polymeric soluble components and intact capsids. The former case concerns the formation of dodecons containing pentons of two types, in some cases including pentons of a serotype which normally cannot produce a soluble polymeric structure of this kind. It has been suggested, mostly from indirect evidence, that the dodecon structure includes a core protein (cf. Norrby, 1968b). The character of this postulated core protein probably is of decisive importance for the kind of soluble polymeric structures produced by individual serotypes. Dodecon core protein of one serotype may allow pentons of a different, normally nondodecon-producing structure to aggregate into a polymeric product of this kind. The suggested preference for mixed assembly of pentons and hexons of relatively closely related serotypes into intact capsids is not unexpected. It has been demonstrated that both vertex capsomers and hexons display a wide range of antigenic specificities including type- (demonstrated for hexons only), subgroup-, intersubgroup-, and groupspecific components. It seems likely that the degree of fitting between capsomers should be higher if they belonged to the same subgroup than if they were more distantly related. It is of interest that a number of successful experiments were made to produce capsid mosaics by double infections with serotypes 3 and 4. Type 4 belongs to Rosen’s subgroup III, but it has been found that with regard to immunological characteristics of both hexons and vertex capsomers this serotype resembles members of subgroup I (Norrby and Wadell, 1969; Wade11 and Norrby 1969), which includes type 3. The aim of the present study was to characterize capsid products derived from simultaneous multiplication of two serotypes of human adenoviruses. No attempts were made to perform subsequent passages of progeny material. Thus virions, phenotypitally mixed in such a way that they contain the nucleic acid (alternatively nucleoprotein) of one type and capsid of another type were not identified. Furthermore, it cannot be concluded to which extent the mixed capsid products identified, were the result of genotypic or phenotypic interactions. However,
GOLLMAR
the high frequency of occurrence of mixed capsids in certain combinations of serotypes strongly suggest a phenomenon of phenotypic mixing. Phenotypic mixing of viruses was first conclusively demonstrated by Streisinger (1956). He showed that the progeny of simultaneous infection with the Escherichia coli phages T2 and T4 contained virions which were neutralized by antisera against both serotypes. On further passage, these virions yielded only T2 and T4 progeny. In later studies phenotypic mixing on the capsid level was also demonstrated with animal viruses; ECHO-virus type 7 and Coxsackievirus type A9 (Ito and Melnick 1959), poliovirus types 1 and 2 (Ledinko and Hirst, 1961) and the simian and human adenoviruses, types SA7 and 2, respectively (Altstein and Dodonova, 1968). In the latter study as much as 90 % of the virions were phenotypically mixed to an extent which allowed them to be neutralized by antisera against both serotypes. Experiments in this laboratory have demonstrated that capsid mosaics of the kinds described above can be identified also among virions from cocultivation of certain human adenoviruses with monkey or dog adenoviruses (Norrby, unpublished). ACKNOWLEDGMENTS Mrs. Halyna Marusyk gave a valuable assistance in electron microscopic studies and Mr. Thamas Varsanyi in chromatographic separation experiments. Excellent technical assistance was provided by Mrs. Margareta Jurstrand. REFERENCES ALTSTEIN, A. D., and DODONOVA, N. N. (1968). Interaction between human and simian adenoviruses in simian cells: Complementation, phenotypic mixing and formation of monkey cell “adapted” virions. Virology 35, 248-254. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167-182. GINSBERG, H. S. (1958). Characteristics of adenoviruses. III. Reproductive cycle of types 1 to 4. J. Ezp. Med. 107, 133-152. ITO, H., and MELNICK, J. L. (1959). Double infection of single cells with ECHO 7 and Coxsackie A9 viruses. J. Exp. Med. 109, 393-406. LEDINICO, N., and HIRST, G. K. (1961). Mixed infection of HeLa cells with polioviruses t,ypes 3 and 2. Virology 14, 207-219.
PRODUCTS
FROM
COCULTIVATION
MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A and 12. Virology 36, 115-125. NOHHBY, E. (1966a). The relationship between the soluble antigens and the virion of adenovirus type 3. II. Identification and characterization of an incomplete hemagglutinin. Virology 30, 608-617. NORHBY, E. (1966b). The relationship between the soluble antigens and the virion of adenovirus t.ype 3. I. Morphological characteristics. Vilirology 28, 236-248. NORRBY, E. (1968a). Biological significance of structural adenovirus components. Curr. Top. Microbial. Immun. 43, l-43. N~R~BY, E. (196810). Comparison of soluble components of adenovirus t,ypes 3 and 11. J. Gen. Viral. 2, 135-142. N~HI~BY, E. (196%). Comparative studies on the soluble components of adenovirus types 9 and 15 and the intermediat.e strain 9-15. J. Viral. 2, 1200-1210. NORRBY, E. (1968d). Identificat,ion of soluble romponents of adenovirus type 11. J. Gen. Viral. 2, 123-134. NORRBY, E. (1969a). The structural and functional diversit,y of adenovirus capsid components. J. Gen. Viral. 5, 221-236. NORRBY, E. (1969b). The relationship between the soluble antigens and the virion of adenovirus fype 3. IV. Immunological complexity of soluble components. Virology 37, 565-576. NCIRRBY, E. (1969c). Capsid mosaics of intermediate strains of human adenoviruses. J. Viral. 4, 657-662. NOHHBY, E. (1970). Adenoviruses. In “Comparative Virology” (K. Maramorosch and E. Kurst.ak, eds.). Academic Press, New York, in press. N~RRBY, Is., and SKAARET, P. (1967). The rela-
OF ADENOVIRUS
SEROTYPES
3%
tiollship between the soluble antigens and the virion of adenovirus type 3. III. Immunological identification of fiber antigen and isolated vertex capsomer antigen. Virology 32, 489-502. NORRRY, E., and SKAARET, P. (1968). Comparison between soluble components of adenovirua types 3 and 16 and of the intermediate strain S-16. (the San Carlos agent). ViroZogv 36, 201-m211. NORRBY, E., and WADELL, G. (1969). IIII~IUIIOlogical relationships between hexons or cchrt ain human adenoviruses. J. ViroZ. 4, 663-670. NORRBY, E., NYI~ERG, B., SKAAXET. P., and LENGYEL, A. (1967). Separation and characsterization of soluble adenovirus type 0 caomponents. J. Viral. 1, 1101-1108. NORRBY, E., W~DI~LI,, G., and MARUS~K, Ii. (1969a). Fiber-associated incomplete and cornplete hemagglutinins of adenovirns t,vpe ii. ,4rch. Ges. Virusjorsch. 28, 239-244. I~., and HAMMARsKJ~~LI), NORHHY, E., MARUSYH, M.-L. (196913). The relationship between the soluble antigens and t,he virion of adenovirns type 3. V. Identification of antigen specificities available at the surface of virions. Virolog,r/ 38, 477482. ROSEN, L. (1960). A hemagglut,ination-inhibition technique for typing adenoviruses. A,,lrr. J. Hyg. 51, 120-128. SIMON, M. (1962). Hemagglutination experiment,s with cert,ain adenovirus type strains. Acfa Microbial. Hung. 9, 45-54. STREISINGER, C. (1956). Phenotypic mixing of host range and serological specificities in bacteriophage T2 and T4. Virology 2, 388-398. E. (1969). ImmunoWADELL, G., and NORRBY, logical and other biological characteristics of pentons of human adenoviruses. J. Viro?. 4, 671-680. WADELL, G., NORRRY, E., and SCH~NNING, I:. (1967). Ultrastructure of soluble ant,igens and the virion of adenovirus type 4. .4rch. Ges. Virusjorsch. 21, 234-242.
44, 383-3%
Mosaics
(1977)
of Capsid Certain ERLING
Department
of Virology,
Components Human
by Cocultivation
Adenoviruses
NORRBY Karolinska
Produced
YLVA
AND
Institufet,
of
in Vitro’ GOLLMAR
School of Medicine,
Stockholm,
Sweden
Accepted December 31, 19YO Products of cocultivation of seven human adenovirus serotypes in different pairwise combinations were characterized. Capsids composed of fibers (or more likely pentons) and hexons of different serotypes were identified by use of different techniques. One evidence for this was the demonstration by electron microscopy of the occurrence of fibers of different lengths, i.e., specific for different serotypes, in single virions. Capsid mosaics also could involve hexons of different types. The frequency of occurrence of mixed capsids was related to the degree of biological relatedness between the serotypes used for mixed infections. Cocultivation products also included mixed polymeric soluble components. Dodecons composed of pentons of two different serotypes or of core component(s) of one type and pentons mainly of another type were identified. Pentons of serotypes, which during single infections do not produce dodecons, e.g., types 6 and 16, were found to be capable of participating in the formation of structures of this kind. Monomeric soluble capsid components were characterized by reference to theil anion exchange chromatography behavior. Cocultivat,ion of types 3 and 11 caused t,he appearance of four different populations of hexons. Two of these corresponded to hexons of types 3 and 11 present in the control mixture. The two other populations of hexons contained type-specific determinants of both serotypes in varying proportions. It is proposed that these hexons represent hybrid soluble components containing polypeptides derived from the virus of two different serotypes. No evidence for amixingbetweenstructural proteins of penton components of different serotypes was found. INTRODUCTIOX
B considerable structural and functional diversity of capsid components of human adenoviruses has been demonstrated (cf. Norrby, 1969a). Various components belonging to individual serotypes can be readi identified by different biological tests. Thus it is possible to test to what extent mixed capsid products may occur after cocult’ivation of different types of adenoviruses. In the present, study a phenotypic characterization of products of cocultivation 1 Supported by grants from the Swedish Medical Research Council (projects Nos. B71-16X-54807C, B71-16X-744-06), and t,he Swedish Cancer Society (Project No. 171-B70-04X) and Lotten Bohmans fund.
of different human adenoviruses was attempted. The progeny was not genetJically analyzed at this stage of the investigat,ion. Various serot’ypes representing the three subgroups of human adenoviruses (Rosen, 1960) mere employed. Product,s specific for two different serotypes were demonstrated to occur together (a) in the capsid of complete and incomplete virus particles and (b) in soluble components, bot,h in their oligomeric forms and as regards hexons also in their monomeric form. The frequency of occurrence of mosaics of capsid components varied depending upon the degree of biological relationship between the k-o types used for mixed infections. 383
384
NORRBY MATERIALS
AND
AND
METHODS
Virus and cell cultures. An iso1at.e of t(ype 3 and prototype strains of human adenovirus types 4, 6, 9, 11, 15, and 16 were used. The serological specificity of strains was controlled in neutralization and hemagglutination-inhibition tests. The different strains were propagated in heteroploid cell lines, MAS-A cells, under conditions described previously (Norrby, 1966a) and KB cells. The latter cells were kindly provided by L. Philipson, Department of Microbiology, Uppsala, Sweden. Eagle’s minimal essential medium supplied with 2% calf serum was used for their maintenance. At an advanced stage of cytopathic degeneration in infected cultures, cells were scraped off with a “rubber policeman” into the medium and then sedimented by low speed centrifugation (200 y). The pellet of cells from one Roux bottle (about lo* cells) was resuspended in 5 ml of phosphate-buffered, 0.067 M, pH 7.2-7.4, physiological saline (PBS) (Dulbecco and Vogt), 1954). The cell-associated virus products were released either by treatment with a nonionic detergent (Nonidet P40, Shell Co.) in a final concentration of 0.5% for 15 minutes on an ice bath, or by 3-5 cycles of freezing and thawing. The material was then centrifuged at, low speed (200 g) in order to remove cell debris. Technique for cocultivation of adenoviruses of differen.t serotypes. Purified virions were used as inocula. These were prepared by centrifugation of detergent extracted tissue culture materials in discont’inuous CsCl gradients as described previously (Norrby et al., 1969a). The purified virions were dialyzed against PBS to remove CsCl immediately before being used for inoculation of cultures. Input multiplicities of ten 50 % tissue culture infectious doses or higher were used. Since the eclipse period for multiplication of members of subgroup III is somewhat longer than that for members of subgroup I (Ginsberg, 1958), the former (as well as members of subgroup II) were inoculat’ed 4 hours before the latter. Two serotypes belonging to the same subgroup were inoculated simultaneously. Cell monolayers were infect.ed with purified virus in 10 ml complete main-
GOLLMAR
tenance medium per Roux bottle. Usually some different concentrations of virions of one type were propagated together with a constant amount of the second type. After incubation for 2 hours at 37”, medium was added to a final volume of 150 ml per bottle. The infected cell monolayer was harvested after incubation of cultures for 4048 hours at 37”. Virus products were released from cells as described above. On the basis of different biological tests, harvests of virus material containing about equal quantities of the two virus types grown together were selected for further experimentation. An,tisera. Techniques for purification of soluble components of different serotypes of adenovirus and the procedure used for immunization of rabbits with these preparations were presented in previous publications (Norrby, 1969b; Norrby and Wadell, 1969; Wade11 and Norrby, 1969). The specificity of hyperimmune sera was controlled as described in the same publications. Biological tests. Techniques for infectivity tit,rations, hemagglutination, hemagglutination-enhancement (HE), group-specific complement fixation (CF) tests were already described (Norrby, 1969b). In addition to these tests a variant’ of t,he hemagglutination inhibition antibody consumption (HIC) test (Norrby and Skaaret, 1967) was used. This test, was designed not to demonstrate fiber structures but to indicate the typespecific antigenic component of free hexons. This hexon HIC test was based upon the finding that antihexon antisera free of antifiber antibodies are capable of causing a type-specific inhibition of the hemagglutinin (HA) activity of komotypic virions, presumably due to an aggregation of these (Norrby, 196913;Norrby and Wadell, 1969). Serial 2-fold dilutions (volume 0.05 ml) of the material to be tested, which has to be devoid of all complete HA activity, were mixed with 0.025 ml antiserum against honzotypic hexons dilut,ed to contain four ant.ivirion HI units. Aft,er incubation for 1 hour at room temperat)ure 0.025 ml containing 4 HA units of purified freshly prepared homotypic virions was added. Erythrocytes were added after incubat.ion for another hour at room temperature. The last
tubal, which, after sediment,ation of cells at 37”C, displayed $1 complete or a clear-cut partial agglutination was considered to contain one HIC unit (HICU) of typespecific hexon ant)igenic components. Separation techJrigues. The techniques previously described for adsorption on and elut.ion from red cells of the complete HAS of serobypes 3 (Norrby, 1966a) and 9 (Yorrby et al., 1967) were employed. Zonal cent,rifugation, discontinuous CsCl gradient centrifugat ion and anion exchange chromatography 1echniyues have also been presented in earlier publications (Norrby, 1966a, b; Korrby and Skaaret, 1967; Xorrby FLal., 1969a). Electron microscopy. Preparations t,o be examined in the tGctron microscope were added dropwise to n carbon-coated copper grid. Aft,er lo-20 set, the grid was rinsed carefully with a 1 ?;, ammonium acetate solution and one drop of a 4 % solution of sodium tungstosilicate was added to the grid. Excess fluid was removed with filter paper after incubation for another minute and the grid was allowed to dr>. in the air. The preparations were examined in a l’hilips Ii:11 -200 electron microscope at :L primnrv magnification of about 46,000. The magnification of t,he microscope was calibrated in terms of a grating spacing (Ladd Research Indusiries, Inc., Burlington, Vermont j.
Chamctekhx ft/’ Capsids Produced by Cocultivatio,,, of Two Aclenovims Xerotypes The possible simultaneous occurrence of $bws (or pen.to?l,s)of two different, serotypes of adenovirus in individual capsids was studied by the use of three different techniques. Virions were purified by centrifugaCsCl gradient tion in a discontinuous (Korrby et al., 1969:~) and then either (a) precipitated with excess amounts of specific ant isrra against fibers of the two serotypes, (b) adsorbed on and eluted from red cells in cases when t,his \vas possible, or (c) analyzed in the electron microscope. Thr precipitation technique (a) was used for annlysis of products of cocultivat’ion of
virus of serotypes agglutinating t w different’ kinds of erythrocytes, e.g., monkey and rat cells. hntisera against fibers of one type gave :I significant (4.fold or higher) reduct,ion in the amount of virion-:LssociBtCd HA of both kinds in certain cases. This concerned products from cocultivat ion of biologically relatively closely related adenovirus serotypes, e.g., 3 and 4, 4 and l(i (Norrbp and Wadell, 1969), or 9 and 15. The adsorption-rlution procedure (b) WL:: used for analysis of cocultivation product :: including either type 3 or type 9 (or both) and in addil ion a acrotgpe agglut inat ilkg rrd cells of :t different kind. Eluates from red cells were analyzed for a possible occurrcnc~~ of agglutinins for two kinds of wd cells. Results obtained in experiments of this kind are exemplified in Tables 1 and 2. I~Xuates of virions from monkey erythrocytest\-pe :i (Simon, 3962; Xorrby, 1966a--\vrre iountl to carry fibtw of types 4 (Table 1) and also in other experiments of the biologically relatively distantly related wrot ype 6, :w demonstrated by the presence of r:tt c~xll HA. It was furthermore found that virions produced in cocultivation of types 13:md !I included particles cluting from monkt~y ~11~ or from human 0 cells b\- trcatmrnt \vith cholera filtrate (Korr’by it al., 19A7j, \vhic+ were capable of agglut&ting rat ant1 morj key erythrocyt es, respectivel\- (TabIt, 2 1. The length of fibers varies from ontl stw)type of adenovirus to another, in :t \~a> correlated with their biological subgrouping (Yorrbl., 1969a). Individual virions cwq+ng fiber:: of distinctly diffrrent fibw 1(,11gt hs were identified by electron microscopy- (c J in a number of different expwimwt:: ~1s exemplified in Icigs. 1 and 2. The i’rc~lntwc!~ of occurrence of mixed particles of this kirltl varied, :M should be expected, ill proport ion to the rclat ive \-ieltl of hcmugglut iniw representing thr t n-o wrotypes inclutlt~cl itI t,he exprriment , but in :tddition :dso 1o tllr) biological relatednew of the t\vo wrotylws concerned. Thus particles carrying fibws of both serotype 1 (17-1s nm long; \\‘:rclcll et al., 1967), which is :\ member of Howl’s subgroup 111 displaying a numbtbr of biological characteristics relating it IO wbgroul) I members (cf. Sorrb>-, 196S:t: Nowh~~ :rntl
386
NORRBY TABLE
AND
GOLLMAR TABLE
1
ADSORPTION TO AND ELUTION ERYTHROCYTES OF PURIFIED MIXED AND SINGLE INFECTIONS VIRUS TYPES 3 AND 4
FROM MONKEY VIRIONS FROM WITH ADENO-
2
ADSORPTION ON AND ELUTION FROM MONKEY AND HUMAN 0 RED CELLS OF PURIFIED VIRIONS FROM MIXED AND SINGLE INFECTIONS WITH ADENOVIRUS TYPES 3 AND 9
HA activity (units per 0.05 ml) in tests with Preparation
of virions
HA activity (units per 0.05 ml) in tests with Preparation
tionkey Rat cells, cells, type 3) (we 4) :
4
Cocultivation products
Control mixture from single infections
I
of virions Monkey , Rat cells cells Ihe 3) (:tse 9) .__-
-
exe&-,
Starting material Supernatant after adsorptiona Eluate from red cellsa Starting material Supernatant aft,er adsorptiona Eluate from red cellsa
320
<8
80
I
80
320
20
640
160
<8
160
320
Cocultivation products
~ <4
Q The samples were absorbed at 37” for 1 hour with packed monkey erythrocytes in a final concentration of 10%. Elution from monkey red cells was brought about by incubation at 4’ for 2 hours (Norrby, 1966a).
Wadell, 1969; Wade11 and Norrby, 1969), and of members of subgroup I, types 3, 11, and 16 (10 nm long; Norrby, 1966b, 1968b) were readily demonstrated. Cocultivation of other representatives for different subgroups (e.g., types 3 and 6,6 and 15,9 and 4) caused the appearance of virions carrying fibers of two different lengths at a frequency of about 5 % or less of all virions in preparations containing good yields of HA of both serotypes. In an attempt to determine whether hexons originating from two different serotypes can appear together in the same capsid, mixtures of virions and specific antisera against hexons were examined in the electron microscope. In previous studies it was found t,hat antibodies against hexons of members of subgroups I (t,ype 3; Norrby et al., 196913) and II (types 9 and 15; Norrby, 1969c) and of a single member of subgroup 111 (type 4;
Control mixture from single infections
Starting material Supernatant after adsorption with monkey cells Eluate from monkey cells” Supernatant after adsorption with human 0 cells Eluate from human 0 cellsa Starting material Supernatant after adsorption with monkey cells Eluate from monkey cellsa Supernat,ant after adsorption with human 0 cells Eluate from human 0 cellsa
32
256 128
<4
8
16
-
8
<4
8
128
128
256
<4
128
64
<4
128
<4
<4
256
-
a Elution from monkey cells (final concentration 5’%) was brought about as described in the legend of Table 1 and from human 0 cells (final concentration 1070) by treatment with a standard reagent cholera filtrate product (N. V. PhilipsRoxane. The Netherlands) diluted 1:8 (Norrbv et al., 1967; Norrby, 1968cj.
PRODUCTS
FROM
COCULTIVATION
OF AI1EXOVIKUK
SEROTYI’ES
i$Si
FIG. 1. Ultrastructure of virions purified from cocultivation products of adenovirus t,ypes 3 and -1. Note the appearance of short (10 nm; short, arrows) and long (li-18 run; long arrows) fibers in almost all pttrfirlrs. Negnrivc contrasting with STS. X 400,000. The hnr represents 30 nm.
388
NORRBY
AND
GOLLMAR
FIG. 2. Ultrastructure of virions isolated from cocultivation products of adenovirus types 3 and 4 (a) and types 3 and 6 (b). In (a) four 10 nm (type 3) and two 17-18 nm (type 4) long fibers can be seen, whereas in (b) two fibers are 10 nm (type 3) and four fibers are 26-28 nm (type 6). Negative contrasting with STS. X 400,000. Bars represent 30 nm.
Norrby and Wadell, unpublished) were capable of coating the surface of homotypic, but not of heterotypic virions. The interaction between virions from cocultivation experiments including various pairwise combinations of type 4 and/or selected members of subgroups I and II and specific antihexon antibodies were examined in the electron microscope. Antihexon antibodies were employed in quantities giving a complete coating of control Cons at a particle concentration corresponding to that of the test sample. In some experiments virion preparations absorbed wit,h red cells of one type or another were studied. The degree of ant’ibody coating of the surface of virions as well as the extent of aggregation of virions was determined. Analyses of the degree of antibody coating revealed that particles in a free-lying form or more frequent’ly in aggregates could display what was interpreted to represent only a partial coating of their surface (Figs. 3a and b). Part.icles of this kind occurred not only in the center of aggregates, in which case they might have represented trapped particles, but also in their periphery (arrows, Fig. 3). Under comparable conditions antibody-coat,ed particles were partially more frequently encountered in studies of virions produced in cocultivation of relatively closely related serotypes of virus, e.g., types 3 and 4, than in studies of other types such as 3 and 9 or 13. The extent of aggregation of virions by
different antihexon sera also indicated a somewhat higher tendency for serotypes belonging to the same subgroup to give hexon mosaics, than for serotypes of two different subgroups. In experiments with cocultivation products of adenovirus types 3 and 4 in about equal yields, antisera against hexons of both types aggregated the majority of all virions. A somewhat smaller percentage of all virions from cocultivation experiments with type 3 and types 9 or 15 were aggregated under the corresponding conditions. Table 3 illustrates findings in an experiment with adenovirus serotypes 3 and 15 in which the contribution to cocultivation product’s is somewhat higher for t,ype 3 than for type 15. After absorption with red cells of one kind, remaining virions were found to associate preferentially with antihexon sera against the serotype agglutinating red cells of another kind. Thus virions (rat HA) remaining after absorpt,ion with monkey red cells were coated and aggregated predominantly by an ant.iadenovirus type 15 antihexon serum (Table 3). This finding suggests that virions containing fibers or probably pentons of only one type tend to contain hexons of the same t,ype. Polymeric Forms of Soluble Components Derived from Mixed Infections with Two Di$erent Serotypes
Polymeric forms of soluble components, e.g., dodecons and dimers of pentons or
PROINCTS
FROM
COCULTIVATION
OF Al~ENOVII:UH
HEROTYI’JW
339
FIG. 3. Electron microscopy of the association between specific antihrxon antibodies and virions plkfic,d from a mixed infection with adenovirus types 3 atld 4 (a and hj and a single infection with type 1 (c). The control (c) displays a complete coating by homotypic antihexon antibodies. Al?tisercml against type 3 hrxolls gave neither antibody coating nor aggregation of the same virions. The reverse> sit elation w:w t YIN for the second control of the experiInerrt, type 3 viriolls (Irot included in the figrlrc%. Cocultivatiou products in (a) and (b) were incubated with sera against hexons of types 3 at~d 1. respcsclively, Imder condition (particle to antibody ratio) identical with t,hose of the c~)ntrols. Note the :ip pearancr of somr poorly antibody-coat,ed particles (arrows) itI (a) and the small nrunber of ant itrod!. moles-rdcs visihlc ill (b). Xegat,ive contrasting with STS. X 130,O~)O.
fibers, occur in characteristic fashion among different serotyprs of human adenoviruses (cf. Sorrby, 196%). In order t,o characterize solublr> components among products from
material barcocult ivat ion experiments, vested from the low density region of discont,inuous CsCl gradients MW dialyzed then ~ubjrctetl to ZOTM~ against I’?%3 and
390
NORRBY
AND
GOLLMAR
TABLE
3
INTERACTIONS BETWEEN PURIFIED VIRIONS FROM A COCULTIVATION EXPERIMENT M~ITH TYPES 3 AND 15 AND FROM SINGLE INFECTIONS WITH EACH SEROTYPE AND ANTIHEXON SERA AGAINST BOTH SEROTYPES Preparation virions
of
Cocultivation products”
Absorption with red cells from-
Addition of antihexon serum against type
-b
3
-
15
Monkey
3
Relative amount of virions in aggregates Almost
all
5(t7570 of all particles Few
Extent of coating of surface of virions with IgG antibodies Some virions only partly Only partial
in aggregates coated coating of
virions in aggregates Only sparse coating
of virions
in aggregates Monkey Rat Rat
15 3 15
All All Few
Partial t’o complete Complete Only partial coating
of vi-
rions in aggregates Type
3 (control)
-
Type
15 (control)
-
3 15 3 15
All No No All
Complete None None Complete
a Absorptions were repeated until all HA for a certain kind of red cells had been removed. b No absorption. c Hemagglutination and virion HIC tests with soluble components from the harvest of cocultivation products examined revealed the presence of about four times more type-specific components of type 3 ihan of type 15.
centrifugation at a preselected time and speed. Cocultivation of serotypes 3 and 11 caused the appearance of dodecons agglutinating monkey erythrocytes at +4” (type 11 HA) which sedimented with a rate (SO-SO S) similar to that of type 3 dodecons (Fig. 4). In the control mixture, type 11 dodecons sedimented with a rate of 100-120 S, as was previously described (Norrby, 1968b). Spontaneously occurring soluble complete HAS of types 4 and 16 predominantly are represented by dodecons (Wade11 et al., 1967) and fiber dimers (Norrby and Skaaret, 1968), respectively. However after cocultivation of these two serotypes it was found that the monkey HA, i.e., type 16 oligomers, sedimented together with type 4 dodecons (Fig. 5). A similar effect was encountered when type 16 was propagated together with type 15, which by itself yields dodecons and fiber dimers (Norrby, 196%). Additional examples of a similar phenomenon were encountered in studies of other serotypes. The possible occurrence of dodecons including pentons of two serotypes was ana-
lyzed by erythroeyte absorption or adsorption-elution experiments and by electron microscopy. Absorption of dodecons from mixed infections, purified by zonal centrifugation, with one kind of red cells in a number of experiments caused a significant (4-fold or greater) reduction in the amount of HA for another kind of red cells. Furthermore under conditions which allowed adsorption-elution, e.g., analyses of cocultivation products of serotypes 3 and 4, it was found that the monkey cell eluate contained rat cell HA as described above for virions. The occurrence of dodecons containing two kinds of pentons, in some cases mainly pentons of a serotype which normally is not capable of forming this kind of polymeric structure (e.g., type 6) was confirmed by electron microscopy (Fig. 6). Properties of Monomeric Soluble Components Produced by Cocullivation of Two Serotypes of Adenovirus Physical characteristics of monomeric soluble components from single and mixed infections, were compared by anion exchange
PRO1 )UCTS
FROM
COCULTIVATION
HAU per 0.05 ml
256 -
B I
12864 32 ;/JJ~a,,,a,L -*-..+-. -- --x__._ <2 5 0 10 FRACTION
15
20
NUMBER
FIG. 4. Zonal centrifugation of soluble complete HBs from a mixed infection with adenovirus types 3 and 11 (A), and as a control the corresponding components from single infections with the same serotypes (B). HA activity at f4” (X----X ; type 11) and at 37” (O--O; types 3 + 11) have been recorded. The samples were centrifuged in a linear 5-20% sucrose gradient at 25,000 rpm for 6 hours (4”) in rotor SW 25 (Spinco). The bottom of the tube is to the left.
chromatography fractionation. In order to allow an analysis of monomeric soluble components, virions, and soluble complete HA were removed by consecutive centrifugations of material in discontinuous CsCl gradients and linear sucrose gradient,s, respectively, as described above. An analysis of hexons was performed with harvests from cult,ures mixedly infected with types 3 and 11, bot’h members of subgroup I, or with types 11 and 15, representing two different subgroups. The reason for choosing these combinations was a pract’ical one. Among different, serotypes characterized in this laborat,ory (cf. Norrby, 1969a) the position of hexons of t)ypes 3 or 15 and 11 in the elution diagram obtained by anion exchange chromatography are dist,inctly different. The results of an experiment with cocultivation products of types 3 and 11 are presented in Fig. 7. Hexons were monitored in CF test,s
OF ADENOVIRUS
SEROTYPES
391
with antiserum against type 2. Furt,hermore the hexon HIC test described under Materials and Methods was used to indicate the Dype-specific part of hexons of both types. After separation of cocultivation products. 4 peaks (NOS. I through IV) of hexon activities could be distinguished. The position of peaks I and IV corresponded to those of type 3 and type 11 hexons in the cont,rol mixture as confirmed by conductomet,ric measurements. The hexon HIC tests revealed the serotype identity of the t,wo peaks in both materials. Hexon peaks II and IT1 were found in cocultivation products, but not in control materials. Peak II, eluting immediately after t,he peak of type 3 hexons, contained mainly type 3 hexon HIC test positive material, but, also some of type 11 origin. The reverse sit)uation was true for hexons in peak III, which eluted closest to type 11 hexons. Fract’ionation and characterization of products of cocultivat’ion of t)ypes 11 and 15 also caused t’he appearance of :I barely detectable fraction of hexons eluting in a posiHAU per 0.05 ml 32~
16. 8. 4: <2
FRACTION NUMBER FIG. 5. Zonal centrifugat,ion of soluble complete HAS from a mixed infection with adenovirus types 4 and 16 (A) and as a control the corresponding components from single infections wit.h the same serotypes (B). The distribution of rat (X----X; type 4) and monkey cell HAS (O-------O; type 16) is illustrated. Conditions of cerrtrifugation as given in the legend of Fig. 1.
AND
GOLLMAR
terials from mixed and single infections could be demonstrated. DISCUSSION
FIG. 6. Ultrastructure of dodecons isolated by aonal centrifugation from cocultivation products ‘of adenovirus types 4 and 16 (a), types 3 and 6 (b), and types 6 and 11 (c). Note the appearance of fibers of varying lengths in individual dodecons as well as the participation of type 16 pentons (10 nm long fibers) and type 6 pentons (26-28 nm long fibers) in the formation of dodecon structures. The latter two serotypes under conditions of single infections produce dimers of pentons, but no dodecons. Negative contrasting with STS. X250,000. Bars represent 30 nm.
tion intermediate between those of “parental” hexons. The fractionated materials in Fig. 7 were also analyzed for the presence of types 3 and 11 pentons. Unfortunately, the latter pentons, as previously experienced (Norrby, 1968d), are labile under the conditions of fractionation and no satisfactory recovery could be obtained. Type 3 pentons, which were readily identified, displayed exactly the same position and distribution in the elution diagram obtained after fractionation of cocultivation products and of control material. A further analysis of the effect of cocultivation on charact’eristics of pentons was performed with products of mixed infections with types 3 and 15. In this system pentons are readily recovered and appear at distinctly different positions. No difference between the distribution of pentons in ma-
A mixed infection with two different adenoviruses provides an opportunity for peptides of these two serotypes to assemble into hybrid capsomers. The present findings demonstrate that cocultivation products of types 3 and 11 included two populations of hexons (peaks II and III in Fig. 7), which were not present in control materials. These hexons carried type-specific antigenic components of both types and eluted at a position intermediate between that of hexons of the two “parental” types. It could be argued t.hat peaks II and III contain aggregates of hexons of types 3 and 11. However, this explanation seems less likely since (a) a similar appearance of hexons in the elution diagram was never encountered in fractionation of control materials and (b) there was a clear-cut preference for hexons occurring only among cocultivation products to appear in two readily distinguishable peaks of intermediate position. This preference may be of some significance in relationship to the structure of hexons. One might speculate that hexons of peak II are composed of two adenovirus type 3 subunits and one type 11 subunit, of the size described by Xaizel (1968) and that for hexons of peak III the relative occurrence of subunits might be the converse. Structural hexon proteins of adenovirus types 11 and 15, which represent two different subgroups, also seemed to have some capacity to form hybrid hexons although in a much lower frequency than those of types 3 and 4. This is of interest since, on the basis of the well pronounced immunological dissimilarities between hexons of types 11 and 15 (Norrby and Wadell, 1969), one would anticipate the occurrence of corresponding distinct differences between the polypeptides of their hexons. Monomeric soluble components of mixed polypeptide origin theoretically might also occur in cocultivation products such as fibers, vertex capsomers or the joint structure, pentons. No evidence for the occurrence of this was found. Unfortunately no
PKOl>UCTS
FROM
CFU per 0.025ml
COCULTIVATION
OF rll)ENOVIlZUS
SlSI~OTYl’I~:S
393
,
Hexon
HICU IV
I ::
32.
FIG. 7. Anion exchange chromatography separation of soluble components (excluding rnost~ dodefrom a mixed infection wit.h adenovirus types 3 and 11 (upper part of diagram) aJJd a corltrol mixture of the corresponding soluble components of the same two serotypes (bottom part of diagram). A linear gradient of 0.1 to 0.3 M N&l in 0.04 M Tris-HCl buffer, pH 8.4, was used for elut’ion of components from t,he column. The following activities were determined: group-specific hexon CF antigen (0 ---O) identified with an ant,iadenovirus type 2 hexon serum and type-specific adenovirus 3 (U---W) and 11 (A-----A) hexon antigens demonstrated in hexon HIC tests (see Materials and Methods). P denotes the position of type 3 pentons and D that of trace amounts of type 3 dodecons remaining in the samples. The position of different peaks in the two fractionations were monitored by conductometric measurements, whereafter the distribution of fractions m-ere adjusted to comparable scales. cons)
mixed infectlion lvith two serotypes belonging to the same subgroup suitable for an analysis of the effect, on both kinds OF pentons was available. However, in case a mixing between vert,ex capsomers and fibers of, for example, types 3 and 11 into penton hybrids could occur one would have anticipated to see some modification of the distribut,ion of penton incomplete HAS in the anion exchange chromatography experiment illustrat’ed in Fig. 7, since type 11 pentons (in cases when this component is detected) elute at rela-
tively low molarity of S&l. This was not found. lcurthermore, experiments employing serotypes representing two different subgroups in no case suggested the occurrence of modified populations of pentons. It is possible that t,he degree of fitting bet,ween polypeptides of different serotypes, i.e., t,he possibilitjy of occurrence of hybrid components, is much lower in pentons than in hexons, since the former represent a more specialiaed structure (cf. Sorrby, 1970). Pentons and hexons of two serotypc+ were
394
NORRBY
AND
found capable of assembling into mixed products of both polymeric soluble components and intact capsids. The former case concerns the formation of dodecons containing pentons of two types, in some cases including pentons of a serotype which normally cannot produce a soluble polymeric structure of this kind. It has been suggested, mostly from indirect evidence, that the dodecon structure includes a core protein (cf. Norrby, 1968b). The character of this postulated core protein probably is of decisive importance for the kind of soluble polymeric structures produced by individual serotypes. Dodecon core protein of one serotype may allow pentons of a different, normally nondodecon-producing structure to aggregate into a polymeric product of this kind. The suggested preference for mixed assembly of pentons and hexons of relatively closely related serotypes into intact capsids is not unexpected. It has been demonstrated that both vertex capsomers and hexons display a wide range of antigenic specificities including type- (demonstrated for hexons only), subgroup-, intersubgroup-, and groupspecific components. It seems likely that the degree of fitting between capsomers should be higher if they belonged to the same subgroup than if they were more distantly related. It is of interest that a number of successful experiments were made to produce capsid mosaics by double infections with serotypes 3 and 4. Type 4 belongs to Rosen’s subgroup III, but it has been found that with regard to immunological characteristics of both hexons and vertex capsomers this serotype resembles members of subgroup I (Norrby and Wadell, 1969; Wade11 and Norrby 1969), which includes type 3. The aim of the present study was to characterize capsid products derived from simultaneous multiplication of two serotypes of human adenoviruses. No attempts were made to perform subsequent passages of progeny material. Thus virions, phenotypitally mixed in such a way that they contain the nucleic acid (alternatively nucleoprotein) of one type and capsid of another type were not identified. Furthermore, it cannot be concluded to which extent the mixed capsid products identified, were the result of genotypic or phenotypic interactions. However,
GOLLMAR
the high frequency of occurrence of mixed capsids in certain combinations of serotypes strongly suggest a phenomenon of phenotypic mixing. Phenotypic mixing of viruses was first conclusively demonstrated by Streisinger (1956). He showed that the progeny of simultaneous infection with the Escherichia coli phages T2 and T4 contained virions which were neutralized by antisera against both serotypes. On further passage, these virions yielded only T2 and T4 progeny. In later studies phenotypic mixing on the capsid level was also demonstrated with animal viruses; ECHO-virus type 7 and Coxsackievirus type A9 (Ito and Melnick 1959), poliovirus types 1 and 2 (Ledinko and Hirst, 1961) and the simian and human adenoviruses, types SA7 and 2, respectively (Altstein and Dodonova, 1968). In the latter study as much as 90 % of the virions were phenotypically mixed to an extent which allowed them to be neutralized by antisera against both serotypes. Experiments in this laboratory have demonstrated that capsid mosaics of the kinds described above can be identified also among virions from cocultivation of certain human adenoviruses with monkey or dog adenoviruses (Norrby, unpublished). ACKNOWLEDGMENTS Mrs. Halyna Marusyk gave a valuable assistance in electron microscopic studies and Mr. Thamas Varsanyi in chromatographic separation experiments. Excellent technical assistance was provided by Mrs. Margareta Jurstrand. REFERENCES ALTSTEIN, A. D., and DODONOVA, N. N. (1968). Interaction between human and simian adenoviruses in simian cells: Complementation, phenotypic mixing and formation of monkey cell “adapted” virions. Virology 35, 248-254. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167-182. GINSBERG, H. S. (1958). Characteristics of adenoviruses. III. Reproductive cycle of types 1 to 4. J. Ezp. Med. 107, 133-152. ITO, H., and MELNICK, J. L. (1959). Double infection of single cells with ECHO 7 and Coxsackie A9 viruses. J. Exp. Med. 109, 393-406. LEDINICO, N., and HIRST, G. K. (1961). Mixed infection of HeLa cells with polioviruses t,ypes 3 and 2. Virology 14, 207-219.
PRODUCTS
FROM
COCULTIVATION
MAIZEL, J. V., JR., WHITE, D. O., and SCHARFF, M. D. (1968). The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A and 12. Virology 36, 115-125. NOHHBY, E. (1966a). The relationship between the soluble antigens and the virion of adenovirus type 3. II. Identification and characterization of an incomplete hemagglutinin. Virology 30, 608-617. NORHBY, E. (1966b). The relationship between the soluble antigens and the virion of adenovirus t.ype 3. I. Morphological characteristics. Vilirology 28, 236-248. NORRBY, E. (1968a). Biological significance of structural adenovirus components. Curr. Top. Microbial. Immun. 43, l-43. N~R~BY, E. (196810). Comparison of soluble components of adenovirus t,ypes 3 and 11. J. Gen. Viral. 2, 135-142. N~HI~BY, E. (196%). Comparative studies on the soluble components of adenovirus types 9 and 15 and the intermediat.e strain 9-15. J. Viral. 2, 1200-1210. NORRBY, E. (1968d). Identificat,ion of soluble romponents of adenovirus type 11. J. Gen. Viral. 2, 123-134. NORRBY, E. (1969a). The structural and functional diversit,y of adenovirus capsid components. J. Gen. Viral. 5, 221-236. NORRBY, E. (1969b). The relationship between the soluble antigens and the virion of adenovirus fype 3. IV. Immunological complexity of soluble components. Virology 37, 565-576. NCIRRBY, E. (1969c). Capsid mosaics of intermediate strains of human adenoviruses. J. Viral. 4, 657-662. NOHHBY, E. (1970). Adenoviruses. In “Comparative Virology” (K. Maramorosch and E. Kurst.ak, eds.). Academic Press, New York, in press. N~RRBY, Is., and SKAARET, P. (1967). The rela-
OF ADENOVIRUS
SEROTYPES
3%
tiollship between the soluble antigens and the virion of adenovirus type 3. III. Immunological identification of fiber antigen and isolated vertex capsomer antigen. Virology 32, 489-502. NORRRY, E., and SKAARET, P. (1968). Comparison between soluble components of adenovirua types 3 and 16 and of the intermediate strain S-16. (the San Carlos agent). ViroZogv 36, 201-m211. NORRBY, E., and WADELL, G. (1969). IIII~IUIIOlogical relationships between hexons or cchrt ain human adenoviruses. J. ViroZ. 4, 663-670. NORRBY, E., NYI~ERG, B., SKAAXET. P., and LENGYEL, A. (1967). Separation and characsterization of soluble adenovirus type 0 caomponents. J. Viral. 1, 1101-1108. NORRBY, E., W~DI~LI,, G., and MARUS~K, Ii. (1969a). Fiber-associated incomplete and cornplete hemagglutinins of adenovirns t,vpe ii. ,4rch. Ges. Virusjorsch. 28, 239-244. I~., and HAMMARsKJ~~LI), NORHHY, E., MARUSYH, M.-L. (196913). The relationship between the soluble antigens and t,he virion of adenovirns type 3. V. Identification of antigen specificities available at the surface of virions. Virolog,r/ 38, 477482. ROSEN, L. (1960). A hemagglut,ination-inhibition technique for typing adenoviruses. A,,lrr. J. Hyg. 51, 120-128. SIMON, M. (1962). Hemagglutination experiment,s with cert,ain adenovirus type strains. Acfa Microbial. Hung. 9, 45-54. STREISINGER, C. (1956). Phenotypic mixing of host range and serological specificities in bacteriophage T2 and T4. Virology 2, 388-398. E. (1969). ImmunoWADELL, G., and NORRBY, logical and other biological characteristics of pentons of human adenoviruses. J. Viro?. 4, 671-680. WADELL, G., NORRRY, E., and SCH~NNING, I:. (1967). Ultrastructure of soluble ant,igens and the virion of adenovirus type 4. .4rch. Ges. Virusjorsch. 21, 234-242.