Download Adenovirus-associated Virus Structural Protein Sequence Homology

J. gen. Virol. (I979), 45, 2o9-216
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Adenovirus-associated Virus Structural Protein Sequence Homology
By M. D. L U B E C K , 1. H. M. LEE,2t M. D. H O G G A N a AND F. B. J O H N S O N I : ~
1Department of Microbiology, Brigham Young University, Provo, Utah, U.S.A.
Department of Microbiology, Mt. Sinai School of Medicine, New York City, N. Y., U.S.A.
a Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, NIH,
Bethesda, Md., U.S.A.
(Accepted 14 May I979)
SUMMARY
Adenovirus-associated virus (AAV) structural proteins (VPI, VP2, and VP3)
have been examined to determine if areas of sequence homology exist between
these three virion proteins. Tryptic and chymotryptic maps have been produced
which demonstrate extensive areas of sequence homology common to all three
proteins. The amino acid compositions of the proteins were also determined and
were found to be very similar. These data are consistent with the hypothesis that
all three virion proteins arise either from a common precursor or similar transcripts.
INTRODUCTION
Adenovirus-associated viruses (AAV) are defective parvoviruses which have been
demonstrated to be antigenically distinct from helper adenoviruses (Hoggan et aL
I966). Three structural proteins have been identified in AAV-3 virions: VPI (mol. wt.
about 66ooo), VP2 (tool. wt. about 80000) and VP 3 (mol. wt. about 92000; Johnson et al.
I97I; Rose et al. I97I). These three virion proteins have also been found to be present
in the AAV top component (Johnson et aL I975) and in AAV dense-band particles (Johnson
et al. I97I). About 7 o ~ of the AAV genome is transcribed into a single messenger RNA
with a mol. wt. of about I.o× io n (Carter, I974; Carter & Rose, I974). This messenger
possesses sufficient information to code for a protein of mol. wt. i2oooo or little more than
the largest virion protein. Thus, the aggregate mol. wt. of the three virion proteins (238 ooo)
exceeds the coding capacity o f the virus genome (I6oooo) and that of the virus messenger
020000). Evidence suggesting the three AAV proteins share common antigens resulted
from a study of the cross-reactivity of antisera prepared against purified sodium dodecyl
sulphate (SDS)-bound virion polypeptides (Johnson et al. t972). Further evidence concerning the structural and antigenic relatedness of these proteins has been obtained and
suggests the possible common origin of all three virion proteins from a I2oooo mol. wt.
precursor protein, VPo (Johnson et al. I977). The present study was undertaken in an
attempt to confirm these findings through the use of peptide mapping techniques and to
compare the amino acid composition of the purified proteins.
METHODS
Viruses and cells. AAV was produced in KB cell suspension cultures with adenovirus type
2 helper and purified as previously described (Johnson et al. I97I).
* Present address: Department of Microbiology, Mt Sinai School of Medicine, New York City, N.Y.,
U.S.A.
i" Present address: American Cyanimid Co., Clifton, N.J., U.S.A.
~: Author to whom reprint requests should be addressed.
oo22-1317/79/oooo-3665 $02.oo
~ 1979 SGM
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2IO
M.D. LUBECK AND OTHERS
Protein determination. The method of Lowry et al. (t95I) was used to determine protein
concentrations. Determinations were performed on SDS-disrupted virions using bovine
serum albumin as the standard protein.
Radioiodination of virion protein. Purified virions were disrupted by treatment in I.O~o
SDS at loo °C for 2 min. Samples containing 3o to 5o/zg of virus protein were suspended
in 5o/zl of o'o5 M-phosphate buffer, pH 7'4 (buffer I), and the proteins iodinated by the
chloramine-T method (McConahey & Dixon, I966). Briefly, 5o/zCi Na 1~5I (obtained from
Industrial Nuclear Co., St. Louis, Mo, U.S.A.) were added to the virus suspension, followed
by Io/zl of chloramine-T in buffer I, resulting in a final chloramine-T concentration of
0.2 mg/ml. The mixture was agitated for 5 min at room temperature, whereupon the reaction was terminated by the addition of 25/tg of sodium metabisulphite dissolved in Io/tl
of buffer I.
Electrophoresis of proteins. Iodinated virion proteins were dialysed exhaustively against
o.ol M-phosphate buffer, pH 7.2 (buffer II) and the iodinated proteins separated by SDSpolyacrylamide gel electrophoresis according to the method of Maizel 0969). The virion
proteins were visualized by staining with Coomassie Blue according to the method of Fairbanks et al. (I97I).
Amino acid analysis. The stained bands were excised (less than 2 mm gel/band) and washed
with distilled H20. Each gel piece was then hydrolysed with t-o ml of constant boiling hydrochloric acid containing o'o5 ~ mercaptoethanol and o.o 5 ~/o phenol. The hydrolysis of the
virus proteins was allowed to proceed at I IO °C in an evacuated, sealed tube for 24 h. Each
sample was then cooled and the polyacrylic acid precipitate removed by centrifugation.
The supernatant was transferred to a clean tube with a Pasteur pipette and the precipitate
washed with 2 × o . 8 ml H~O and combined with the previous supernatant. The protein
hydrolysate was dried in a heating vacuum desiccator at 45 °C and then dissolved in 8o/tl
of sample dilution buffer (o.2 M-sodium formate, pH 2.2). Twenty/zl of each sample was
routinely analysed.
Amino acid analyses were performed with the fluorometric microbore analyser, using
O-phthalaldehyde (OPA) as the detection agent (Lee et aL 1978, 1979).
Peptide mapping. The stained purified proteins were eluted from the gel fragments by
incubation in buffer II containing o-I ~ SDS. The dark blue eluates were then briefly
centrifuged at 60o g for 15 min, filtered, and exhaustively dialysed at 24 °C against buffer
II to remove unbound SDS. Fifty/zg of bovine serum albumin were then added as a carrier
and the proteins co-precipitated by the addition of 0-2 vol. of cold 5o ~ trichloroacetic acid
(TCA). After incubation on ice for 4 h the precipitate was pelleted at 2 5 o o o g for 30 min.
The pellet was then solubilized in cold I N-NaOH and re-precipitated with cold 20 ~ TCA.
After washing sequentially in cold 2 o ~ TCA, acetone: I N-HC1 (40: i, v/v; --20 °C), and
acetone (--2o °C), the protein precipitate was dried under a fine stream of nitrogen gas.
The proteins were then suspended in o'o5 M-NH4HCO3, pH 8.6 and digested with either
diphenyl carbamoyI chloride-treated trypsin (Calbiochem Inc., San Diego, Ca, U.S.A.) or
chymotrypsin (fi-chymotrypsin, Sigma Chemical Company, St. Louis, Mo., U.S.A.). The
ratio of enzyme to virion protein was i/IOO in each instance. The proteolytic hydrolyses were
allowed to proceed for 6 h at room temperature with intermittent agitation, after which the
digests were lyophilized. The peptides were dissolved in electrophoresis buffer (acetic acid:
formic acid: H20, 8o: 20: 9oo, v/v, pH 2. I) and applied to cellulose thin-layer chromatographic sheets (o.i mm thickness, 2o x 2o cm, EM laboratories, Elmsford, N.Y., U.S.A.) by
repeated spotting under a current of warm air. Electrophoresis was conducted for about I h
at I kV. The plates were then dried under a current of warm air until opaque and allowed to
equilibrate with the chromatography solvent before development in the second dimension.
The chromatography solvent consisted of butanol:pyridine:acetic acid: HzO in the ratio
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AA V structural protein analysis
211
Table I. The amino acid composition of the three AA I"-3 virion proteins*
Residues
/k
f
Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
Ileu
Leu
Tyr
Phe
VP3
VP2
VPI
1t 7"o
62"5
86.8
99"7
I oo-8
66"3
49"9
1o.2
32"6
52"9
2o'5
26.2
95"9
58"8
69"2
82'7
86"9
49"o
43"2
1o-8
24"7
45"7
I9'4
25"3
90"3
54"5
63"4
75'5
59'8
4o'4
33 "5
8.2
23"4
38"6
195
23"4
* The virus utilized was the major-band virus (relative specific density in CsCI -- 1"394 g/ml).
Table 2. The amino acid composition of the three AA I/-2 virion proteins from the
major-band virus*
VP3
Mol ~
Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
lieu
Leu
Tyr
Phe
His
VP2
A
r
16.68
8'35
I 1-59
12"33
13' I I
8.06
6'23
1"39
4"23
6.62
2-61
3"57
5"87
~,
Residues
127"9
64'0
88-9
94'6
1OO'6
61.8
47'8
1o'7
32"4
5o'7
20"0
27"4
45'0
VPI
A
g
Mol ~
15'50
9"4o
11-36
13'1o
12'90
7"41
6'38
1.6o
3 "47
6"99
2-86
4"Ol
5"o3
-~
f
-
-
~
,
-
-
Residues
Mol %
Residues
996
6o4
73"0
84-2
82"9
476
41-o
1o3
22 "3
44"9
18"4
25-8
32"3
I6.48
9"85
I 1'95
13'54
10"4I
6"7I
5"78
1"65
3 '7°
6"44
3"35
4"61
5"53
90"9
54"3
65"9
74'7
57'4
37"0
31'9
9'I
20"4
35'5
18'5
25'4
3o'5
h
* Relative specific density in CsCI = 1.388 g/ml.
of 97:75: x5:6o (v/v). Radioactive peptides were located by autoradiography on G A F H R
iooo high resolution medical X-ray film.
RESULTS
Amino acid analyses of virion proteins
During the hydrolysis of the virion proteins, a relatively large amount of ammonia is
released from the hydrolysis of the polyacrylamide gel. The ammonia is not removed during
the subsequent handling of the hydrolysate (Houston, I971) and later reacts with O-phthalaldehyde (OPA) forming a fluorescent adduct. This results in a large ammonia peak that
frequently obscures the basic amino acid peaks (Lys and Arg) and interferes with their
analysis and measurement. The OPA analyser has a detection sensitivity at the IOO pmol
level and, therefore, generally requires less than I/zg protein. Since the amount of protein
loaded on a single gel is in the/zg range, a band in a single gel is normally enough for several
analyses.
Table t shows the amino acid compositions of the virus proteins, VPI, VP2, and VP3
from AAV type 3. These compositions are based on mol. wt. of the respective proteins: that
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M.D. LUBECK AND OTHERS
Table 3. The amino acid composition of the three AA V-2 virion proteins from the
dense-band virus*
Residues
A
Asp
Thr
Set
Glu
Gly
Ala
Val
Met
Ileu
Leu
Tyr
Phe
VP3
I21.I
66.6
9o'8
96"9
98' I
6o-5
48"4
7'6
31'5
53"3
24"2
27.8
VP2
97"I
6o.2
77'7
82-6
82'6
58'6
4o'8
9"7
VPI
90"3
54"2
65"9
75"8
56"9
37"6
3I'6
9-o
22"3
20"8
44"7
19.4
27"2
36q
18.i
25"3
* Relative specific density in CsCI = 1.47 g/ml.
is, about 830 residues for VP3, 720 residues for VP2 and 600 residues for VPI. Differences,
as well as similarities, in the amino acid compositions of these three proteins were observed.
VP3, the largest protein, contained a relatively large amount of aspartate, glutamate and
glycine, whereas VPI had much less aspartate, glycine and glutamate than VP3. The
threonine, methionine, tyrosine and phenylalanine contents of these three polypeptides are
quite similar. It is interesting to note that the amino acid compositions of VPI and VP2 are
contained within that of VP3, and the amino acid composition of VPI is contained within
that of VP2.
The amino acid composition of major-band AAV-2 is shown in Table 2, and that of
dense-band AAV-2 is shown in Table 3. Not surprisingly, the amino acid compositions
of AAV-2 and AAV-3 were very similar as were the compositions of virus of both densities.
Peptide mapping of AA V-3 proteins
Tryptic peptide maps were initially produced for each of the three structural proteins,
VPI, VP2 and VP 3 (Fig. I). Comparisons of the radioactively-labelled peptides produced
from each of the digests indicated that at least 19 highly-labelled peptides were found
common to all three proteins. A number of poorly-labelled peptides (about 3I peptides)
were also found common to all three maps. As tyrosine is more readily iodinated than
histidine using this iodination technique, the darker spots in the autoradiographs most
likely represent tyrosine-containing peptides. VP 3, the largest virion protein, was found to
possess at least three unique highly-labelled tryptic peptides, whereas the tryptic maps of
VP2 and VP~ contained no unique peptides. Peptides that appeared to be c o m m o n to the
VP2 and VPI maps were also identified. However, these peptides were poorly labelled and
restricted to the more obscure and difficult to interpret areas of the maps. It thus appeared
that the only peptides in these tryptic maps that could be clearly identified as unique to a
single protein were the highly-labelled peptides unique to the VP3 map.
Chymotryptic maps of the three structural proteins were also produced to further compare the sequence homology of the three structural proteins (Fig. 2). Comparisons of the
chymotryptic maps indicated that there are at least I7 highly-labelled peptides common to
the three maps. Numerous (23) poorly-labelled peptides were also found, common to all
three. The VP3 map was observed to possess at least four highly-labelled and five poorlylabelled unique chymotryptic peptides. A single poorly-labelled peptide was observed to be
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AA V structural protein analysis
213
Q
%
.o
r,
6 v P27lWl,2,9~O
~')-IVP3 "~°~VP2, 3.9
4
Electrophoresis
Fig. t. Tryptic maps of AAV-3 structural proteins. Purified virion proteins were digested enzymically for 6 h at 24 °C and then electrophoresed at pH 2.I on cellulose thin-layer plates. The maps
were developed in the second dimension by ascending chromatography at pH 5"6. The diagrammatic representation in this figure and in Fig. 2 shows the VPI spots enclosed within a solid line, the
VP2 spots are stippled and the VP3 spots are shown by a series of diagonal lines. Thus, peptides
shared by all three polypeptides are enclosed by a solid line and contain dots and diagonal lines.
u n i q u e to the VPI map. N o peptides u n i q u e to the VP2 m a p were identified; however, a
single poorly-labelled peptide was observed that was c o m m o n to VP2 a n d VP3. It thus
appears that the p r e p o n d e r a n c e of labelled peptides in both the tryptic a n d chymotryptic
digests are c o m m o n to all three proteins. These maps thus provide considerable evidence for
the concept that these three proteins are largely encoded in the same sequences of virus D N A .
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214
M. D. L U B E C K A N D O T H E R S
@
@
~@
~
o
E
'~VP3?
@.VP2,3/~
vP3""
.-,¢
s
@
@
Electrophoresis
Fig. 2. Chymotryptic maps of AAV-3 structural proteins. Purified virion proteins were digested
enzymically for 6 h at 24 °C and then electrophoresed at pH 2.1 on cellulose thin-layer plates.
The maps were developed in the second dimension by ascending chromatography at pH 5"6.
DISCUSSION
It has previously been demonstrated that the structural proteins of adenovirus-associated
viruses possess similar biochemical and immunological characteristics. The labelling of
AAV- 3 structural proteins with selected amino acids (leucine, lysine and aspartate) has
previously demonstrated that, with respect to these amino acids, the amino acid compositions of these three proteins are similar (Johnson et al. 1977). Rose et al. (1970 have similarly
demonstrated that none of the AAV-2 virion proteins is selectively enriched with arginine
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AA V structural protein analysis
2I 5
(unlike the helper adenovirus proteins). The amino acid composition data presented here
provide more complete evidence demonstrating the similarity of the amino acid compositions
of the three virion proteins. The relative concentration (mole percentage) of each amino acid
is also similar for each of the virion proteins. The amino acid compositions of the three
proteins are such that VP2 and VPI could be derived from VP3, and VPI could be derived
from VP2. Data obtained from the purified polypeptides of dense-band AAV particles
indicate that there are no apparent differences between the amino acid compositions of
dense-band and major-band virion polypeptides.
The peptide mapping data indicate that the three AAV- 3 structural proteins possess extensive sequence homology. The chymotryptic peptide maps indicate that a single peptide is
unique to VPI. That a unique peptide is not identified in the VPI trypsin digest suggests that
the precursor molecule is cleaved by an enzyme with a trypsin-like specificity. The tryptic
maps as well as the chymotryptic peptide maps of VP2 and VPI are very similar. It is
not clear at this point why a larger number of peptides in the VP2 digests were not identified,
but may be due to tyrosine- and histidine-poor regions of the molecule. The amino acid
composition data indicate that VP2 and VPI possess an identical number oftyrosine residues,
so that sequences unique to VP2 would be detected by these techniques only if they contained histidine residues. The tryptic and chymotryptic maps clearly demonstrated numerous
peptides unique to VP 3. Peptides unique to two of the three peptides were few in number and
poorly labelled, so that definitive identification of these peptides was not possible. Peptide
maps of the three virion proteins using 35S-methionine and 14C-mixed amino acids did not
provide improved resolution of these peptides (data not shown). The above data suggest that
the VPI sequence may be contained in VP2 and that the VP2 sequence may be contained in
VP 3. It would seem possible from these data that VP3 could serve as the direct precursor to
both VPI and VP2. Alternatively, it may be that all three proteins are derived from VPo,
the I2OOOO mol. wt. precursor observed in a previously described pulse-chase experiment
(Johnson et al. I977).
Using peptide mapping techniques, Tattersall & Shatkin (t977) have demonstrated that
the two smallest proteins (proteins B and C) of MVM are completely contained within the
largest virion protein (protein A). Protein C, the smallest virion protein, was similarly
found to be contained within protein B. As the combined tool. wt. of most parvovirus proteins exceed the coding capacity of their respective genomes (see Rose, t974, for review)
the processing of a precursor protein to two or three structural proteins may then represent
a general mechanism used in the replication process of most parvoviruses.
This work was supported by gifts from Brigham Young University, the ASBYU College
Council, an Intergovernmental Personnel Agreement to F. B. J. and Research Contract No.
DADA I7-69-C-9137 from the U.S. Army Medical Research and Development Command
t o l l . M. U
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