VIROLOGY
9.3, 198-208
(1979)
A Maturation
Protein
in Adenovirus
Morphogenesis
HAKAN PERSSON,’ BERIT MATHISEN, LENNART AND ULF PETTERSSON Department
of Microbiology,
University
PHILIPSON,
of Uppsala, The Biomedical Center, Box 581, S-751 23 Uppsala, Sweden Accepted October 17, 1978
Adenovirus type 2 (ad2) assembly intermediates have been isolated by sucrose-gradient centrifugation. Such intermediates are distinguished from mature v&ions by the presence of uncleaved precursors to polypeptides VI, VII, and VIII as well as a prominent polypeptide with a molecular weight of 50,000 (50K) which disappears during maturation (B. Edvardsson, E. Eve&t, H. Jornvall, L. Prage, and L. Philipson, 1976, J. Viral. 19, 533-547; J.-C. D’Halluin, G. Martin, G. Torpier, and P. Boulanger, 1978, J. Vilirol. 26, 357-363). In the present communication we demonstrate that the 50K polypeptide is encoded by the viral genome and its gene is located on the l-strand of fragment SmaI-F (map coordinates 11.3 to 18.1).
in the assembly pathway. Through genetic and biochemical studies a wealth of knowlThe adenoviruses provide a convenient edge has been accumulated, in particular model system to study virus structure and concerning the morphogenesis of phage T4 morphogenesis, since they unlike most other viruses, have structural proteins which are of Escherichia coli and phage P22 of SalIn both these sysmonella typhimirium. soluble under nondenaturing conditions. tems the DNA has been found to enter The adenovirus type 2 (adz) virion contains preassembled capsids and both scaffolding at least nine different polypeptides, most of and maturation proteins have been identified which have been purified and characterized (for a review see Casjens and King, 1975). in considerable detail (for a review see Studies on the assembly of animal DNA Philipson et al. , 1975). Some virion polypepviruses have been hampered by the lack of tides are synthesized as precursors which nonsense mutants with lesions in the assuffer posttranslational cleavage during sembly pathway. Several temperaturevirus maturation (Anderson et al., 1973; Edvardsson et al., 1976). The assembly sensitive mutants which cause defective intermediates are distinguished from mature assembly have been described (Weber, 1976; virions by the presence of precursors to Edvardsson et al., 1978; D’Halluin et al., 1978b) but only one of them yields infectious polypeptides VI, VII, and VIII (Edvardsson progeny upon shift down (Weber, 1976). et al., 1976). In addition, analysis by SDSStudies on adenovirus morphogenesis have polyacrylamide gel electrophoresis (SDStherefore mainly been approached through PAGE) has revealed the presence of a 50K a biochemical characterization of the assembly polypeptide in assembly intermediates which intermediates. In the present communication is absent or present in reduced amounts in we demonstrate that intermediates in adenomature virions (Edvardsson et al., 1976; D’Halluin et al., 1978a). Nothing is how- virus assembly are associated with a 50K ever known concerning the origin of this polypeptide which is encoded by the l-strand of ad2 DNA and which has the properties of polypeptide. The morphogenesis of bacteriophages has a maturation protein. been studied in detail. These studies were facilitated by the availability of nonsense MATERIALS AND METHODS mutants which are defective in defined steps Infection and labeling conditions. Ad2 1 To whom reprint requests should be addressed. was propagated in suspension cultures of INTRODUCTION
0042-6822/79/030198-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
198
ADENOVIRUS
HeLa S3 cells in Eagle’s spinner medium as described before (Pettersson et al., 1976). Cells were infected at a multiplicity of 2000 particles per cell as described by Everitt et al. (1971). In all labeling experiments the medium contained l/50 of the normal concentration of methionine. [35S]Methionine was added 14 hr postinfection at a concentration of 200 &i/ml in pulse-chase experiments and at 1 $.X/ml for production of labeled virus. The labeling procedure and the virus purification scheme has been described in detail elsewhere (Edvardsson et al., 1976). Cell fractionation. Cytoplasmic and nuclear extracts were prepared as described before (Edvardsson et al., 1976). The extracts were analyzed by sucrose-gradient centrifugation. Sucrose-gradient centrifugation. Linear 25-40% sucrose gradients containing 0.2 M NaCl, 0.02 M sodium phosphate buffer, pH 8.0, and 0.01 M EDTA were centrifuged at 85,000 g for 2 hr. The gradients were fractionated from the bottom and individual fractions were analyzed by scintillation counting and SDS-PAGE. SDS -PAGE. Polyacrylamide gel electrophoresis was performed according to the method of Maize1 (1971). Exponential lo18% gradient slab gels (McGuire et al. ,1974; Edvardsson et al., 1976) or homogeneous slab gels (10 or 13%) were used. Electrophoresis was carried out at 140 V for 20 hr for the gradient gels and at 100 V for 10 hr for the other gels. The gels were analyzed by fluorography (Bonner and Laskey, 1974). Radioisotopes and counting procedures. [35S]Methionine (600 Wmmol) was obtained from New England Nuclear Corporation (Boston, Mass.). Radioactive samples were precipitated on Whatman 3MM filters with 10% trichloroacetic acid, washed with ethanol, and air dried. The filters were counted in a toluene-based scintillation liquid. Preparation of complementary strands of ad2 DNA. Adenovirus DNA was obtained from purified virions by the method of Tibbetts et al. (1974). Separated strands of ad2 DNA were prepared by equilibrium centrifugation of denatured DNA in CsCl in the presence of ribopoly (U,G) as described by Tibbetts et al. (1974).
ASSEMBLY
199
Selection of RNAIDNA hybrids. Cytoplasmic RNA was extracted from ad2infected HeLa cells (2000 virus particles/ cell) 18 hr postinfection as described by Brawerman et al. (1972). The conditions for hybridization between messenger RNA (mRNA) and the complementary strands of ad2 DNA, selection of RNA-DNA hybrids by exclusion chromatography on a Sepharose 2B column, and the isolation of the hybridized poly(A)-containing mRNA by poly(U)Sepharose chromatography are described in detail in a separate communication (H. Persson et aE., in preparation). Cell free translation. Unfractionated late cytoplasmic RNA or mRNA selected by hybridization to the viral l-strand was translated in vitro in a micrococcal nucleasetreated rabbit reticulocyte lysate (Pelham and Jackson, 1976). The translation mixtures were incubated for 2 hr at 35”, treated with pancreatic RNase and sodium hydroxide (Persson et al., 1978) and an equal volume of twice concentrated gel sample buffer was added (Maize& 1971) before analysis by SDS-PAGE. Hybrid arrested cell-free translation (HART) (Paterson et al., 1977). Restriction
fragments of ad2 DNA were denatured by incubation at room temperature in 0.3 M NaOH for 10 min. After neutralization with HCl the DNA was mixed with selected or unselected RNA. Thirty micrograms of unselected RNA or RNA selected on 0.5 pug of complementary strand DNA was mixed with l-3 pg of a restriction fragment. After adjusting the final volume to 5 ~1 with HzO, the samples were heated to 100” for 30 see, quickly chilled, and brought to a final volume of 100 ~1 containing 80% (v/v) deionized formamide, 20 mM l,kpiperazinediethanesulfonic acid (Pipes) buffer, pH 6.4, 1 m&f EDTA, and 0.4 M NaCl. The samples were incubated at 48” for 2 hr and the hybridization terminated by the addition of 300 ~1 ice-cold water containing 15 pg (in the case of hybrid selecting RNA) E. coli ribosomal RNA. The samples were then precipitated with 2.5 vol of ethanol at -20” overnight and centrifuged at 8000 g for 20 min at 4”. The pellet was dissolved in 0.15 M potassium acetate, pH 5.6, and reprecipitated with 2.5 vol of ethanol at
200
PERSSON
-20” overnight. The precipitates were finally collected by centrifugation as above, ethanol removed by lyophilization, and the samples dissolved in the appropriate amount of cold distilled water. Selected RNA was dissolved in 4 ~1of H,O and unselected RNA in 25 ~1 of H,O. The entire sample from the selected RNA was added to the in vitro translation system as described above while 3 ~1 of the unselected hybridized RNA was added per 25 ~1 of reaction mixture. Tryptic peptide analysis. Fractions from the sucrose gradient which contained 35Slabeled ad2 assembly intermediates (Fig. 2) were precipitated with ice-cold 10% trichloroacetic acid (TCA), washed once in 5% TCA, twice in acetone, dried, and dissolved in gel sample buffer (Maize& 1971). RNA selected on l-stand DNA was translated in vitro in the presence of [35S]methionine. Both samples were analyzed by SDS-PAGE. The gel was stained with Comassie brilliant blue (2% in 10% acetic acid, 30% methanol) for 30 min and destained for l-2 hr in 10% acetic acid 30% methanol. The gel was then dried and autoradiographed polypeptides were excised, eluted from the gel, washed, oxidized with performic acid, washed again, and digested with ~-l-tosylamide-Z-phenylethyl chloromethyl ketone (TPCK)-trypsin as described previously (Persson et al., 1978). The digests were finally lyophilized, dissolved in pyridine-acetate buffer, pH 5.5 (acetic acid:pyridine:water ‘7:20:970), and fractionated in two dimensions on Whatman CC41 cellulose thinlayer plates as previously described (Linne et al., 1977; Persson et al., 1978). Methionine-containing peptide were visualized by autoradiography. RESULTS
Polypeptide Composition Intermediates
of Assembly
Previous results have shown that a heterogenous class of assembly intermediates which sediment slower than mature virions can be isolated from nuclei 20 hr after ad2 infection (Edvardsson et al., 1976). In order to study the polypeptide composition of assembly intermediates, a nuclear extract was prepared from cells
ET AL.
labeled with [35S]methionine between 15 and 20 hr after infection. The supernatant was sedimented through a 25-40% sucrose gradient at 85000 g for 120 min as described by D’Halluin et al. (1978a) and the radioactivity profile of the fractionated gradient is shown in Fig. 1A. The fast sedimenting peak has previously (Edvardsson et al., 1976) been characterized as nuclear or young virions (NV) and the slowly sedimenting peak as assembly intermediates (IM). Figure 1A also shows the polypeptide composition of IM, NV, and mature virus. The precursor polypeptides 27K (pVI), 26K (presumably pVIII), and pVI1 are present as prominent bands in the IM fraction and also to some extent in the NV fraction but not in the mature virions which instead contain polypeptides, VI, VII, and VIII. In addition a polypeptide with a molecular weight of 50,000 (50K) is present in the IM fraction, in trace amounts in the NV fraction, and the ad2 virus marker. To ascertain that the peak of the 50K polypeptide coincided with the peak of the assembly intermediates, individual fractions from the sucrose gradient were analyzed by SDSPAGE. A fluorogram of the gel is shown in Fig. 1B. The label in the 50K polypeptide peaks in fractions 19-23 which corresponds to the IM peak. Since the 50K polypeptide is not present in the mature virions and since it is not known to be precursor of any other polypeptide it has the properties of a maturation protein in virus assembly. Fate of the 50K Polypeptide Chase Experiments
in Pulse-
In phage P22 it has been shown that the product of gene 8, which is a scaffolding protein, fails to chase out of the proheads in pulse-chase experiments which indicates that the protein is recycled (King and Casjens, 1974). On the other hand, the maturation protein p22 in phage T4 is extensively degraded during phage assembly (Kurtz and Champe, 1977). In order to establish which of these pathways the 50K polypeptide follows, pulse-chase experiments were performed and the fate of the 50K polypeptide was determined. Infected
ADENOVIRUS
201
ASSEMBLY
A
Ad2 NV
i c-4 “4 1’2
IM
FRACTION NUMBER c-4
14
16
18
20
22
24
26
28
2
B
FIG. 1. Analysis of ad2 assembly intermediates. A nuclear extract from ad2 infected KB cells, labeled with [3sS]methionine from 15 to 20 hr after infection was analyzed on a 25-40% sucrose gradient. (A) Radioactivity profile of the gradient and polypeptide composition of pooled NV and IM fractions as determined from a lo-18% SDS-polyacrylamide gradient gel. (B) The distribution of polypeptides in individual fractions of the sucrose gradient. Fluorograms of lo-18% SDSpolyacrylamide gradient gels are shown.
HeLa cells were pulse-labeled with [35S]methionine at 14 hr after infection for 15 min followed by a 6-hr chase and nuclear and cytoplasmic extracts were sedimented through sucrose gradients. Figure 2 shows the distribution of label in the sucrose gradients. The radioactivity in the IM area shows considerable decrease when chased for more than 1.5 hr and large amounts of
label enter both the nuclear and cytoplasmic virus. The IM and NV fractions were separately pooled as indicated in Fig. 2 and analyzed by SDS-PAGE (Fig.3). The labeled 50K polypeptide band is prominent after 1.5 hr but disappears almost completely after a 6-hr chase. No detectable 50K polypeptide was observed in the cytoplasmic virus. The precursor polypeptides
202
PERSSON ET AL. lSh
L,5h
3,oh
p? 50. a ito-
NV I” ct----(
NV l
6.0h
NV
IM :
t
20
30
IM
NV
It.4
I
!O P-i. 10
20
30
LO
10
40
10
20
30
40
10 20 30 LO FRACTION NUMBER
FIG. 2. Sucrose gradient analysis of a pulse-chase experiment. Cells were pulse-labeled with [T3]methionine for 15 min followed by a chase at 14 hr after ad2 infection. Samples were analyzed after indicated time periods. Nuclear (top panels) and cytoplasmic (bottom panels) extracts were centrifuged separately. Fractions from the NV and IM peaks were pooled separately as indicated and analyzed by SDS-PAGE.
suggest that the mRNA is transcribed from the viral l-strand since the messenger RNA map of the ad2 genome (Pettersson et al., 1976) shows that a gene block is located on the viral l-strand in this position. In order to establish the relationship between the 50K polypeptide in assembly intermediates and the virus coded 50K polypeptide we decided to isolate the two polypeptides and The ad2 Genome Encodes a 5OK Polypeptide compare them by tryptic fingerprint analysis. A 50-56K polypeptide has previously We have recently designed a method for been detected as a minor constituent of selection of viral mRNA utilizing Sepharose adenovirus type 2 (Maizel, 1971; Eve&t 2B chromatography for separation of DNAet al., 1973; Anderson et al., 1973). A poly- RNA hybrids from unhybridized RNA peptide with the same molecular weight in (Persson et al., in preparation). By utilizing SDS-polyacrylamide gels has also been large single stranded DNA to select RNA a observed from cells labeled with [35S]methi- differential exclusion between hybrids and onine late after ad2 infection (Anderson nonhybridized RNA can be achieved. To et al., 1973). Furthermore cell-free translaisolate the 50K mRNA, late ad2 cytoplasmic tion of late adenovirus mRNA selected RNA was hybridized to viral l-strand DNA by hybridization to restriction fragment in solution. The RNA-DNA hybrids were HindIII-C of the ad2 genome (map isolated by chromatography on Sepharose coordinates 7.5-17.0) directs the synthesis 2B. The hybrids were melted in 90% of this polypeptide (Lewis et al., 19781, formamide at 65” for 15 min and the mRNA which has been designated IV&. The map isolated by poly(U)-Sepharose chromatogcoordinates given for the IVa, polypeptide raphy whereupon it was translated in an 27K, 26K, and pVI1 chase efficiently and their mature counterparts are recovered in nuclear (Fig. 3) and cytoplasmic virus (not shown, see Fig. 12 in Edvardsson et al., 1976) during the same chase period. These results suggest that the 50K polypeptide turns over during virus maturation.
ADENOVIRUS
--II
-m 7gp -P
50K
--PI -pII FIG. 3. SDS-PAGE of the pulse-chase experiment. Analysis by SDS-PAGE of fractions from the pulsechase experiment in Fig. 2. The slots in the fluorogram contain the following samples: (1) and (10) ad2 virus marker; (2), (3), (4), and (5) correspond to IM fractions from 1.5, 3, 4.5, and 6 hr of chase; and (6), (‘7), (8), and (9) correspond to NV fractions from the same time points.
in vitro protein-synthesizing
system. Messenger RNA selected on the viral l-strand directed the synthesis of four polypeptides: a 75K polypeptide, corresponding to the adenovirus DNA-binding protein (Van der Vliet and Levin, 19’73)and a 19K polypeptide (Fig. 4). These two polypeptides have previously been characterized as ad2 early polypeptides which are synthesized in small or reduced amount also late after infection (Saborio and Oberg, 1976; Persson et al., 1978). In addition late mRNA selected on the l-strand directed the synthesis of a prominent 50K polypeptide as well as small amounts of a 60K polypeptide. The 50K polypeptide, synthesized from mRNA selected by the viral l-strand, migrated slightly faster on SDS-PAGE than the 50K polypeptide, previously detected in ad2 assembly intermediates (Fig. 4). Tryptic Fingerprint Polypeptide
Analysis
of the 50K
The relationship between the 50K polypeptide detected in ad2 assembly intermediates and the 50K polypeptide coded by
ASSEMBLY
203
the l-strand of the genome was investigated by tryptic fingerprint analysis. The two 50K polypeptides were purified by SDS-PAGE, eluted from the gel, and digested with TPCK-trypsin. Tryptic peptides were then separated in two dimensions on cellulose thin-layer plates and the plates analyzed by autoradiography. At least six [35S]methionine-labeled tryptic peptides with identical mobilities in the two dimensions were observed in both cases (Fig. 5). Thus the 50K polypeptide present in ad2 assembly intermediates and the 50K polypeptide programmed by mRNA from the l-strand of ad2 DNA have indistinguishable primary structures. Localization of the Gene for the 50K Polypeptide on the ad2 Genome
Hybridization of mRNA to a complementary DNA fragment has recently been shown to make the mRNA untranslatable in vitro (Paterson et al., 1977). This method was used to localize the gene for the 50K polypeptide on the ad2 genome and thereby verify its relationship to the IVa, polypeptide. Messenger RNA selected by hybridization to the viral l-strand was hybridized to different restriction enzyme fragments of ad2 DNA under conditions which allow formation of RNA-DNA hybrids while DNA-DNA renaturation is prevented (Casey and Davidson, 1977). The BamHI restriction fragments of ad2 DNA were hybridized to l-strand-selected mRNA. After hybridization, the samples were precipitated with ethanol and the mRNA translated in vitro in a reticulocyte cell-free system (Pelham and Jackson, 1976). The BamHI-B fragment (map coordinates O29.1) completely arrested the translation of the 50K polypeptide (Fig. 6). In contrast hybridization to restriction fragments BamHI-C and D (map coordinates 29.1-40.9 and 40.9-59.0) had no effect on the in vitro synthesis of the 50K polypeptide, as was also true for restriction fragments derived from the right-hand end of the ad2 genome (BarnHI-A, EcoRI-C and D, data not shown). To locate the gene for the 50K polypeptide more precisely, within the
204
PERSSON ET AL.
D
B
D
E 75K 60K 50K
27 .27K -26~
FIG. 4. In vitro synthesis of the 50K polypeptide. Ad2-infected HeLa cells were harvested 18 hr postinfection and cytoplasmic RNA was prepared. The RNA was hybridized in solution to the viral l-strand and the RNA-DNA hybrids separated from the unhybridized RNA by exclusion chromatography on a Sepharose 2B column (H. Persson et al., in preparation). The hybrids were melted, polyadenylated RNA isolated by poly(U)-Sepharose chromatography, and the selected RNA precipitated several times with ethanol. Unfractionated late RNA (5 @g/25 ~1 of reaction volume) or mRNA selected by hybridization to 0.5 fig of the viral l-strand was translated in vitro in a micrococcal nuclease treated reticulocyte lysate and the translational products analyzed in a 10% SDS-polyacrylamide gel. Electrophoresis was carried out for 10 hr at 100 V whereupon the gel was analyzed by fluorography. Slot A: late unfractionated RNA. Slot B: RNA selected on the viral l-strand. Slot C: no RNA added. Slot D: Pooled fractions, containing adenovirus assembly intermediates, from the sucrose gradient shown in Fig. 1. A [35S]methionine-labeled virus marker (ad2) was included.
BamHI-B fragment we hybridized the l-strand-selected mRNA to two SmaI restriction enzyme fragments, one of which (SmaI-F) is known to be transcribed late after infection (Pettersson et al., 1976). The SmaI-F fragments (map coordinates 11.318.1) completely arrested the translation of the 50K polypeptide while the SmuI-E fragment (map coordinates 2.9-10.7) had no effect on its synthesis (Fig. 6). Control experiments with unselected cytoplasmic RNA revealed that the DNA fragments themselves were not inhibitory for trans-
lation. The results suggest that the major part of the gene for the 50K polypeptide in assembly intermediates is located on the lstrand of fragment SmuI-F. The results do not exclude that a small portion of the 50K gene extends into the neighboring SmaI-B fragment. DISCUSSION
The morphogenesis of the adenovirion follows a complex pathway. The hexon capsomeresmay first assemble into ninemers (Pereira and Wrigley, 1974) which become
ADENOVIRUS
-
E LECTROPHORESIS
+
FIG. 5. Tryptic peptide analysis of the 50K polypeptide present in ad2 assembly intermediates and the 50K polypeptide encoded by the viral l-strand. Fractions from a sucrose gradient (Fig. 1) containing ad2 assembly intermediates were precipitated with TCA and the washed precipitates dissolved in gel sample buffer. RNA selected by hybridization to the viral l-strand was translated in vitro in a reticulocyte cell-free system in the presence of [35S]methionine. Both samples were analyzed on a 10% SDS-polyacrylamide gel. The polypeptide band corresponding to the 50K polypeptide in both sampleswas eluted from the gel and their tryptic pep tides were analyzed as described under Materials and Methods. Top panel (IM 50K), digest of the 50K polypeptide present in ad2 assembly intermediates. Bottom panel (IV%), digest of the 50K polypeptide translated from RNA selected on viral l-strand DNA.
associated with pentons and uncleaved precursors for polypeptides VI and VIII (pV1 and pVII1). In this way a procapsid is formed. The DNA, presumably covered
ASSEMBLY
205
with polypeptides V and pVI1, enters the procapsid shortly thereafter generating the assembly intermediates (Edvardsson et al., 1976, 1978). Alternatively the core proteins may enter the capsid after the DNA (D’Halluin et aZ., 1978a, b). The ad2 assembly intermediates contain a 50K polypeptide which is almost absent in nuclear vii-ions (Fig. 1, Ishibashi and Maize& 1974; Edvardsson et al., 1976, 1978; D’Halluin et al., 1978a). The final step in the assembly pathway involves the cleavage of precursor polypeptides pV1, pVI1, and pVII1 (Weber, 1976), probably by a protease which is associated with the virion (Ishibashi and Maizel, 1974; Weber, 1976). The experimental evidence for this tentative pathway is almost entirely based on pulse-chase labeling kinetics of infected cells and polypeptide composition of different classes of assembly intermediates. Due to the lack of suitable nonsense mutants in the adenovirus system it has been difficult to accumulate different classes of intermediates in sufficient quantities to characterize their structures. Temperature-sensitive mutants have only in one case (ad2 tsl, Weber, 1976) been demonstrated to accumulate young virions which mature into virions upon shift-down conditions. In other cases (Edvardsson et al., 1978) the tsmutants accumulate intermediates which cannot be chased into mature virions after shift-down, probably due to assembly of defective particles at the nonpermissive temperature. In several phage systems well-defined nonsense mutations are available which have made it possible to identify both scaffolding and maturation proteins required in different steps of the assembly pathway. The scaffolding proteins are defined as proteins involved in a matrix network for the assembly of the procapsid (King and Casjens, 1974) and the maturation proteins may have specific functions in subsequent steps, such as the introduction of DNA into preformed capsids or the rearrangement of the DNA or the capsomeres within the particles (Laemmli et al., 1974). In the adenovirus system pV1 and pVII1, which are located internally in the
PERSSON ET AZ,.
206
-5OK 50K-
FIG. 6. Hybrid arrested cell-free translation of RNA selected by hybridization to the viral l-strand. Late ad2 cytoplasmic RNA was hybridized in solution to the viral I-strand and the hybridized RNA isolated as described under Materials and Methods and in the legend to Fig. 4. RNA selected from 0.5 pg of the viral l-strand DNA was hybridized in the presence of 80% formamide to I pg of the indicated restriction fragments of ad2 DNA. After hybridization for 2 hr at 48” the reactions were terminated by addition of cold distilled water and the samples subsequently precipitated with ethanol. The precipitates were collected and the entire sample was translated in vitro. Ten microliters of the translation mixtures were analyzed on a 10% SDS-polyacrylamide gel (left panel) or on a 13% SDS-polyacrylamide gel (right panel). Electrophoresis was for 10 hr at 100 V and the gels were analyzed by fluorography. The restriction enzyme fragments used for individual hybridizations are indicated at the top of the figure. No RNA added indicates endogenous translation in the cell-free system. The fluorograms were exposed for 2 days (left panel) or 8 days (right panel); [Wlmethioninelabeled virus marker was also included in the analysis (Ad2).
capsids (Everitt et al., 19’75), may have a scaffolding function but if so they differ from the scaffolding proteins of phage P22 in S. typhimurium since they remain in a cleaved form in the mature virion. Several investigators (Edvardsson et al., 1976; D’Halluin et al., 1978a) have previously reported the presence of polypeptides in assembly intermediates which are absent or present in reduced amounts in mature virions. One such polypeptide is the 50K polypeptide which is further investigated in the present communication. The peak of the 50K polypeptide is shown to coincide with the peak of assembly intermediates in sucrose gradients (Fig. 1B) and it thus appears
established that the 50K polypeptide is associated with the assembly intermediates and not fortuituously present in semipurified preparations of assembly intermediates. Pulse-chase experiments further demonstrate that the 50K polypeptide disappears from the peak of assembly intermediates during chase without being recovered in the peak of mature virions. The 50K polypeptide thus has the properties of a maturation protein which unlike the gene 8 product of phage P22 (King and Casjens, 1974) fails to be reutilized during virus assembly. In vitro translation of mRNA selected on the l-strand of ad2 DNA demonstrates that
ADENOVIRUS
the viral genome specifies a polypeptide which has a similar electrophoretic mobility in SDS-PAGE as the 50K maturation protein. The identity between the in vitro synthesized polypeptide and the maturation protein is furthermore established by tryptic fingerprint analysis (Fig. 5). It is not known why the in vitro and in vivo synthesized polypeptides have slightly different electrophoretic mobilities in SDS-PAGE but the 50K polypeptide presumably becomes modified by phosphorylation after translation (D’Halluin et al., 1978a) which may alter its mobility in SDS-polyacrylamide gels. The major part of the gene for the 50K maturation protein is located within fragment SmaI-F (map coordinates 11.3 to 18.1) as revealed by hybrid arrested cell free translation (Fig. 6). Thus the gene for the 50K maturation protein is located in the left of the two late gene blocks on the l-strand which were identified by hybridization between late mRNA and separated strands of several restriction fragments (Pettersson et al., 1976). The location of the gene for the 50K polypeptide is interesting in the sense that it is controlled by a different promoter than the late genes which specify the structural polypeptides of the adenovirus particle. Lewis et al. (1978) have previously mapped the gene for polypeptide IVa, within fragment HindIII-C (map coordinates 7.5-17.0) by in vitro translation of mRNA which was selected by hybridization to restriction fragments of ad2 DNA. Because of its size and map position it is likely that polypeptide IVa, is identical to the 50K maturation protein. It is, however, unclear whether the presence of the IVa, polypeptide in purified virus preparations is caused by trace amounts of immature particles in purified virus or whether trace amounts of the 50K polypeptide remain in virions during maturation and play a functional role also in the mature particle. ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council and the Swedish Cancer Society. We thank M. Gustafson for excellent
ASSEMBLY
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secretarial help and S. Andersson and P. Alestrom for excellent technical assistance. REFERENCES ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. (1973). Processing of adenovirus e-induced proteins. J. Viral. 12, 241-252. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem.
46, 83-88.
BRAWERMAN, G., MENDECKI, J., and LEE, S. Y. (1972). A procedure for the isolation of mammalian messenger ribonucleic acid. Biochemistry 11, 637-641. CASEY, J. and DAVIDSON, N. (1977). Rates of formation and thermal stabilities of RNA-DNA and DNA-DNA duplexes at high concentrations of formamlde. Nucl. Acids. Res. 4, 1539-1552. CASJENS, S., and KING, J. (1975). Virus assembly. Ann. Rev. Biochem. 44, 555-611. D’HALLUIN, J.-C., MARTIN, G., TORPIER, G., and BOULANGER,P. (1978a). Adenovirus type 2 assembly analyzed by reversible cross-linking of labile intermediates. J. Virol. 26, 357-363. D’HALLUIN, J.-C., MILLEVILLE, M., BOULANGER, P. A., and MARTIN, G. R. (197813).Temperaturesensitive mutant of adenovirus type 2 blocked in virion assembly: Accumulation of light intermediate particles. J. Viral. 26, 344-356. EDVARDSSON, B., EVERITT, E., J~RNVALL, H., PRAGE,L., and PHILIPSON,L. (1976). Intermediates in adenovirus assembly. J. Viral. 19, 533-547. EDVARDSSON,B., USTACELEBI, S., WILLIAMS, J., and PHILIPSON, L. (1978). Assembly intermediates among adenovirus type 5 temperature-sensitive mutants. J. Viral. 25, 641-651. EVERITT, E., SUNDQUIST, B., and PHILIPSON, L. (1971). Mechanism of the arginine requirement for adenovirus synthesis: I, Synthesis of structural proteins. J. Viral. 8, 742-753. EVERITT, E., LUTTER, L., and PHILIPSON, L. (1975). Structural proteins of adenoviruses: XII, Location and neighbor relationship among proteins of adenok-ion type 2 as revealed by enzymatic iodination, immunoprecipitation and chemical cross-linking. Virology 67, 197-208. EVERITT, E., SUNDQUIST, B., PETTERSSON, U., and PHILIPSON, L. (1973). Structural proteins of adenoviruses: X, Isolation and topography of low molecular weight antigens from the virion of adenovirus type 2. Virology 52, 130-147. ISHIBASHI, M., and MAIZEL, J. V., JR. (1974). The polypeptides of adenovirus: V, Young vii-ions, structural intermediates between top components and aged virions. Virology 57, 409-424.
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KING, J., and CASJENS, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature (London) 251, 112-119. KURTZ, M. B., and CHAMPE, S. (1977). Precursors of the T4 internal peptides. J. Viral. 22, 412-419. LAEMMLI, U. K., PAULSON,J. R., and HITCHINS, V. (1974). Maturation of the head of bacteriophage T4. J. Supramol. Struct. 2, 276-301. LEWIS, J. B., ANDERSON,C. W., and ATKINS, J. F. (1978). Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments Cell 12, 37-44. LINN& T., J~RNVALL, H., and PHILIPSON, L. (1977). Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-einfected cells. Eur. J. Biochem. 76, 481-490. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosh and H. Koprowski, eds.), Vol. V, pp. 179-246. Academic Press, New York. MCGUIRE, J. C., PINE, J. J., and BARROWCARRAWAY,J. (1974). Gene expression during the development of bacteriophage +29:III, Analysis of viral-specific protein synthesis with suppressible mutants. J. Viral. 13, 690-698. PATERSON,B. M., ROBERTS,B. E., and KUFF, E. L. (1977). Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free transiation. Proc. Nat. Acad. Sci. USA 74, 4370-4374. PELHAM, H. R. B., and JACKSON, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256.
PEREIRA, H. G., and WRIGLEY, N. G. (1974). In vitro reconstitution, hexon bonding and handedness of incomplete adenovirns capsid. J. Mol. Biol. 85, 617-631. PERSSON,H., PETTERSSON,U., and MATHEWS,M. B. (1978). Synthesis of a structural adenovirus polypeptide in the absence of viral DNA replication. Virology
90, 67-79.
PETTERSSON,U., TIBBETTS, C., and PHILIPSON, L. (1976). Hybridization maps of early and late messenger RNA sequences on the adenovirus type 2 genome. J. Mol. Biol. 101, 479-501. PHILIPSON, L., PE?TERSSON,U., and LINDBERG, U. (1975). In “Molecular Biology of Adenoviruses. Virology Monographs 14” (S. Gard and C. Hallauer, eds.), Springer-Verlag, Wien/New York. SABORIO, J., and GBERG, B. (1976). Zn viva and in vitro synthesis of adenovirus type 2 early proteins. J. Viral. 17, 865-875. TIBBE?TS, C., PETTERSSON,U., JOHANSSON,K., and PHILIPSON, L. (1974). Relationship of mRNA from productivity infected cells to the complementary strands of adenovirns type 2 DNA. J. Viral. 13, 370-377. VAN DER VLIET, P. C., and LEVINE, A. J. (1973). DNA-binding proteins specific for cells infected by adenovirnses. Nature New Biol. 246, 170-174. WEBER, J. (1976). Genetic analysis of adenovirns type 2: III, Temperature sensitivity of processing of viral proteins. J. Viral. 17, 462-471.
9.3, 198-208
(1979)
A Maturation
Protein
in Adenovirus
Morphogenesis
HAKAN PERSSON,’ BERIT MATHISEN, LENNART AND ULF PETTERSSON Department
of Microbiology,
University
PHILIPSON,
of Uppsala, The Biomedical Center, Box 581, S-751 23 Uppsala, Sweden Accepted October 17, 1978
Adenovirus type 2 (ad2) assembly intermediates have been isolated by sucrose-gradient centrifugation. Such intermediates are distinguished from mature v&ions by the presence of uncleaved precursors to polypeptides VI, VII, and VIII as well as a prominent polypeptide with a molecular weight of 50,000 (50K) which disappears during maturation (B. Edvardsson, E. Eve&t, H. Jornvall, L. Prage, and L. Philipson, 1976, J. Viral. 19, 533-547; J.-C. D’Halluin, G. Martin, G. Torpier, and P. Boulanger, 1978, J. Vilirol. 26, 357-363). In the present communication we demonstrate that the 50K polypeptide is encoded by the viral genome and its gene is located on the l-strand of fragment SmaI-F (map coordinates 11.3 to 18.1).
in the assembly pathway. Through genetic and biochemical studies a wealth of knowlThe adenoviruses provide a convenient edge has been accumulated, in particular model system to study virus structure and concerning the morphogenesis of phage T4 morphogenesis, since they unlike most other viruses, have structural proteins which are of Escherichia coli and phage P22 of SalIn both these sysmonella typhimirium. soluble under nondenaturing conditions. tems the DNA has been found to enter The adenovirus type 2 (adz) virion contains preassembled capsids and both scaffolding at least nine different polypeptides, most of and maturation proteins have been identified which have been purified and characterized (for a review see Casjens and King, 1975). in considerable detail (for a review see Studies on the assembly of animal DNA Philipson et al. , 1975). Some virion polypepviruses have been hampered by the lack of tides are synthesized as precursors which nonsense mutants with lesions in the assuffer posttranslational cleavage during sembly pathway. Several temperaturevirus maturation (Anderson et al., 1973; Edvardsson et al., 1976). The assembly sensitive mutants which cause defective intermediates are distinguished from mature assembly have been described (Weber, 1976; virions by the presence of precursors to Edvardsson et al., 1978; D’Halluin et al., 1978b) but only one of them yields infectious polypeptides VI, VII, and VIII (Edvardsson progeny upon shift down (Weber, 1976). et al., 1976). In addition, analysis by SDSStudies on adenovirus morphogenesis have polyacrylamide gel electrophoresis (SDStherefore mainly been approached through PAGE) has revealed the presence of a 50K a biochemical characterization of the assembly polypeptide in assembly intermediates which intermediates. In the present communication is absent or present in reduced amounts in we demonstrate that intermediates in adenomature virions (Edvardsson et al., 1976; D’Halluin et al., 1978a). Nothing is how- virus assembly are associated with a 50K ever known concerning the origin of this polypeptide which is encoded by the l-strand of ad2 DNA and which has the properties of polypeptide. The morphogenesis of bacteriophages has a maturation protein. been studied in detail. These studies were facilitated by the availability of nonsense MATERIALS AND METHODS mutants which are defective in defined steps Infection and labeling conditions. Ad2 1 To whom reprint requests should be addressed. was propagated in suspension cultures of INTRODUCTION
0042-6822/79/030198-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
198
ADENOVIRUS
HeLa S3 cells in Eagle’s spinner medium as described before (Pettersson et al., 1976). Cells were infected at a multiplicity of 2000 particles per cell as described by Everitt et al. (1971). In all labeling experiments the medium contained l/50 of the normal concentration of methionine. [35S]Methionine was added 14 hr postinfection at a concentration of 200 &i/ml in pulse-chase experiments and at 1 $.X/ml for production of labeled virus. The labeling procedure and the virus purification scheme has been described in detail elsewhere (Edvardsson et al., 1976). Cell fractionation. Cytoplasmic and nuclear extracts were prepared as described before (Edvardsson et al., 1976). The extracts were analyzed by sucrose-gradient centrifugation. Sucrose-gradient centrifugation. Linear 25-40% sucrose gradients containing 0.2 M NaCl, 0.02 M sodium phosphate buffer, pH 8.0, and 0.01 M EDTA were centrifuged at 85,000 g for 2 hr. The gradients were fractionated from the bottom and individual fractions were analyzed by scintillation counting and SDS-PAGE. SDS -PAGE. Polyacrylamide gel electrophoresis was performed according to the method of Maize1 (1971). Exponential lo18% gradient slab gels (McGuire et al. ,1974; Edvardsson et al., 1976) or homogeneous slab gels (10 or 13%) were used. Electrophoresis was carried out at 140 V for 20 hr for the gradient gels and at 100 V for 10 hr for the other gels. The gels were analyzed by fluorography (Bonner and Laskey, 1974). Radioisotopes and counting procedures. [35S]Methionine (600 Wmmol) was obtained from New England Nuclear Corporation (Boston, Mass.). Radioactive samples were precipitated on Whatman 3MM filters with 10% trichloroacetic acid, washed with ethanol, and air dried. The filters were counted in a toluene-based scintillation liquid. Preparation of complementary strands of ad2 DNA. Adenovirus DNA was obtained from purified virions by the method of Tibbetts et al. (1974). Separated strands of ad2 DNA were prepared by equilibrium centrifugation of denatured DNA in CsCl in the presence of ribopoly (U,G) as described by Tibbetts et al. (1974).
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199
Selection of RNAIDNA hybrids. Cytoplasmic RNA was extracted from ad2infected HeLa cells (2000 virus particles/ cell) 18 hr postinfection as described by Brawerman et al. (1972). The conditions for hybridization between messenger RNA (mRNA) and the complementary strands of ad2 DNA, selection of RNA-DNA hybrids by exclusion chromatography on a Sepharose 2B column, and the isolation of the hybridized poly(A)-containing mRNA by poly(U)Sepharose chromatography are described in detail in a separate communication (H. Persson et aE., in preparation). Cell free translation. Unfractionated late cytoplasmic RNA or mRNA selected by hybridization to the viral l-strand was translated in vitro in a micrococcal nucleasetreated rabbit reticulocyte lysate (Pelham and Jackson, 1976). The translation mixtures were incubated for 2 hr at 35”, treated with pancreatic RNase and sodium hydroxide (Persson et al., 1978) and an equal volume of twice concentrated gel sample buffer was added (Maize& 1971) before analysis by SDS-PAGE. Hybrid arrested cell-free translation (HART) (Paterson et al., 1977). Restriction
fragments of ad2 DNA were denatured by incubation at room temperature in 0.3 M NaOH for 10 min. After neutralization with HCl the DNA was mixed with selected or unselected RNA. Thirty micrograms of unselected RNA or RNA selected on 0.5 pug of complementary strand DNA was mixed with l-3 pg of a restriction fragment. After adjusting the final volume to 5 ~1 with HzO, the samples were heated to 100” for 30 see, quickly chilled, and brought to a final volume of 100 ~1 containing 80% (v/v) deionized formamide, 20 mM l,kpiperazinediethanesulfonic acid (Pipes) buffer, pH 6.4, 1 m&f EDTA, and 0.4 M NaCl. The samples were incubated at 48” for 2 hr and the hybridization terminated by the addition of 300 ~1 ice-cold water containing 15 pg (in the case of hybrid selecting RNA) E. coli ribosomal RNA. The samples were then precipitated with 2.5 vol of ethanol at -20” overnight and centrifuged at 8000 g for 20 min at 4”. The pellet was dissolved in 0.15 M potassium acetate, pH 5.6, and reprecipitated with 2.5 vol of ethanol at
200
PERSSON
-20” overnight. The precipitates were finally collected by centrifugation as above, ethanol removed by lyophilization, and the samples dissolved in the appropriate amount of cold distilled water. Selected RNA was dissolved in 4 ~1of H,O and unselected RNA in 25 ~1 of H,O. The entire sample from the selected RNA was added to the in vitro translation system as described above while 3 ~1 of the unselected hybridized RNA was added per 25 ~1 of reaction mixture. Tryptic peptide analysis. Fractions from the sucrose gradient which contained 35Slabeled ad2 assembly intermediates (Fig. 2) were precipitated with ice-cold 10% trichloroacetic acid (TCA), washed once in 5% TCA, twice in acetone, dried, and dissolved in gel sample buffer (Maize& 1971). RNA selected on l-stand DNA was translated in vitro in the presence of [35S]methionine. Both samples were analyzed by SDS-PAGE. The gel was stained with Comassie brilliant blue (2% in 10% acetic acid, 30% methanol) for 30 min and destained for l-2 hr in 10% acetic acid 30% methanol. The gel was then dried and autoradiographed polypeptides were excised, eluted from the gel, washed, oxidized with performic acid, washed again, and digested with ~-l-tosylamide-Z-phenylethyl chloromethyl ketone (TPCK)-trypsin as described previously (Persson et al., 1978). The digests were finally lyophilized, dissolved in pyridine-acetate buffer, pH 5.5 (acetic acid:pyridine:water ‘7:20:970), and fractionated in two dimensions on Whatman CC41 cellulose thinlayer plates as previously described (Linne et al., 1977; Persson et al., 1978). Methionine-containing peptide were visualized by autoradiography. RESULTS
Polypeptide Composition Intermediates
of Assembly
Previous results have shown that a heterogenous class of assembly intermediates which sediment slower than mature virions can be isolated from nuclei 20 hr after ad2 infection (Edvardsson et al., 1976). In order to study the polypeptide composition of assembly intermediates, a nuclear extract was prepared from cells
ET AL.
labeled with [35S]methionine between 15 and 20 hr after infection. The supernatant was sedimented through a 25-40% sucrose gradient at 85000 g for 120 min as described by D’Halluin et al. (1978a) and the radioactivity profile of the fractionated gradient is shown in Fig. 1A. The fast sedimenting peak has previously (Edvardsson et al., 1976) been characterized as nuclear or young virions (NV) and the slowly sedimenting peak as assembly intermediates (IM). Figure 1A also shows the polypeptide composition of IM, NV, and mature virus. The precursor polypeptides 27K (pVI), 26K (presumably pVIII), and pVI1 are present as prominent bands in the IM fraction and also to some extent in the NV fraction but not in the mature virions which instead contain polypeptides, VI, VII, and VIII. In addition a polypeptide with a molecular weight of 50,000 (50K) is present in the IM fraction, in trace amounts in the NV fraction, and the ad2 virus marker. To ascertain that the peak of the 50K polypeptide coincided with the peak of the assembly intermediates, individual fractions from the sucrose gradient were analyzed by SDSPAGE. A fluorogram of the gel is shown in Fig. 1B. The label in the 50K polypeptide peaks in fractions 19-23 which corresponds to the IM peak. Since the 50K polypeptide is not present in the mature virions and since it is not known to be precursor of any other polypeptide it has the properties of a maturation protein in virus assembly. Fate of the 50K Polypeptide Chase Experiments
in Pulse-
In phage P22 it has been shown that the product of gene 8, which is a scaffolding protein, fails to chase out of the proheads in pulse-chase experiments which indicates that the protein is recycled (King and Casjens, 1974). On the other hand, the maturation protein p22 in phage T4 is extensively degraded during phage assembly (Kurtz and Champe, 1977). In order to establish which of these pathways the 50K polypeptide follows, pulse-chase experiments were performed and the fate of the 50K polypeptide was determined. Infected
ADENOVIRUS
201
ASSEMBLY
A
Ad2 NV
i c-4 “4 1’2
IM
FRACTION NUMBER c-4
14
16
18
20
22
24
26
28
2
B
FIG. 1. Analysis of ad2 assembly intermediates. A nuclear extract from ad2 infected KB cells, labeled with [3sS]methionine from 15 to 20 hr after infection was analyzed on a 25-40% sucrose gradient. (A) Radioactivity profile of the gradient and polypeptide composition of pooled NV and IM fractions as determined from a lo-18% SDS-polyacrylamide gradient gel. (B) The distribution of polypeptides in individual fractions of the sucrose gradient. Fluorograms of lo-18% SDSpolyacrylamide gradient gels are shown.
HeLa cells were pulse-labeled with [35S]methionine at 14 hr after infection for 15 min followed by a 6-hr chase and nuclear and cytoplasmic extracts were sedimented through sucrose gradients. Figure 2 shows the distribution of label in the sucrose gradients. The radioactivity in the IM area shows considerable decrease when chased for more than 1.5 hr and large amounts of
label enter both the nuclear and cytoplasmic virus. The IM and NV fractions were separately pooled as indicated in Fig. 2 and analyzed by SDS-PAGE (Fig.3). The labeled 50K polypeptide band is prominent after 1.5 hr but disappears almost completely after a 6-hr chase. No detectable 50K polypeptide was observed in the cytoplasmic virus. The precursor polypeptides
202
PERSSON ET AL. lSh
L,5h
3,oh
p? 50. a ito-
NV I” ct----(
NV l
6.0h
NV
IM :
t
20
30
IM
NV
It.4
I
!O P-i. 10
20
30
LO
10
40
10
20
30
40
10 20 30 LO FRACTION NUMBER
FIG. 2. Sucrose gradient analysis of a pulse-chase experiment. Cells were pulse-labeled with [T3]methionine for 15 min followed by a chase at 14 hr after ad2 infection. Samples were analyzed after indicated time periods. Nuclear (top panels) and cytoplasmic (bottom panels) extracts were centrifuged separately. Fractions from the NV and IM peaks were pooled separately as indicated and analyzed by SDS-PAGE.
suggest that the mRNA is transcribed from the viral l-strand since the messenger RNA map of the ad2 genome (Pettersson et al., 1976) shows that a gene block is located on the viral l-strand in this position. In order to establish the relationship between the 50K polypeptide in assembly intermediates and the virus coded 50K polypeptide we decided to isolate the two polypeptides and The ad2 Genome Encodes a 5OK Polypeptide compare them by tryptic fingerprint analysis. A 50-56K polypeptide has previously We have recently designed a method for been detected as a minor constituent of selection of viral mRNA utilizing Sepharose adenovirus type 2 (Maizel, 1971; Eve&t 2B chromatography for separation of DNAet al., 1973; Anderson et al., 1973). A poly- RNA hybrids from unhybridized RNA peptide with the same molecular weight in (Persson et al., in preparation). By utilizing SDS-polyacrylamide gels has also been large single stranded DNA to select RNA a observed from cells labeled with [35S]methi- differential exclusion between hybrids and onine late after ad2 infection (Anderson nonhybridized RNA can be achieved. To et al., 1973). Furthermore cell-free translaisolate the 50K mRNA, late ad2 cytoplasmic tion of late adenovirus mRNA selected RNA was hybridized to viral l-strand DNA by hybridization to restriction fragment in solution. The RNA-DNA hybrids were HindIII-C of the ad2 genome (map isolated by chromatography on Sepharose coordinates 7.5-17.0) directs the synthesis 2B. The hybrids were melted in 90% of this polypeptide (Lewis et al., 19781, formamide at 65” for 15 min and the mRNA which has been designated IV&. The map isolated by poly(U)-Sepharose chromatogcoordinates given for the IVa, polypeptide raphy whereupon it was translated in an 27K, 26K, and pVI1 chase efficiently and their mature counterparts are recovered in nuclear (Fig. 3) and cytoplasmic virus (not shown, see Fig. 12 in Edvardsson et al., 1976) during the same chase period. These results suggest that the 50K polypeptide turns over during virus maturation.
ADENOVIRUS
--II
-m 7gp -P
50K
--PI -pII FIG. 3. SDS-PAGE of the pulse-chase experiment. Analysis by SDS-PAGE of fractions from the pulsechase experiment in Fig. 2. The slots in the fluorogram contain the following samples: (1) and (10) ad2 virus marker; (2), (3), (4), and (5) correspond to IM fractions from 1.5, 3, 4.5, and 6 hr of chase; and (6), (‘7), (8), and (9) correspond to NV fractions from the same time points.
in vitro protein-synthesizing
system. Messenger RNA selected on the viral l-strand directed the synthesis of four polypeptides: a 75K polypeptide, corresponding to the adenovirus DNA-binding protein (Van der Vliet and Levin, 19’73)and a 19K polypeptide (Fig. 4). These two polypeptides have previously been characterized as ad2 early polypeptides which are synthesized in small or reduced amount also late after infection (Saborio and Oberg, 1976; Persson et al., 1978). In addition late mRNA selected on the l-strand directed the synthesis of a prominent 50K polypeptide as well as small amounts of a 60K polypeptide. The 50K polypeptide, synthesized from mRNA selected by the viral l-strand, migrated slightly faster on SDS-PAGE than the 50K polypeptide, previously detected in ad2 assembly intermediates (Fig. 4). Tryptic Fingerprint Polypeptide
Analysis
of the 50K
The relationship between the 50K polypeptide detected in ad2 assembly intermediates and the 50K polypeptide coded by
ASSEMBLY
203
the l-strand of the genome was investigated by tryptic fingerprint analysis. The two 50K polypeptides were purified by SDS-PAGE, eluted from the gel, and digested with TPCK-trypsin. Tryptic peptides were then separated in two dimensions on cellulose thin-layer plates and the plates analyzed by autoradiography. At least six [35S]methionine-labeled tryptic peptides with identical mobilities in the two dimensions were observed in both cases (Fig. 5). Thus the 50K polypeptide present in ad2 assembly intermediates and the 50K polypeptide programmed by mRNA from the l-strand of ad2 DNA have indistinguishable primary structures. Localization of the Gene for the 50K Polypeptide on the ad2 Genome
Hybridization of mRNA to a complementary DNA fragment has recently been shown to make the mRNA untranslatable in vitro (Paterson et al., 1977). This method was used to localize the gene for the 50K polypeptide on the ad2 genome and thereby verify its relationship to the IVa, polypeptide. Messenger RNA selected by hybridization to the viral l-strand was hybridized to different restriction enzyme fragments of ad2 DNA under conditions which allow formation of RNA-DNA hybrids while DNA-DNA renaturation is prevented (Casey and Davidson, 1977). The BamHI restriction fragments of ad2 DNA were hybridized to l-strand-selected mRNA. After hybridization, the samples were precipitated with ethanol and the mRNA translated in vitro in a reticulocyte cell-free system (Pelham and Jackson, 1976). The BamHI-B fragment (map coordinates O29.1) completely arrested the translation of the 50K polypeptide (Fig. 6). In contrast hybridization to restriction fragments BamHI-C and D (map coordinates 29.1-40.9 and 40.9-59.0) had no effect on the in vitro synthesis of the 50K polypeptide, as was also true for restriction fragments derived from the right-hand end of the ad2 genome (BarnHI-A, EcoRI-C and D, data not shown). To locate the gene for the 50K polypeptide more precisely, within the
204
PERSSON ET AL.
D
B
D
E 75K 60K 50K
27 .27K -26~
FIG. 4. In vitro synthesis of the 50K polypeptide. Ad2-infected HeLa cells were harvested 18 hr postinfection and cytoplasmic RNA was prepared. The RNA was hybridized in solution to the viral l-strand and the RNA-DNA hybrids separated from the unhybridized RNA by exclusion chromatography on a Sepharose 2B column (H. Persson et al., in preparation). The hybrids were melted, polyadenylated RNA isolated by poly(U)-Sepharose chromatography, and the selected RNA precipitated several times with ethanol. Unfractionated late RNA (5 @g/25 ~1 of reaction volume) or mRNA selected by hybridization to 0.5 fig of the viral l-strand was translated in vitro in a micrococcal nuclease treated reticulocyte lysate and the translational products analyzed in a 10% SDS-polyacrylamide gel. Electrophoresis was carried out for 10 hr at 100 V whereupon the gel was analyzed by fluorography. Slot A: late unfractionated RNA. Slot B: RNA selected on the viral l-strand. Slot C: no RNA added. Slot D: Pooled fractions, containing adenovirus assembly intermediates, from the sucrose gradient shown in Fig. 1. A [35S]methionine-labeled virus marker (ad2) was included.
BamHI-B fragment we hybridized the l-strand-selected mRNA to two SmaI restriction enzyme fragments, one of which (SmaI-F) is known to be transcribed late after infection (Pettersson et al., 1976). The SmaI-F fragments (map coordinates 11.318.1) completely arrested the translation of the 50K polypeptide while the SmuI-E fragment (map coordinates 2.9-10.7) had no effect on its synthesis (Fig. 6). Control experiments with unselected cytoplasmic RNA revealed that the DNA fragments themselves were not inhibitory for trans-
lation. The results suggest that the major part of the gene for the 50K polypeptide in assembly intermediates is located on the lstrand of fragment SmuI-F. The results do not exclude that a small portion of the 50K gene extends into the neighboring SmaI-B fragment. DISCUSSION
The morphogenesis of the adenovirion follows a complex pathway. The hexon capsomeresmay first assemble into ninemers (Pereira and Wrigley, 1974) which become
ADENOVIRUS
-
E LECTROPHORESIS
+
FIG. 5. Tryptic peptide analysis of the 50K polypeptide present in ad2 assembly intermediates and the 50K polypeptide encoded by the viral l-strand. Fractions from a sucrose gradient (Fig. 1) containing ad2 assembly intermediates were precipitated with TCA and the washed precipitates dissolved in gel sample buffer. RNA selected by hybridization to the viral l-strand was translated in vitro in a reticulocyte cell-free system in the presence of [35S]methionine. Both samples were analyzed on a 10% SDS-polyacrylamide gel. The polypeptide band corresponding to the 50K polypeptide in both sampleswas eluted from the gel and their tryptic pep tides were analyzed as described under Materials and Methods. Top panel (IM 50K), digest of the 50K polypeptide present in ad2 assembly intermediates. Bottom panel (IV%), digest of the 50K polypeptide translated from RNA selected on viral l-strand DNA.
associated with pentons and uncleaved precursors for polypeptides VI and VIII (pV1 and pVII1). In this way a procapsid is formed. The DNA, presumably covered
ASSEMBLY
205
with polypeptides V and pVI1, enters the procapsid shortly thereafter generating the assembly intermediates (Edvardsson et al., 1976, 1978). Alternatively the core proteins may enter the capsid after the DNA (D’Halluin et aZ., 1978a, b). The ad2 assembly intermediates contain a 50K polypeptide which is almost absent in nuclear vii-ions (Fig. 1, Ishibashi and Maize& 1974; Edvardsson et al., 1976, 1978; D’Halluin et al., 1978a). The final step in the assembly pathway involves the cleavage of precursor polypeptides pV1, pVI1, and pVII1 (Weber, 1976), probably by a protease which is associated with the virion (Ishibashi and Maizel, 1974; Weber, 1976). The experimental evidence for this tentative pathway is almost entirely based on pulse-chase labeling kinetics of infected cells and polypeptide composition of different classes of assembly intermediates. Due to the lack of suitable nonsense mutants in the adenovirus system it has been difficult to accumulate different classes of intermediates in sufficient quantities to characterize their structures. Temperature-sensitive mutants have only in one case (ad2 tsl, Weber, 1976) been demonstrated to accumulate young virions which mature into virions upon shift-down conditions. In other cases (Edvardsson et al., 1978) the tsmutants accumulate intermediates which cannot be chased into mature virions after shift-down, probably due to assembly of defective particles at the nonpermissive temperature. In several phage systems well-defined nonsense mutations are available which have made it possible to identify both scaffolding and maturation proteins required in different steps of the assembly pathway. The scaffolding proteins are defined as proteins involved in a matrix network for the assembly of the procapsid (King and Casjens, 1974) and the maturation proteins may have specific functions in subsequent steps, such as the introduction of DNA into preformed capsids or the rearrangement of the DNA or the capsomeres within the particles (Laemmli et al., 1974). In the adenovirus system pV1 and pVII1, which are located internally in the
PERSSON ET AZ,.
206
-5OK 50K-
FIG. 6. Hybrid arrested cell-free translation of RNA selected by hybridization to the viral l-strand. Late ad2 cytoplasmic RNA was hybridized in solution to the viral I-strand and the hybridized RNA isolated as described under Materials and Methods and in the legend to Fig. 4. RNA selected from 0.5 pg of the viral l-strand DNA was hybridized in the presence of 80% formamide to I pg of the indicated restriction fragments of ad2 DNA. After hybridization for 2 hr at 48” the reactions were terminated by addition of cold distilled water and the samples subsequently precipitated with ethanol. The precipitates were collected and the entire sample was translated in vitro. Ten microliters of the translation mixtures were analyzed on a 10% SDS-polyacrylamide gel (left panel) or on a 13% SDS-polyacrylamide gel (right panel). Electrophoresis was for 10 hr at 100 V and the gels were analyzed by fluorography. The restriction enzyme fragments used for individual hybridizations are indicated at the top of the figure. No RNA added indicates endogenous translation in the cell-free system. The fluorograms were exposed for 2 days (left panel) or 8 days (right panel); [Wlmethioninelabeled virus marker was also included in the analysis (Ad2).
capsids (Everitt et al., 19’75), may have a scaffolding function but if so they differ from the scaffolding proteins of phage P22 in S. typhimurium since they remain in a cleaved form in the mature virion. Several investigators (Edvardsson et al., 1976; D’Halluin et al., 1978a) have previously reported the presence of polypeptides in assembly intermediates which are absent or present in reduced amounts in mature virions. One such polypeptide is the 50K polypeptide which is further investigated in the present communication. The peak of the 50K polypeptide is shown to coincide with the peak of assembly intermediates in sucrose gradients (Fig. 1B) and it thus appears
established that the 50K polypeptide is associated with the assembly intermediates and not fortuituously present in semipurified preparations of assembly intermediates. Pulse-chase experiments further demonstrate that the 50K polypeptide disappears from the peak of assembly intermediates during chase without being recovered in the peak of mature virions. The 50K polypeptide thus has the properties of a maturation protein which unlike the gene 8 product of phage P22 (King and Casjens, 1974) fails to be reutilized during virus assembly. In vitro translation of mRNA selected on the l-strand of ad2 DNA demonstrates that
ADENOVIRUS
the viral genome specifies a polypeptide which has a similar electrophoretic mobility in SDS-PAGE as the 50K maturation protein. The identity between the in vitro synthesized polypeptide and the maturation protein is furthermore established by tryptic fingerprint analysis (Fig. 5). It is not known why the in vitro and in vivo synthesized polypeptides have slightly different electrophoretic mobilities in SDS-PAGE but the 50K polypeptide presumably becomes modified by phosphorylation after translation (D’Halluin et al., 1978a) which may alter its mobility in SDS-polyacrylamide gels. The major part of the gene for the 50K maturation protein is located within fragment SmaI-F (map coordinates 11.3 to 18.1) as revealed by hybrid arrested cell free translation (Fig. 6). Thus the gene for the 50K maturation protein is located in the left of the two late gene blocks on the l-strand which were identified by hybridization between late mRNA and separated strands of several restriction fragments (Pettersson et al., 1976). The location of the gene for the 50K polypeptide is interesting in the sense that it is controlled by a different promoter than the late genes which specify the structural polypeptides of the adenovirus particle. Lewis et al. (1978) have previously mapped the gene for polypeptide IVa, within fragment HindIII-C (map coordinates 7.5-17.0) by in vitro translation of mRNA which was selected by hybridization to restriction fragments of ad2 DNA. Because of its size and map position it is likely that polypeptide IVa, is identical to the 50K maturation protein. It is, however, unclear whether the presence of the IVa, polypeptide in purified virus preparations is caused by trace amounts of immature particles in purified virus or whether trace amounts of the 50K polypeptide remain in virions during maturation and play a functional role also in the mature particle. ACKNOWLEDGMENTS This study was supported by grants from the Swedish Medical Research Council and the Swedish Cancer Society. We thank M. Gustafson for excellent
ASSEMBLY
207
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