Physical mapping of the genes controlling adenovirus paracrystal formation1 JOSEPHWEBER
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L)c;prrrlet?~etrlrle Microbiologie, Cetrrre Ho.\pirtrlic,r Utri~~ersi~rrit~e rle I'Utril~ersiritle Sherhrooke, P.Q., Ctrtrtrtltr J I H jN4 Accepted August 8. 1978 WEBER,J. 1978. Physical mapping of the genes controlling adenovirus paracrystal form;~tion. Can. J. Microbiol. 24: 1381-1386. The genetic properties and physical mapping of the genes controlling adenovirus-induced paracrystals were examined by means of a new, simplified technique. The induction of paracrystals was virus-strain dependent and blocked by several temper:ttl~re-sensitivemutations. Using interserotypic recombinants between a paracrystal-producing and n non-producing stlain of :~denovirus,the principal pztlacrystal determining factor was mapped between coordinates 30 and 44 on the 100-unit adenovirus genome. WEBER.J . 1978. Physical mapping of the genes controlling adenovirus pal-:tcrystal fol-mation. Can. J . Microbiol. 24: 1381-1386. Les PI-oprietesgenetiq~reset la cartographie physique des genes controlants des pamcristaux induits par I'adenovir~~s ont ete examinees au moyen d'une nouvelle technique simplifiee. Nous avons determine que I'induction des par-acristaux etait contr6lee par la souche virale et etait bloquee par plusieurs mutations thermosensibles. En utilisant des I-ecombinants interserotypiques entre une souche productrice de p:u.acristaux e t une souche non-productrice, le detelminant principal contr-6lant de In production des paracristaux a ete localise entre les coordonnes 30et 44 sur le genome d':rdenovirus comprennent 100 unites.
Introduction Infection by adenoviruses is frequently accompanied by the appearance of intl-anuclear paracrystalline inclusion bodies during the late phase of the lytic cycle. Paracrystals have been reported to be associated with human adenovirus types 2, 4, 6, 18 (Weber and Liao 1969);type 5 (Morgan et rrl. 1960); type 12 (Martinez-Palomo et 01. 1967); and the canine adenovirus ICHV (Givan er rrl. 1967; Matsui and Bernhard 1967). Weber and Liao ( 1969) have shown that paracrystal morphology is related to the subgroup of the inducing adenovirus. The Ad5induced pal-acrystals have been studied by the combined techniques of electron microscopy and optical diffraction (Carstens and Marusy k 1975) and crystallography (Lifchits et ul. 1975), showing that the basic repeating unit consists of a helical polyhedral tubule with an external diameter of 550A and an internal tubular fiber, arranged in rectangular symmetry. The induction of the paracrystals is probably independent of the host cell as they have been observed in HeLa, KB, HEp-2, Vero, LLC-MK2, hamster embryo, bovine kidney, and dog kidney cells (for references, see Weber and Liao 1969; Henry and Atchison 1971) and furthermore they are inte~feron-sensitive (Henry and Atchison, 1971). However, not every strain of the same serotype is capable of inducing the paracrystals, an observation potentially useful for subtyping 'This is paper XI1 in a series on "the genetic analysis of adenovirus type 2."
adenovirus isolates (Weber and Stich 1969). Paracrystal formation was found to be independent of virion assembly by studies using proflavin, arginine deprivation (Marusyk et 01. 1972), and ts mutants (Wills et rrl. 1973). The fragility of the paracrystals have precluded their purification thus far (Carstens and Ma]-usyk 1977), and for that reason most reports concerning their chemical nature were based on immunofluorescent-staining techniques with a variety of antisera. Such studies have suggested the presence of P-antigen and core protein VII (Marusyk et (11. 1972). Earlier reports postulated the involvement of the principal excess capsid proteins hexon, penton, and fiber (Boulanger et al. 1970; Henry and Atchison 1971). This study was undertaken to obtain additional information on the nature of the paracrystals using temperaturesensitive mutants and to attempt t o map it physically on the adenovirus genome. Materials and Methods Cplls rrtrd Viruses All experiments were conducted in HEp-2 cells grown in monolayer culture with Dulbecco modified minimal essential medium (DMEM). Wild-type (WT) :rdenovirus type 2 (Ad?) and its tempelxture-sensitive mutants have been described previously (Weber er (11. 1975). Adj-WT and jtsl, 5ts2,5ts22,5ts36, wer-e obtained from Dr. James Williams (Carnegie-Mellon University, Pittsburgh) and have been described (Russell er (11. 1974). The isolation of 1.ecombinont viruses fr-om crosses between Ad2 and Ad5 ts mutants has been described elsewhere as also the characterization of their DNA molecules (Hassell and Weber 1978). Stocks of all viruses were obt;~inedfrom plaques picked at 33°C and passaged in HEp-2cells thr-ee to four times at 33°C.
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C A N . J. MICROBIOL. VOL. 24, 1978
Sttritlitlg of Prrr.crcr.v.strr1.s A method previously described for the staining of virusinduced inclusion bodies was used (Weber 1972). HEp-2 cells (2 x lo6)were seeded into 80-mm pl;~sticpetri dishes containing 24 circular (12-mm diameter) coverslips. On the following day the coverslips were placed individually into 35-mm bacte~.iological grade petri dishes and 0.025 ml of a virus suspension was pipetted on top of the coverslip, taking care that s u ~ f r ~ tension ce kept the liquid on the coverslip. The petri dishes were incubated for 60min at 33'C. then 2 ml DMEM was added and the incubation continued for 3 days at 33°C or 2 days at 39°C. The coverslips were then rinsed with phosphate-buffered saline (PBS), fixed for lOmin in ethanol - acetic acid (3:1), air-dried, stained for 2-5 min with 2% aceto-orcein (GIBCO) and rinsed successively in 70% ethanol, 100% ethanol, ethanol-xylene ( \ : I ) , 100Yo xylene, and finally mounted in pernlount. By this procedure the int~inuclear paracrystals become cle~irly visible by phasecontrast microscopy.
TABLE1. The effect of mutations and temperature on paracrystal formation"
Electt~o~r Micr.o.scol~.v Infected cells were incubated :it 33"C, washed, and fixed in sitrr at 4'C with 3.5% glutal-aldehyde in phosphate buffer. The cells were then scraped off the petri dishes, pelleted, and postfixed in 1% osmium tetroxide for 1 h. Samples were dehydrated in acetone, embedded in Epon 812, polymerized at 80°C, secyl followed by lead citrate tioned, and stained with u ~ ~ n acetate (Carstens and Marusyk 1975).
synthesis, cells were infected with several temperature-sensitive mutants at the permissive (33°C) and the nonpermissive (39°C) temperature and the formation of PC was scored. The results are shown in Table 1. Adenovil-us type 2 and its temperature-sensitive mutants which are in different complementation groups (Begin and Weber 1975) do not induce PC. Adenovirus type 5 induces PC at both temperatures. The ts mutants show that the expression of late functions is necessary for PC formation. In particular, tsl, ts2, and ts22 gene functions are all required for PC formation. This implies that fiber, loOK, and hexon are involved either directly or indirectly in PC synthesis, and defects in any one of these three genes blocks PC synthesis. Evidently the fiber-defective ts22 has the most direct effect on PC formation as the mutation is sufficiently expressed to block PC even at the permissive temperature. Interestingly, reversion restores PC even at the pel-missive temperature. Interestingly, reversion restores PC formation at the nonpermissive temperature.
Results Properties of Pcrrcrcrystcrls The demonstration of paracrystals (PC) until now has been based on electron microscopy of thin sections (Fig. 1). Because the present mapping technique involved the analysis of a large number of samples, a more rapid and simplified technique was developed for the demonstration of PC. The new technique (see Materials and Methods for details) which was checked by electron microscopy and is based on a simple cytological staining procedure of fixed cells with acetoorcein requires few cells, very little virus, and a phase-contrast microscope. Figures 2, 3, and 4 show the appearance of adenovirus-infected cells with virus strains which do not induce PC or induce small 01-large PC, respectively. Using this technique, the following general observations could be made: (i) the formation of PC is temperature-sensitive, a greater proportion of cells forms PC at 33°C than at 39°C (see also Table l), (ii) cells may contain several PC, in various orientations and even in overlapping array, (iii) large PC deform the nucleus, (iv) PC-positive nuclei generally contain less densely staining inclusions (see Fig. 4), (v) PC formation has no significant effect on virus production, as the yields of paracrystal-forming recombinants and Ad5 were of the same magnitude as the paracrystal-negative recombinants and Ad2 (results not shown). The efyect of ts Mritcrtions To determine which genes are required for PC
pl, Paracrystals -
Virus
33°C
39°C
Phenotype of mutant DNA-negative Fi bre-negative Assembly negative Hexon-negative W T revertant of ts36 -
Various late mutants "Both small- and large-type paracrystals were counted. The percentage was determined from the infected population o f cells.
Mcrpping oftlze Pcrrcrcrystul The general procedure for the mapping of adenovil-us mutants and proteins has been described elsewhere (Hassell and Weber 1978). Briefly, interserotypic recombinants are isolated from crosses between Ad5 and Ad2 ts mutants and theirgenomes are mapped with site-specific restriction endonucleases which recognize unique, serotype-specific cleavage sites on the recombinant molecules. Thirty-six recombinants from 10 different crosses have been mapped by this technique (Hassell and Weber 1978). The physical maps of these recombinants are shown in Fig. 5 . T h e serotype-specificity of five viral-coded proteins has also been determined (Weber and Hassell, manuscript in preparation) and is shown in Figure 5. The percentage of PC-containing nuclei among the in-
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'OSZ x .(MO.I.IB) S I ~ ? ] S / ( . I ~UI ?! B. I B U O~~slla3jo %OS l n o q v .sn.l!Aouapl?jo U ! I ? J I ~ Su!tu.~o~-ll?ls/(.~x?.~~d B q l ! pa)3aju! ~ slla3Jo asul?.~l?addl? [es!d/(] aqL ' 5 sn.l!Aouapl? q ] ! palsaju! ~ sllas z - d g 'P ~ '913 x '(SM~.I.II?) !alsnu atuos u! sll?)s/(.~sl?.red IIl?wsaqq)a ~ ' 1 Io-[ sa.1 ~ ~ I ! Mp a l s a p ! sllas Z - d g 'f~ ,513.OSZx .uo!)saju! . I ~ I J sAlzp ~? 2 sllas pau!l?ls u!as.~o-olasv .p 'f 'sS!g u! !a13nu u! uaas sll?)s/(~se.~l?d aq] q l ! pasnjuos ~ aq lou plnoqs salonsl?A s!tusl?ldo]As aqL 'alqaas!lou aJu suo!snlsu! .~l?a[snu~u]u! ~ ~ ' s ! d aq] / ( l Lluo 's[els/(.lsl?.~ed JO U O ! I I ? ~ IaOqJ asnpu! ~ I O U saop 2 sn.l!Aouapl?jo u!e.~lss ! q ~ .s[las 2 - d palsaju! ~ ~ 2 sn.l!Aouapo J O qd~?.rSo.~s!tuo]oqd ]sl?~)uos-asl?qd .Z '513' 0 0 s ~x 'auo ]saZ.~l?laq] 01 Zu!lu!od Mo.l.ra aq] ' s I I ? ) s / ( . I ~ I ? . I u~ ?! I~? ] u ~lal3nu ~ q]oa .uo!)saju! . I ~ I J ~s/(l?p ? z S I B ) S / ( . I ~ \ JO ? . Iasulr.ll?adde B~ aqL q a 3 2 - d palsaju! ~ ~ s sn.l!Aouapr?Jo qdn.~So.ls!muo.11salg '1 .513
.
. . . . .. ... . .. . ... . .. . . . ~ .. .
...... . .. . . ..
C A N . J . MICROUIOL. VOL.. 24. 1978
0
Enzyme map position Proteins
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Cross
rec
30
43
44
55
57
59
69
71
80
81
90
V
111
I1 lOOK I V
Paracrystal
%
Morph.
FIG.5. Mapping of the paracrystal. For details of the mapping method see text and reference (Hassell and Weber 1978). The enzyme map positions unique to Ad2 or Ad5 were obtained with Bam H1, EcoRI, Hind 111, and SmaI. The proteins appear in the same order as on the genome. The percentage of palxcrystals was determined from among the inclusion body containing cells only. The morphology of the paracrystals was scored as L, large: S, small; SL, both large and small typesobserved.
fected cells was scored (second last column, Fig. 5). The correlation of the presence o r absence of paracrystals with particular map positions and viral proteins was examined (Table 2). It is apparent that PC synthesis cannot be related to a single map position or viral protein. Nevertheless when the DNA between map positions 30 and 44 contains Ad5 sequences there is a greaterchance that PC are expressed, and conversely when these sequences are of Ad2 origin the probability that no PC are formed is greater. Among the five proteins examined the best correlation with paracrystals is
found with protein V, which has been mapped into the region between 30 and 44, thereby corroborating the above conclusion. Discussion By means of a new, simplified technique this paper has shown that the synthesis of adenovirusinduced intranuclear paracrystals is genetically controlled. The formation of paracrystals at the nonpermissive temperature is blocked by temperature-sensitive mutations in genes coding for the lOOK protein (5tsl), hexon (5ts21, and fiber
WEBER
TABLE2. Correlation between the presence of the paracrystal a n d recombinant virus properties
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Recombinant property
Correlation. 'Z
M a p position 30 43 30+43 44 43 44 55 57 59 55 57+ 59 69 71 69+71 80 81 80+81 90
+
+
Proteins V 111 11 IOOK IV NOTE:All recombinants which induced either large or small o r boll1 types of paracrystals were scored positive. When two o r three map positions were ehamined as a group, a single Ad5 map position and the presence of paracrystals \\.as scored as a positive correlation while a negative correlation would be one of the following: paracrystal-positive but both map positions Adz; paracrystal-negative but at least one map position Ads. Each map position may contain either Ad2 o r Ad5 seqilences as each protein may be either Ad2- o r Ad5-specific. The primary data from which this table derives appears in the summary Fig. 5.
(5ts22). In agreement with previous findings (Wills et (11. 1973), paracrystal formation is evidently a late
gene function, because, in addition to these late mutants, the early, DNA-negative mutant 5ts36 also blocks their synthesis. These results do not imply, however, that the gene-products of 5ts1, 5ts2, and 5ts22 are necessarily integral components of the paracrystals. The finding that the fiberdefective mutant 5ts22 blocks paracrystal formation even at the pel-missive temperature confirms a previous report (Wills et (11. 1973) and suggests that the complete integrity of this protein may be an absolute requirement for paracrystal formation. The mapping data showed that the region of the genome between 30 and 44 has relatively greater importance than other I-egions of the genome in determining the formation of paracrystals. However, since the data were not unequivocal, it appears that paracrystal formation may be controlled by several genes. We have already seen the importance of the fiber gene. Marusyk et 01. (1972) claimed tha't anti-VII serum and phenanthrenequinone (see also Henry and Atchison 1971), an
1385
arginine-specific stain, react with paracrystals. We have shown that protein VII is derived from its PI-ecursorby proteolytic cleavage during the maturation of young virions to mature virions (Weber 1976; Bhatti and Weber 1978). I t is likely therefore that the paracrystals in fact contain the precursor PVII which is known to react with antiserum to VII. Protein PVII cannot be the only protein present in the paracrystals, because there doesn't appear to be a sufficient quantity in infected cells to provide for the assembly of such large crystals without severely restricting virion assembly. Hexon and fiber, which are synthesized in huge excess by infected cells, appear to be ideal candidates as paracrystal constituents. Genetically both appear to be necessary for paracrystal formation (Table 1). Unfortunately antiserum to neither protein reacts with paracrystals (Henry and Atchison 1971; Marusyk et (11. 1972). Non-reactivity does not prove these proteins are absent from the paracrystals. It is conceivable that the antigenic determinants are buried within the crystal and are consequently unable to react with antibody molecules. Furthermore if hexon were organized as monomers within the crystal, antibody prepared against the native trirner antigen would not cross-react (Stinski and Ginsberg 1974). The coding region of protein PVII has been mapped near or into the interval most important for paracrystal formation (i.e. 30-44). The observed ambiguity in the mapping may be due to a complex interaction of several gene-products such as PVII, hexon, and fiber in the formation of paracrystals. To test whether certain combinations of Ad2 and Ad5 genes may result in paracrystal formation would require more map positions and knowledge of the precise location of structural genes. The present data are insufficient in this regard. A definite determination of paracrystal protein composition will have to await the development of a technique for their selective extraction and purification. Acknowledgments The technical assistance of Lise Clavet is gratefully acknowledged. Dr. J. Weber is a Research Scholar of the National Cancer Institute of Canada (N.C.I.C.). This work was supported by a grant from the N.C.I.C. BEGIN,M., and J. WEBER.1975. Genetic analysis of adenovirus type 2 1. Isolation and genetic characterization of ternperature-sensitive mutants. J. Virol. 15: 1-7. BHATTI,A. R.. and J. WEBER.1978. Protease ofadenovirustype 2. In vitro processingofcore protein. Biochem. Biophys. Res. Commun. 81: 973-979.
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BOULANGER, P. A., G. TORPIER, and G. BISERTE. 1970. Lnvesti- MORGAN, C.. G. G O D M A NP., B R E I T E N F E Land D , H. ROSE. gation on intlxnuclear paracrystalline inclusions induced by 1960. A co~.relativestudy by electron microscopy and light adenovirus 5 in KB cells. J . Gen. Virol. 6: 329-332. mic~.oscopyof the development of type 5 adenovirus. J. Exp. Med. 112: 373-381. CARSTENS, E.. and R. MARUSYK. 1975. An image analysis study , J. F. W I L L I A M S1974. . of adenovirus type 5-induced crystalline inclusions. J. Gen. RUSSELL,W. C., J . J. S K E H E Land Virol. 29: 249-256. Chalxcterization of tempelxture-sensitive mutants of aden1977. Crystallization and reaggregation of adenovirus ovirus type 5: synthesis of polypeptides in infected cells. J . type 5 structul-al components from infected cell extracts. J. Gen. Virol. 24: 247-259. Gen. Virol. 34: 541-545. S I - I N S KM. I , F., and H. S. GINSBERG. 1974. Antibody to the type 5 adenovirus hexon polypeptide: detection of nascent GIVAN.K.. C. T U R N B U Land L , A. M. JESEQUEL. 1967. Pepsin digestion of virus p>u.ticles in canine hepatitis using eponpolypeptides in the cytoplasm of infected KB cells. Intervirology, 4: 226-236. embedded material. J. Histochem. Cytochem. 15: 688-694. HASSELL,J . A,. and J. WEBER.1978. Genetic analysis of WEBER,J. 1972. Titration of adenovirus by counting cells conadenovirus type 2 VIII. The physical locations of temtaining virus-induced inclusion bodies. Appl. Microbiol. 23: pelxture-sensitive mutations. J . Virol. 28(3). In press. 1025- 1026. 1976. Genetic analysis of adenovirus type 2. 111. H E N R YC, . J., and R. W. ATCHISON. 1971. Paracrystal formaTemperature-sensitivity of processing of viral proteins. J. tion in cell cultures infected with adenovirus type 2. J. Virol. 8: 842-849. Virol. 17: 462-471. , G. KHITTOO.1975. Genetic analysis L I F C H I ' ~AS , , A. R I M S K YG. , TORPIER, and P. BOULANCER.WEBER,J., M. B E G I Nand of adenovirus type 2.11. Preliminary phenotypiccharacteriz;i1975. Crystallographic study of an adenovirus-induced protein crystal in KB cells: n structural model. Acta Cryst;lllogr. tion of tempelxture-sensitive mutants. J . Virol. 15: 1049B31: 441-445. 1056. MARTINEZ-PALOMO, A,, J . LE BUIS,and W. BERNHARD. 1967. WEBER, J . and S. K . L I A O .1969. Light and electron microscopy of virus-associated intranuclear paracrystals in cultured cells Electron microscopy of adenovirus 12 replication. I. Fine infected with type 2.4.6, and 18 human adenovil.uses. Can. J. structul-al changes in the nucleus of infected KB cells. J. Virol. 1: 817-829. Microbiol. 15: 841-845. MARUSYK, R., E. NORRBY, and H. MARUSYK. 1972. The rela- WEBER, J . . and H. F. S.I.ICH.1969. Electron microscopy ofcells tionship of adenovirus-induced pzu.acrystalline structure to infected with adenovirus type 2. J. Vil.01. 3: 198-204. the virus core protein(s). J. Gen. Virol. 14: 261-270. WILLS, E. J., W. C . RUSSELL,J. F. W I L L I A M S1973. . Adenovirus-induced cryst;ils: studies with temperatureMATSUI,K., and W. B E R N H A R D 1967. . Modifications ~rltl-astructurales induites par le virus de I'hepatite canine dans les sensitive mutants. J. Gen. Virol. 20: 407-412. cellules de rein dechien. Ann. Inst. Pasteur, Paris, 112: 773.
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L)c;prrrlet?~etrlrle Microbiologie, Cetrrre Ho.\pirtrlic,r Utri~~ersi~rrit~e rle I'Utril~ersiritle Sherhrooke, P.Q., Ctrtrtrtltr J I H jN4 Accepted August 8. 1978 WEBER,J. 1978. Physical mapping of the genes controlling adenovirus paracrystal form;~tion. Can. J. Microbiol. 24: 1381-1386. The genetic properties and physical mapping of the genes controlling adenovirus-induced paracrystals were examined by means of a new, simplified technique. The induction of paracrystals was virus-strain dependent and blocked by several temper:ttl~re-sensitivemutations. Using interserotypic recombinants between a paracrystal-producing and n non-producing stlain of :~denovirus,the principal pztlacrystal determining factor was mapped between coordinates 30 and 44 on the 100-unit adenovirus genome. WEBER.J . 1978. Physical mapping of the genes controlling adenovirus pal-:tcrystal fol-mation. Can. J . Microbiol. 24: 1381-1386. Les PI-oprietesgenetiq~reset la cartographie physique des genes controlants des pamcristaux induits par I'adenovir~~s ont ete examinees au moyen d'une nouvelle technique simplifiee. Nous avons determine que I'induction des par-acristaux etait contr6lee par la souche virale et etait bloquee par plusieurs mutations thermosensibles. En utilisant des I-ecombinants interserotypiques entre une souche productrice de p:u.acristaux e t une souche non-productrice, le detelminant principal contr-6lant de In production des paracristaux a ete localise entre les coordonnes 30et 44 sur le genome d':rdenovirus comprennent 100 unites.
Introduction Infection by adenoviruses is frequently accompanied by the appearance of intl-anuclear paracrystalline inclusion bodies during the late phase of the lytic cycle. Paracrystals have been reported to be associated with human adenovirus types 2, 4, 6, 18 (Weber and Liao 1969);type 5 (Morgan et rrl. 1960); type 12 (Martinez-Palomo et 01. 1967); and the canine adenovirus ICHV (Givan er rrl. 1967; Matsui and Bernhard 1967). Weber and Liao ( 1969) have shown that paracrystal morphology is related to the subgroup of the inducing adenovirus. The Ad5induced pal-acrystals have been studied by the combined techniques of electron microscopy and optical diffraction (Carstens and Marusy k 1975) and crystallography (Lifchits et ul. 1975), showing that the basic repeating unit consists of a helical polyhedral tubule with an external diameter of 550A and an internal tubular fiber, arranged in rectangular symmetry. The induction of the paracrystals is probably independent of the host cell as they have been observed in HeLa, KB, HEp-2, Vero, LLC-MK2, hamster embryo, bovine kidney, and dog kidney cells (for references, see Weber and Liao 1969; Henry and Atchison 1971) and furthermore they are inte~feron-sensitive (Henry and Atchison, 1971). However, not every strain of the same serotype is capable of inducing the paracrystals, an observation potentially useful for subtyping 'This is paper XI1 in a series on "the genetic analysis of adenovirus type 2."
adenovirus isolates (Weber and Stich 1969). Paracrystal formation was found to be independent of virion assembly by studies using proflavin, arginine deprivation (Marusyk et 01. 1972), and ts mutants (Wills et rrl. 1973). The fragility of the paracrystals have precluded their purification thus far (Carstens and Ma]-usyk 1977), and for that reason most reports concerning their chemical nature were based on immunofluorescent-staining techniques with a variety of antisera. Such studies have suggested the presence of P-antigen and core protein VII (Marusyk et (11. 1972). Earlier reports postulated the involvement of the principal excess capsid proteins hexon, penton, and fiber (Boulanger et al. 1970; Henry and Atchison 1971). This study was undertaken to obtain additional information on the nature of the paracrystals using temperaturesensitive mutants and to attempt t o map it physically on the adenovirus genome. Materials and Methods Cplls rrtrd Viruses All experiments were conducted in HEp-2 cells grown in monolayer culture with Dulbecco modified minimal essential medium (DMEM). Wild-type (WT) :rdenovirus type 2 (Ad?) and its tempelxture-sensitive mutants have been described previously (Weber er (11. 1975). Adj-WT and jtsl, 5ts2,5ts22,5ts36, wer-e obtained from Dr. James Williams (Carnegie-Mellon University, Pittsburgh) and have been described (Russell er (11. 1974). The isolation of 1.ecombinont viruses fr-om crosses between Ad2 and Ad5 ts mutants has been described elsewhere as also the characterization of their DNA molecules (Hassell and Weber 1978). Stocks of all viruses were obt;~inedfrom plaques picked at 33°C and passaged in HEp-2cells thr-ee to four times at 33°C.
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C A N . J. MICROBIOL. VOL. 24, 1978
Sttritlitlg of Prrr.crcr.v.strr1.s A method previously described for the staining of virusinduced inclusion bodies was used (Weber 1972). HEp-2 cells (2 x lo6)were seeded into 80-mm pl;~sticpetri dishes containing 24 circular (12-mm diameter) coverslips. On the following day the coverslips were placed individually into 35-mm bacte~.iological grade petri dishes and 0.025 ml of a virus suspension was pipetted on top of the coverslip, taking care that s u ~ f r ~ tension ce kept the liquid on the coverslip. The petri dishes were incubated for 60min at 33'C. then 2 ml DMEM was added and the incubation continued for 3 days at 33°C or 2 days at 39°C. The coverslips were then rinsed with phosphate-buffered saline (PBS), fixed for lOmin in ethanol - acetic acid (3:1), air-dried, stained for 2-5 min with 2% aceto-orcein (GIBCO) and rinsed successively in 70% ethanol, 100% ethanol, ethanol-xylene ( \ : I ) , 100Yo xylene, and finally mounted in pernlount. By this procedure the int~inuclear paracrystals become cle~irly visible by phasecontrast microscopy.
TABLE1. The effect of mutations and temperature on paracrystal formation"
Electt~o~r Micr.o.scol~.v Infected cells were incubated :it 33"C, washed, and fixed in sitrr at 4'C with 3.5% glutal-aldehyde in phosphate buffer. The cells were then scraped off the petri dishes, pelleted, and postfixed in 1% osmium tetroxide for 1 h. Samples were dehydrated in acetone, embedded in Epon 812, polymerized at 80°C, secyl followed by lead citrate tioned, and stained with u ~ ~ n acetate (Carstens and Marusyk 1975).
synthesis, cells were infected with several temperature-sensitive mutants at the permissive (33°C) and the nonpermissive (39°C) temperature and the formation of PC was scored. The results are shown in Table 1. Adenovil-us type 2 and its temperature-sensitive mutants which are in different complementation groups (Begin and Weber 1975) do not induce PC. Adenovirus type 5 induces PC at both temperatures. The ts mutants show that the expression of late functions is necessary for PC formation. In particular, tsl, ts2, and ts22 gene functions are all required for PC formation. This implies that fiber, loOK, and hexon are involved either directly or indirectly in PC synthesis, and defects in any one of these three genes blocks PC synthesis. Evidently the fiber-defective ts22 has the most direct effect on PC formation as the mutation is sufficiently expressed to block PC even at the permissive temperature. Interestingly, reversion restores PC even at the pel-missive temperature. Interestingly, reversion restores PC formation at the nonpermissive temperature.
Results Properties of Pcrrcrcrystcrls The demonstration of paracrystals (PC) until now has been based on electron microscopy of thin sections (Fig. 1). Because the present mapping technique involved the analysis of a large number of samples, a more rapid and simplified technique was developed for the demonstration of PC. The new technique (see Materials and Methods for details) which was checked by electron microscopy and is based on a simple cytological staining procedure of fixed cells with acetoorcein requires few cells, very little virus, and a phase-contrast microscope. Figures 2, 3, and 4 show the appearance of adenovirus-infected cells with virus strains which do not induce PC or induce small 01-large PC, respectively. Using this technique, the following general observations could be made: (i) the formation of PC is temperature-sensitive, a greater proportion of cells forms PC at 33°C than at 39°C (see also Table l), (ii) cells may contain several PC, in various orientations and even in overlapping array, (iii) large PC deform the nucleus, (iv) PC-positive nuclei generally contain less densely staining inclusions (see Fig. 4), (v) PC formation has no significant effect on virus production, as the yields of paracrystal-forming recombinants and Ad5 were of the same magnitude as the paracrystal-negative recombinants and Ad2 (results not shown). The efyect of ts Mritcrtions To determine which genes are required for PC
pl, Paracrystals -
Virus
33°C
39°C
Phenotype of mutant DNA-negative Fi bre-negative Assembly negative Hexon-negative W T revertant of ts36 -
Various late mutants "Both small- and large-type paracrystals were counted. The percentage was determined from the infected population o f cells.
Mcrpping oftlze Pcrrcrcrystul The general procedure for the mapping of adenovil-us mutants and proteins has been described elsewhere (Hassell and Weber 1978). Briefly, interserotypic recombinants are isolated from crosses between Ad5 and Ad2 ts mutants and theirgenomes are mapped with site-specific restriction endonucleases which recognize unique, serotype-specific cleavage sites on the recombinant molecules. Thirty-six recombinants from 10 different crosses have been mapped by this technique (Hassell and Weber 1978). The physical maps of these recombinants are shown in Fig. 5 . T h e serotype-specificity of five viral-coded proteins has also been determined (Weber and Hassell, manuscript in preparation) and is shown in Figure 5. The percentage of PC-containing nuclei among the in-
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'OSZ x .(MO.I.IB) S I ~ ? ] S / ( . I ~UI ?! B. I B U O~~slla3jo %OS l n o q v .sn.l!Aouapl?jo U ! I ? J I ~ Su!tu.~o~-ll?ls/(.~x?.~~d B q l ! pa)3aju! ~ slla3Jo asul?.~l?addl? [es!d/(] aqL ' 5 sn.l!Aouapl? q ] ! palsaju! ~ sllas z - d g 'P ~ '913 x '(SM~.I.II?) !alsnu atuos u! sll?)s/(.~sl?.red IIl?wsaqq)a ~ ' 1 Io-[ sa.1 ~ ~ I ! Mp a l s a p ! sllas Z - d g 'f~ ,513.OSZx .uo!)saju! . I ~ I J sAlzp ~? 2 sllas pau!l?ls u!as.~o-olasv .p 'f 'sS!g u! !a13nu u! uaas sll?)s/(~se.~l?d aq] q l ! pasnjuos ~ aq lou plnoqs salonsl?A s!tusl?ldo]As aqL 'alqaas!lou aJu suo!snlsu! .~l?a[snu~u]u! ~ ~ ' s ! d aq] / ( l Lluo 's[els/(.lsl?.~ed JO U O ! I I ? ~ IaOqJ asnpu! ~ I O U saop 2 sn.l!Aouapl?jo u!e.~lss ! q ~ .s[las 2 - d palsaju! ~ ~ 2 sn.l!Aouapo J O qd~?.rSo.~s!tuo]oqd ]sl?~)uos-asl?qd .Z '513' 0 0 s ~x 'auo ]saZ.~l?laq] 01 Zu!lu!od Mo.l.ra aq] ' s I I ? ) s / ( . I ~ I ? . I u~ ?! I~? ] u ~lal3nu ~ q]oa .uo!)saju! . I ~ I J ~s/(l?p ? z S I B ) S / ( . I ~ \ JO ? . Iasulr.ll?adde B~ aqL q a 3 2 - d palsaju! ~ ~ s sn.l!Aouapr?Jo qdn.~So.ls!muo.11salg '1 .513
.
. . . . .. ... . .. . ... . .. . . . ~ .. .
...... . .. . . ..
C A N . J . MICROUIOL. VOL.. 24. 1978
0
Enzyme map position Proteins
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Cross
rec
30
43
44
55
57
59
69
71
80
81
90
V
111
I1 lOOK I V
Paracrystal
%
Morph.
FIG.5. Mapping of the paracrystal. For details of the mapping method see text and reference (Hassell and Weber 1978). The enzyme map positions unique to Ad2 or Ad5 were obtained with Bam H1, EcoRI, Hind 111, and SmaI. The proteins appear in the same order as on the genome. The percentage of palxcrystals was determined from among the inclusion body containing cells only. The morphology of the paracrystals was scored as L, large: S, small; SL, both large and small typesobserved.
fected cells was scored (second last column, Fig. 5). The correlation of the presence o r absence of paracrystals with particular map positions and viral proteins was examined (Table 2). It is apparent that PC synthesis cannot be related to a single map position or viral protein. Nevertheless when the DNA between map positions 30 and 44 contains Ad5 sequences there is a greaterchance that PC are expressed, and conversely when these sequences are of Ad2 origin the probability that no PC are formed is greater. Among the five proteins examined the best correlation with paracrystals is
found with protein V, which has been mapped into the region between 30 and 44, thereby corroborating the above conclusion. Discussion By means of a new, simplified technique this paper has shown that the synthesis of adenovirusinduced intranuclear paracrystals is genetically controlled. The formation of paracrystals at the nonpermissive temperature is blocked by temperature-sensitive mutations in genes coding for the lOOK protein (5tsl), hexon (5ts21, and fiber
WEBER
TABLE2. Correlation between the presence of the paracrystal a n d recombinant virus properties
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Recombinant property
Correlation. 'Z
M a p position 30 43 30+43 44 43 44 55 57 59 55 57+ 59 69 71 69+71 80 81 80+81 90
+
+
Proteins V 111 11 IOOK IV NOTE:All recombinants which induced either large or small o r boll1 types of paracrystals were scored positive. When two o r three map positions were ehamined as a group, a single Ad5 map position and the presence of paracrystals \\.as scored as a positive correlation while a negative correlation would be one of the following: paracrystal-positive but both map positions Adz; paracrystal-negative but at least one map position Ads. Each map position may contain either Ad2 o r Ad5 seqilences as each protein may be either Ad2- o r Ad5-specific. The primary data from which this table derives appears in the summary Fig. 5.
(5ts22). In agreement with previous findings (Wills et (11. 1973), paracrystal formation is evidently a late
gene function, because, in addition to these late mutants, the early, DNA-negative mutant 5ts36 also blocks their synthesis. These results do not imply, however, that the gene-products of 5ts1, 5ts2, and 5ts22 are necessarily integral components of the paracrystals. The finding that the fiberdefective mutant 5ts22 blocks paracrystal formation even at the pel-missive temperature confirms a previous report (Wills et (11. 1973) and suggests that the complete integrity of this protein may be an absolute requirement for paracrystal formation. The mapping data showed that the region of the genome between 30 and 44 has relatively greater importance than other I-egions of the genome in determining the formation of paracrystals. However, since the data were not unequivocal, it appears that paracrystal formation may be controlled by several genes. We have already seen the importance of the fiber gene. Marusyk et 01. (1972) claimed tha't anti-VII serum and phenanthrenequinone (see also Henry and Atchison 1971), an
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arginine-specific stain, react with paracrystals. We have shown that protein VII is derived from its PI-ecursorby proteolytic cleavage during the maturation of young virions to mature virions (Weber 1976; Bhatti and Weber 1978). I t is likely therefore that the paracrystals in fact contain the precursor PVII which is known to react with antiserum to VII. Protein PVII cannot be the only protein present in the paracrystals, because there doesn't appear to be a sufficient quantity in infected cells to provide for the assembly of such large crystals without severely restricting virion assembly. Hexon and fiber, which are synthesized in huge excess by infected cells, appear to be ideal candidates as paracrystal constituents. Genetically both appear to be necessary for paracrystal formation (Table 1). Unfortunately antiserum to neither protein reacts with paracrystals (Henry and Atchison 1971; Marusyk et (11. 1972). Non-reactivity does not prove these proteins are absent from the paracrystals. It is conceivable that the antigenic determinants are buried within the crystal and are consequently unable to react with antibody molecules. Furthermore if hexon were organized as monomers within the crystal, antibody prepared against the native trirner antigen would not cross-react (Stinski and Ginsberg 1974). The coding region of protein PVII has been mapped near or into the interval most important for paracrystal formation (i.e. 30-44). The observed ambiguity in the mapping may be due to a complex interaction of several gene-products such as PVII, hexon, and fiber in the formation of paracrystals. To test whether certain combinations of Ad2 and Ad5 genes may result in paracrystal formation would require more map positions and knowledge of the precise location of structural genes. The present data are insufficient in this regard. A definite determination of paracrystal protein composition will have to await the development of a technique for their selective extraction and purification. Acknowledgments The technical assistance of Lise Clavet is gratefully acknowledged. Dr. J. Weber is a Research Scholar of the National Cancer Institute of Canada (N.C.I.C.). This work was supported by a grant from the N.C.I.C. BEGIN,M., and J. WEBER.1975. Genetic analysis of adenovirus type 2 1. Isolation and genetic characterization of ternperature-sensitive mutants. J. Virol. 15: 1-7. BHATTI,A. R.. and J. WEBER.1978. Protease ofadenovirustype 2. In vitro processingofcore protein. Biochem. Biophys. Res. Commun. 81: 973-979.
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CAN. J . MICROBIOL. VOL. 24. 1978
BOULANGER, P. A., G. TORPIER, and G. BISERTE. 1970. Lnvesti- MORGAN, C.. G. G O D M A NP., B R E I T E N F E Land D , H. ROSE. gation on intlxnuclear paracrystalline inclusions induced by 1960. A co~.relativestudy by electron microscopy and light adenovirus 5 in KB cells. J . Gen. Virol. 6: 329-332. mic~.oscopyof the development of type 5 adenovirus. J. Exp. Med. 112: 373-381. CARSTENS, E.. and R. MARUSYK. 1975. An image analysis study , J. F. W I L L I A M S1974. . of adenovirus type 5-induced crystalline inclusions. J. Gen. RUSSELL,W. C., J . J. S K E H E Land Virol. 29: 249-256. Chalxcterization of tempelxture-sensitive mutants of aden1977. Crystallization and reaggregation of adenovirus ovirus type 5: synthesis of polypeptides in infected cells. J . type 5 structul-al components from infected cell extracts. J. Gen. Virol. 24: 247-259. Gen. Virol. 34: 541-545. S I - I N S KM. I , F., and H. S. GINSBERG. 1974. Antibody to the type 5 adenovirus hexon polypeptide: detection of nascent GIVAN.K.. C. T U R N B U Land L , A. M. JESEQUEL. 1967. Pepsin digestion of virus p>u.ticles in canine hepatitis using eponpolypeptides in the cytoplasm of infected KB cells. Intervirology, 4: 226-236. embedded material. J. Histochem. Cytochem. 15: 688-694. HASSELL,J . A,. and J. WEBER.1978. Genetic analysis of WEBER,J. 1972. Titration of adenovirus by counting cells conadenovirus type 2 VIII. The physical locations of temtaining virus-induced inclusion bodies. Appl. Microbiol. 23: pelxture-sensitive mutations. J . Virol. 28(3). In press. 1025- 1026. 1976. Genetic analysis of adenovirus type 2. 111. H E N R YC, . J., and R. W. ATCHISON. 1971. Paracrystal formaTemperature-sensitivity of processing of viral proteins. J. tion in cell cultures infected with adenovirus type 2. J. Virol. 8: 842-849. Virol. 17: 462-471. , G. KHITTOO.1975. Genetic analysis L I F C H I ' ~AS , , A. R I M S K YG. , TORPIER, and P. BOULANCER.WEBER,J., M. B E G I Nand of adenovirus type 2.11. Preliminary phenotypiccharacteriz;i1975. Crystallographic study of an adenovirus-induced protein crystal in KB cells: n structural model. Acta Cryst;lllogr. tion of tempelxture-sensitive mutants. J . Virol. 15: 1049B31: 441-445. 1056. MARTINEZ-PALOMO, A,, J . LE BUIS,and W. BERNHARD. 1967. WEBER, J . and S. K . L I A O .1969. Light and electron microscopy of virus-associated intranuclear paracrystals in cultured cells Electron microscopy of adenovirus 12 replication. I. Fine infected with type 2.4.6, and 18 human adenovil.uses. Can. J. structul-al changes in the nucleus of infected KB cells. J. Virol. 1: 817-829. Microbiol. 15: 841-845. MARUSYK, R., E. NORRBY, and H. MARUSYK. 1972. The rela- WEBER, J . . and H. F. S.I.ICH.1969. Electron microscopy ofcells tionship of adenovirus-induced pzu.acrystalline structure to infected with adenovirus type 2. J. Vil.01. 3: 198-204. the virus core protein(s). J. Gen. Virol. 14: 261-270. WILLS, E. J., W. C . RUSSELL,J. F. W I L L I A M S1973. . Adenovirus-induced cryst;ils: studies with temperatureMATSUI,K., and W. B E R N H A R D 1967. . Modifications ~rltl-astructurales induites par le virus de I'hepatite canine dans les sensitive mutants. J. Gen. Virol. 20: 407-412. cellules de rein dechien. Ann. Inst. Pasteur, Paris, 112: 773.
