The Molecular Biology of Yaba Tumour Pox Virus

J. gen. Firol. (1982), 62, 207-218.
Printed in Great Britain
207
Key words: lipids/proteins/DNA hybridization
The Molecular Biology of Yaba Tumour Pox Virus: Analysis of Lipids,
Proteins and DNA
By H. R O U H A N D E H , *
D. K I L P A T R I C K
AND A. V A F A I
Laboratory of Molecular and Cancer Virology, Department of Microbiology, Southern
Illinois University, Carbondale, Illinois 6290i, U.S.A.
(Accepted 31 March 1982)
SUMMARY
Cytopathological studies have shown that Yaba tumour pox virus (Yaba virus)
infection leads to the accumulation of large lipid vacuoles. The rate of accumulation
of these vacuoles increased as the infection proceeded. These lipid vacuoles were not
seen in control cells or in cells infected with monkeypox virus (MPV) but were seen
during Yaba virus infection in the presence of cytosine arabinofuranoside (100
~g/ml). Yaba virus also failed to inhibit host protein synthesis as infection proceeded
for prolonged periods. Yaba virus proteins were shown to be substantially different
from those of MPV when analysed by two-dimensional electrophoresis. The genome
of Yaba virus gave restriction enzyme fragments which differed from those of the
MPV genome when cleaved with the enzymes HindlII and XhoI. However, Yaba
virus DNA hybridized to the HindlII fragments K, L and M and to the XhoI
fragments A, B, C, E and G ofMPV DNA.
INTRODUCTION
Yaba tumour pox virus (Yaba virus) is an unclassified poxvirus capable of inducing benign
histiocytomas in man and monkey (Bearcroft & Jamieson, 1958; Andrewes et al., 1959;
Ambrus et al., 1963; Sproul et aL, 1963). In rhesus monkeys inoculated with Yaba virus,
histiocytes migrate into the infected area and rapidly proliferate leading to tumour formation
(Sproul et al., 1963). Multinucleate cells with many cytoplasmic inclusion bodies, vacuoles
and globules of neutral fat are common within the tumours. Tumours induced in man and
monkeys never become malignant and regress spontaneously, probably as the result of
cytopathic effects of the virus and degeneration of the individual cells (Grace & Mirand,
1963). Although Yaba virus is morphologically similar to other viruses of the poxvirus group,
there are differences between this virus and other poxviruses.
Immunological studies of Yaba virus did not show any cross-reactivity with vaccinia, off
(Niven et al., 1961) and monkeypox viruses (Behbehani et al., 1968). The rate of growth of
Yaba virus is much slower than that of other poxviruses. The complete cycle of vaccinia virus
synthesis has been shown to be around 5 to 10 h (Dales & Kajioka, 1964; Appleyard &
Westwood, 1964). However, electron microscopic observations of Yaba virus in infected cells
showed mature virus particles 5 days after infection (DeHarven & Yohn, 1966). Although
markedly altered in morphology, the virus-infected cells could be maintained for extended
periods of time (Tsuchiya et al., 1969). Recent studies have also shown that the results of
SDS-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of Yaba virus polypeptides
differed from other poxviruses, with the exception of a few proteins that were common to all
of the poxviruses analysed (Arita & Tagaya, 1980).
Since Yaba virus causes tumours, the interaction of this virus with its host during infection
is of interest, These studies report two of the interesting properties seen during infection with
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208
H. ROUHANDEH,
D. KILPATRICK
AND A. VAFAI
Yaba virus: lipid accumulation in infected cells and the lack of inhibition of host proteins
during infection. Monkeypox virus (MPV) was used in these studies as a means of
comparison because (i) it is a well-established member of the Orthopoxvirus group and is
non-tumourigenic and (ii) it has the same natural host and will grow in the same permissive
cells as Yaba virus. Several properties of the virions of both viruses were also analysed to
elucidate any further differences that have not been previously reported.
METHODS
Virus and cells. MPV Copenhagen strain 10001 and Yaba virus were grown in
monolayers of a cynomolgus line of monkey kidney cells (Tsuchiya et al., 1969). The
monolayers were infected at an input multiplicity of infection (m.o.i.) of 0.1 to 1. Adsorption
was allowed to continue for 2 h at 37 °C. Eagle's modified minimal essential medium (MEM)
containing 2 % calf serum was then added. MPV was routinely harvested at 3 days
post-infection. Yaba virus was harvested at 6 to 7 days post-infection.
Purification of virus. MPV and Yaba virus were purified from tissue culture fluid as
described previously (Kilpatrick & Rouhandeh, 1981).
Detection of lipid droplets in the infected cells. Cells were grown on coverslips and infected
with either Yaba virus or MPV (1 p.f.u./cell) in the presence or absence of cytosine
arabinofuranoside (100 #g/ml). Three days post-infection the coverslips were washed with
phosphate-buffered saline (PBS), fixed with 4% glutaraldehyde for 2 h and 1% osmium
tetroxide for 1 h. The coverslips were washed with PBS and stained with 5 % Giemsa stain for
10 min. After washing with PBS, the coverslips were mounted on slides with PBS and
examined for lipid droplets.
Radioactive labelling of cells. Cells grown in 75 cm 2 tissue culture flasks were infected at
an input m.o.i, of 10 with either MPV or Yaba virus and allowed to adsorb for 2 h at 37 °C.
The infected and control cells were washed with serum-free MEM, and 10 ml 2% MEM
containing 10% unlabelled methionine and 10/~Ci/ml [3SS]methionine (Schwarz/Mann; sp.
act. 1046 Ci/mmol) was added. The samples were harvested after 3 days. The radioactive
medium was poured off and the cells were washed three times with PBS. A 5 ml amount of
buffer was then added and the cells scraped into the medium and pelleted at 800 g for 8 rain.
The pellet was allowed to drain and 50/A sonication buffer (0.01 i - T r i s - H C l pH 7.4, 5
mM-MgC12) was added and the pellet sonicated at low force (10/~m, peak-peak power) three
times for 15 s each and stored at - 7 5 °C. A 5 ~I amount of each sample was trichloroacetic
acid (TCA)-precipitated and counted for 10 min or 1% efficiency using a Beckman liquid
scintillation counter.
Two-dimensional electrophoresis. Control cells, infected cells and purified virus were
analysed by O'Farrell's (1975) technique as described previously (Kilpatrick & Rouhandeh,
1981).
Isolation of virus DNA. DNA was isolated from purified Yaba virus and MPV by
treatment of the virus with 5% SDS in TNE buffer (0.01 M-Tris-HC1 pH 7.4, 0.1 M-NaC1,
0-001 M-EDTA) and autodigested Pronase (500/~g/ml) overnight at 37 °C. The DNA was
extracted twice with 80% phenol, twice with chloroform-n-butanol (24: 1), and precipitated
from the aqueous phase by 100% ethanol. The DNA was washed twice with ethanol,
dissolved in TNE buffer and precipitated with ethanol. The resulting DNA was dissolved in
TE buffer (0.05 M-Tris-HC1 pH 7.8 and 0-001 M-EDTA) at a concentration of 1 ~tg/5 ~tl.
Restriction of the DNA. Digestion of DNAs with HindIII (Bethesda Research
Laboratories) restriction endonuclease was carried out in 20 mM-Tris-HC1 pH 7.4, 7
mM-MgC12 and 60 mM-NaC1. XhoI (Bethesda Research Laboratories) digestion was done in
150 mM-NaCl, 6 mM-Tris-HCl pH 7.9, 6 mM-MgC12 and 6 mM-2-mercaptoethanol.
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Yaba tumour pox virus
209
Incubation was for 2 h at 37 °C. SmaI (New England Biolabs) digestion was done in 20
mM-KCI, 6 mM-Tris-HC1 pH 8, 6 mM-MgC12 and 6 mM-2-mercaptoethanol. Incubation was
at 30 °C for 1 h. The reactions were stopped by the addition of an equal volume of agarose
suspension (Schaffner et al., 1976).
Gel electrophoresis. After heating the samples for 5 min at 60 o C and quick cooling in an
ice-bath, the samples were applied to a 0.7% horizontal agarose gel (20 × 15 x 0.3 cm).
Lambda phage DNA digested with HindlII was run in parallel as a size marker.
Electrophoresis was in a Tris-buffered system (Loening, 1969; 4.84 g Tris pH 7-8, 2.82 g
sodium acetate, 0.74 g EDTA, 1.8 ml acetic acid in 1 1) at 1.5 V/cm for 15 h. After
electrophoresis the gel was stained for 30 min with 1 pg/ml ethidium bromide, placed under a
u.v. lamp and photographed through a red filter with Polaroid type 55 film.
Evaluation of photographs. For comparison of migration profiles, the number of bands and
the migration distances were determined by enlarging the negatives. The mol. wt. of the
poxvirus DNA cleavage products were determined by comparison to the lambda phage DNA
fragments of known size. To determine greater than molar amounts of DNA in a restriction
fragment, the negatives were scanned using a laser densitometer.
Preparation of labelled Yaba virus DNA probe. A 1 /tg amount of Yaba virus DNA or
MPV DNA was labelled with [~J2p]dCTP to a specific activity of 6 x 107 to 8 × 107
ct/min/pg as described by Rigby et al. (1977). The nick-translation system was purchased
from New England Nuclear.
Analysis of virus DNA by the Southern technique. MPV DNA (2 pg) was cleaved with
HindlII and XhoI restriction enzymes. The fragments were separated by electrophoresis on a
0.7% agarose gel. After electrophoresis the DNA was denatured by submerging the gel in
0.5 ~a-NaOH, 1.5 M-NaC1 at room temperature for 30 min. Subsequently, the gel was
neutralized in 3 M-sodium acetate pH 5.5 for 30 min and the denatured DNA fragments were
then transferred to BA 85 nitrocellulose filters (Schleicher & Schuell, Keene, N.H., U.S.A.) by
Southern's (1975) technique. The filters were rinsed in 2 x SSC, dried at 80 °C for 2 h, and
sealed in plastic bags.
DNA-DNA hybridization and autoradiography. The D N A - D N A hybridization was
performed as described by Wahl et al. (1979) and Stabel et al. (1980). The filters were
preincubated in 70 ml of a solution containing 50% formamide, fivefold concentrated
Denhardt reagent (1 x reagent contains 20 mg each of bovine serum albumin, Ficoll-400 and
polyvinylpyrrolidone in 100 ml), and 300 pg of heat-denatured calf thymus DNA per ml in
0.05 M-sodium phosphate pH 6.5 and 5 x SSC. The filters were preincubated at 41 °C for 24
h. The preincubation buffer was replaced by 50 ml of a solution containing the heat-denatured
32p-labelled virus DNA probe (6 x 107 to 7 × 107 ct/min) in 50% formamide, 1 x Denhardt
reagent and 100 #g of heat-denatured calf thymus DNA per ml in 5 x SSC, 0.02 M-sodium
phosphate pH 6.5, and 10% sodium dextran sulphate 500. The filters were incubated at
41 °C for 12 to 14 h. The filters were then washed successively with several changes of 2 ×
SSC and 0.5% SDS at 65 °C for a total of 5 h. After air-drying, the filters were exposed to
Kodak X-Omat AR film with an intensifying screen at - 7 6 °C for 1 to 5 days.
RESULTS
Virus-specific lipid formation
The cytopathological analysis of monkey kidney cells during Yaba virus infection shows
the presence of many cytoplasmic granules. These granules were stained with a lipid-specific
stain, Oil red-O, and were easily removed with lipid solvents such as chloroform. Further
enhancement of these granules in Yaba virus-infected cells was seen by fixing the cells with
glutaraldehyde and staining with osmium tetroxide (Fig. 1). These granules accumulated as
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H. ROUHANDEH,
D. KILPATRICK
AND
A. VAFAI
Fig. 1. Lipid granules shown by fixing monkey kidney cells with glutaraldehyde and osmium tetroxide
and staining with Giemsa as described in Methods. (a) Control cells; (b) MPV-infected cells; (c) cells
infected with Yaba virus while in the presence of 100 #g/ml cytosine arabinofuranoside; (d) cells
infected with Yaba virus. The arrows to the right and left of centre in (c) show large lipid droplets.
Numerous small lipid granules or droplets were found in the Yaba virus-infected cells but none could be
detected in the control or MPV-infected cells.
the infection proceeded. No such granules (lipid droplets) were detected in normal monkey
kidney cells and none could be seen during the infection of these cells with MPV (Fig. i). The
presence of cytosine arabinofuranoside (100 #g/ml) during infection with Yaba virus did not
stop the production of the granules (Fig. 1 c). Although poxviruses contain up to 2.7 % lipids,
and in the case of fowlpox up to 34% in the envelope membrane (Moss, 1978), no alteration
in host cell lipid metabolism has been shown in vaccinia virus infection (Stern & Dales, 1974).
The stimulation of the production of these lipids in Yaba virus-infected cells may be another
difference between Yaba virus and other poxviruses.
Protein synthesis in infected cells
The inhibition of host protein synthesis during vaccinia virus infection is fairly quick and
efficient (Pennington, 1974; Harper et al., 1979). A few other orthopoxviruses such as
monkeypox and fowlpox inhibit host protein synthesis to a lesser degree than does vaccinia
(Kilpatrick & Rouhandeh, 1981; Harper et al., 1979). Virus production during Yaba virus
infection was much slower and almost no inhibition of host protein synthesis was seen. Fig. 2
shows uninfected cells, Yaba virus-infected cells and MPV-infected cells analysed by
O'Farrell's (1975) two-dimensional gel electrophoresis method. Infected cells were labelled
for 3 days by adding [35S]methionine to the medium. The degree of alteration of host protein
synthesis can easily be seen by using this method of analysis. A random counting of host
proteins shows around 400 proteins present in the control cells, 380 in the Yaba virus-infected
cells and 50 in the MPV-infected cells. The rectangle in Fig. 2 (a, b) shows the selective
inhibition of a group of host proteins with an isoelectric point of 5-0 and a mol. wt. of about
40 × 103. The synthesis of at least three host proteins is also shown to be stimulated in Yaba
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Yaba tumour p o x virus
6.5
6.3
-,~lt
pH
6.0
5.6
5.0
4.5
•
L
13-(a) ,~
130-93--
~
~2
40-29 - -
130--
i" ~ % "r
"~
* ;;.
i
93--
13--
Fig. 2. Autoradiograms of a two-dimensional analysis of (a) uninfected cells, (b) Yaba virus-infected
cells and (c) MPV-infected cells. The gels contained approx. 300000 ct/min each and were exposed for
30 days. The largest inhibition of host proteins in Yaba virus-infected cells is shown by the rectangle
around those proteins in (a) and (b); however, most of the host proteins are still present. The majority of
host proteins were inhibited when infected with MPV (c). The cells were infected with an rn.o.i, of 10 in
the ease of MPV, or with 10 focus-forming units (f.f.u.) of Yaba virus. Open arrows represent the same
host proteins in (a) or (b). Solid arrows represent virus-specific proteins, Numbers on the left of the gels
indicate mol. wt. x 10-3.
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212
H. R O U H A N D E H ,
IF ~
D. K I L P A T R I C K
6.6
pH
6.0
6.3
A N D A. V A F A I
5.7
5.0
4.5
13o-y
77--~
~i~I, '
•
40-29--
O~
O
................ ~ .... ......
(a)
130--
61--
40-29--
136.6
6.3
6.0
5.7
5.0
4.5
Fig. 3. Autoradiograms of a two-dimensional analysis of either (a) purified Yaba virus proteins or (b)
purified MPV proteins. Both viruses are shown to have at least two proteins possessing the same mol.
wt. and isoelectric point (solid arrows). In both cases 100000 ct/min of purified virus was analysed.
Electrophoresis in the isoelectric focusing (IF) dimension was for a total of 7000 V.h. Electrophoresis
in the SDS dimension was for 5 h at 2.5 W/gel. The dried gels were exposed to X ray film for 4 weeks.
Numbers on the left of the gels indicate mol. wt. x 10 3.
virus-infected cells (open arrows, Fig. 2b) w h e n c o m p a r e d to uninfected cells (open arrows,
Fig. 2a). Cells infected with M P V , however, show that synthesis o f m o s t of the m a j o r host
proteins is inhibited d u r i n g infection.
Several o f the m a j o r proteins of either virus are identified in Fig. 3. A previous analysis o f
both of these viruses b y S D S - P A G E showed that three proteins (mol. wt. 122 × 103, 97 ×
103 and 25 x 103) were c o m m o n (Arita & T a g a y a , 1980). The analysis of these viruses by
O ' F a r r e l l ' s (1975) t w o - d i m e n s i o n a l technique showed few proteins having the s a m e mol. wt.
a n d isoelectric points between the two viruses, with the possible exception of two proteins
with mol. wt. of 77 x 103 a n d 63 x 103 and c o r r e s p o n d i n g isoelectric points of 5.7 a n d 5.5
(Fig. 3). Over 140 polypeptides could be seen with Y a b a virus and 115 polypeptides with
MPV.
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Yaba tumour p o x virus
(a)
1
(b)
2
1
2
14.9--
5.94--
4.18--
2.67--
1.41-1.23 ~
Fig. 4. Patterns obtained after the electrophoretic separation of the cleavage fragments produced by the
digestion of MPV (lanes 1) and Yaba virus (lanes 2) DNA with (a) HindllI and (b) XhoI restriction
endonucleases. Electrophoresis was on either 0.6% or 0.7% agarose gels for 15 h at 30 V. Numbers
indicate tool. wt, ofHindlII-cleaved lambda phage DNA fragments used as size markers.
Analysis of Yaba virus DNA
The differences seen between Yaba virus and MPV lie ultimately within their genomic
organizations. The D N A genome of both viruses was analysed using the restriction
endonucleases HindIII and XhoI. There were major differences in the size of their restriction
fragments. Yaba virus lacked fragments of > 10 megadaltons when cleaved with HindIII (Fig.
4). The restriction fragments shown of MPV are the same as those described by Mackett &
Archard (1979). The mol. wt. of Yaba virus and MPV D N A cleaved with HindIII were 94.3
× 106 and 127 x 106 respectively (Table 1). The analysis of these two genomes with XhoI is
even more interesting. Yaba virus D N A was infrequently cut with this enzyme. Two large
fragments and four smaller fragments were seen (Fig. 4). Orthopoxvirus D N A is cleaved
more often with this enzyme and has been used in mapping studies of several poxvirus D N A s
(Mackett & Archard, 1979). The tool. wt. calculated by using XhoI were 91.8 x 106 and 128
x 106 for Yaba virus and MPV D N A respectively (Table 1). Yaba virus HindIII restriction
fragments A, H, I and K were shown to have similar migration rates to MPV HindIII
fragments E, K, L and M respectively (Table 1). Similar migration rates, however, do not
necessarily mean that they possess similar D N A sequences.
In order to see if MPV and Yaba virus have any sequences in common, 32p-labelled Yaba
virus D N A was hybridized to MPV D N A cleaved with HindIII and XhoI. The Yaba virus
D N A was nick-translated according to the procedure of Rigby et al. (1977) and the transfer
of D N A to nitrocellulose filters was by Southern's (1975) technique. Yaba probe D N A
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H. ROUHANDEH,
D. KILPATRICK
AND
A. VAFAI
Table 1. Restriction fragments of monkeypox virus and Yaba tumour pox virus cleaved with
HindIII and XhoI*
HindIll
XhoI
f
~
Fragment
A
B
C
D
E
F
G
H
I
J
Kt
L+
M'I"
N
O
P
Q
Total
MPV
30.00
17.50
11.70
10.30
9.50
8-90
5.80
5.60
4.30
3.85 × 2
3.40
3-20
2.60
1.80 x 2
1.43
1.05
0.90
127.48
Yaba
9.50
6.80
6.40
5.50
4-85
4.40
4.00
3-40
3.20
2.90
2.60
2.50
2.00
× 3
x 2
×4
× 2
f
Fragment
A~"
B~"
C'~
D
Et
F
G~
H
I
J
K
L
94.30
MPV
18.00 x 2
13.50 × 2
10.00
8.70
8.10
7.20
6.80
4.85
4.70
3.50
2.60 x 3
1-80
128.70
Yaba
30.0
25.0
18.5
11.0
3.7
3.6
91.80
* Mol. wt. x 10-6 obtained by comparison to 2/HindlIl distances in agarose gels,
~"Monkeypox virus D N A fragments which hybridized to 32P-labelled Yaba virus D N A probe.
hybridized to the restriction fragments K, L and M of MPV cleaved with HindIII, and to
fragments A, B, C, E and G when cleaved with XhoI (Fig. 5). The reciprocal of this
experiment, i.e. using MPV DNA as a probe and hybridizing this with HindIII-digested Yaba
virus DNA, showed three bands corresponding to restriction fragments H, I and K. Since
these three bands showed similar migration rates with K, L and M in the HindIII digest of
MPV DNA, as well as hybridization with a DNA probe of either virus, there is a very high
degree of sequence homology between these fragments in either virus. These three fragments
had a combined mol. wt. of 9.2 x 106. Thus, assuming complete homology between these
fragments, there could be a maximum of 7.2 % homology between the genomes of Yaba virus
and MPV.
DISCUSSION
These studies used MPV as a representative of the Orthopoxvirus group as a model of
comparison in order to understand more about the oncogenic virus, Yaba virus. The analysis
of both of these viruses in the same cell line revealed their distinct differences. The
accumulation of lipid droplets in the cytoplasm of Yaba virus-infected cells is of particular
importance. Globules of neutral fat have been shown to be common within tumours induced
by Yaba virus (Metzgar et al., 1962; Sproul et al., 1963). The presence of such lipids in tissue
culture cells infected with other poxviruses has not been shown, with the possible exception of
fowlpox. But even in the case of fowlpox the lipid build-up was not as extensive and was
confined to the intracytoplasmic inclusion bodies where it was incorporated into the virion
(Lyles et al., 1975). The continued development of these lipid droplets in the Yaba
virus-infected cells in the presence of cytosine arabinofuranoside suggests that if the virus is
responsible for the stimulation and/or control of these lipids, it is an 'early' function of the
virus genome.
This continued build-up of lipid droplets as Yaba virus infection proceeds may in part be
related to the lack of inhibition of host macromolecular synthesis which results in a prolonged
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Y a b a t u m o u r p o x virus
(a)
O)
1
2
215
(c)
C~
G~
i!i~i!i~!!ii!ii!~iii!::iill~::!!•/:'i •:!i~: i:•••::
Fig. 5. (a) Etectrophoretic patterns of monkeypox virus D N A cleaved with Xhol (lane 1) and/qindlII
(lane 2). This D N A was transferred to nitrocellulose filters and hybridized to a nick-translated
~2P-labelled Yaba virus D N A probe. The dried filters were exposed to X-ray film using intensifying
screens for 4 days. (b) Autoradiogram of the filter showing Yaba virus D N A hybridized to MPV D N A
cleaved with XhoI (lane 1) or HindllI (lane 2). The ethidium bromide-stained D N A fragments
corresponding to the autoradiogram to which Yaba virus D N A hybridized are indicated by their
appropriate letter sequences. (c) Yaba virus D N A cleaved with HindlII (lane 1) and then transferred to
a taitrocellulose filter and hybridized using 32P-labelled MPV D N A as the probe (lane 2).
infection cycle. Our studies show that unlike MPV and other related poxviruses, where the
onset of inhibition is well advanced by 12 to 16 h after infection (Kilpatrick & Rouhandeh,
1981; Harper et al., 1979), the extent of inhibition seen during Yaba virus infection is low in
comparison. The lack of inhibition, even when using high m.o.i.s, is one of the key differences
between this oncogenic virus and other poxviruses. The synthesis of several host proteins
which appeared to be stimulated during Yaba virus infection along with the accumulation of
lipids and the ability to propagate infected cells for over 2 weeks, reveals the uniqueness Yaba
virus has with regard to the control of the host metabolism.
The analysis of the structural proteins of these two viruses demonstrates that the
differences in the polypeptide profile are very substantial when the two-dimensional technique
is used. While previous work has shown at least three proteins in common between Yaba
virus and MPV (Arita & Tagaya, 1980), we were not able to detect two of these proteins
using O'FarreU's (1975) technique. It is often difficult to resolve higher tool. wt. proteins with
this technique and the proteins were possibly outside the pH range analysed. The lower mol.
wt. protein (25 × 103) was detected but had an isoelectric point of 4-5 in MPV and about 5.0
in Yaba virus. We were, however, able to show over 100 proteins for each virus and very few
of these possessed the same mol. wt. and isoelectric point. Further analysis is needed to
determine whether two proteins we detected (mol. wt. 77 × 10 3 and 63 × 103) are indeed
common to both virions (see Fig. 3).
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H. R O U H A N D E H ~ D. K I L P A T R I C K AND A. V A F A I
1
2
I
~.
3
M
t
L
/
4
5
I
I
JN F O P
I I
!
I
C
t
E QI a O', L ,H
~il
k
K
I
t
I
D
I
A
B
I
I
I
I
IKtNJ
I|1
I
Fig. 6. Electrophoretic patterns of MPV DNA restriction fragments produced by cleavage with the
following restrictinn endonucleases. Lane 1, XhoI; lane 2, XhoI/SmaI double-digest;lane 3, Sinai; lane
4, HindIlI/Smal double-digest; lane 5, HindIII. This restriction analysis is identical to the one
performed by Mackett & Archard (1979) on their Denmark strain of MPV. Therefore, their physical
map locations of HindIII restriction fragments were used to determine the locations on the MPV
genome which hybridize with Yaba virus DNA. These are shown as K, L and M.
Yohn & Gallagher (1969) showed that Yaba virus D N A was different to that of cowpox,
rabbitpox and fowlpox. They showed that the T m value for Yaba D N A was around 82.3 ° C
and that its b u o y a n t density was 1.695 g/ml in CsC1. The percentage G + C ratio of Yaba
virus D N A was also found to be lower (32.5) than the other poxvirus D N A s (35 to 36-5).
The use of restriction enzymes has furthered the analysis of poxvirus D N A by determining
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Yaba tumour pox virus
217
the relatedness of different poxviruses to individual groups and among themselves (Muller et
al., 1977). The differences between Yaba virus D N A and MPV D N A are demonstrated by
their specific cleavage patterns. It is interesting that the cleavage of many orthopoxvirus
genomes with XhoI generates many fragments and that the Yaba virus genome is infrequently
cut with this enzyme (Fig. 4). This characteristic may prove useful in the future analysis of
the Yaba virus genome.
The fact that Yaba virus D N A hybridizes to MPV D N A was expected since they share
common structural proteins and enzymes. However, the regions on the restriction map of
MPV to which Yaba virus D N A hybridizes are of interest. The hybridization of Yaba virus
D N A to the central region of the MPV genome (adjacent fragments L and M, Fig. 6) shows a
degree of sequence conservation similar to that shown by Mackett & Archard (1979). These
authors reported a high degree of sequence conservation in the central region among a wide
range of orthopoxviruses. The conserved sequences in the central region are assumed to
encode functions common between the viruses.
Yaba virus D N A did not hybridize to the terminal fragments of MPV HindlII J and N, or
XhoI K. Since these repetitive end termini have been found among most other poxviruses,
this suggests that part, if not all, of the repetitive sequences at the termini of Yaba virus may
be lacking, or that its termini contain unique sequences. The absence of common termini may
also be important in explaining some of the differences seen between these two viruses. It has
been proposed that the individual characteristics of a virus are generated by the near terminal
sequences which show extensive type-specific variation (Mackett & Archard, 1979). Further
analysis of the terminal fragments of Yaba virus should prove of interest.
We thank Professor R. Bablanian for his critical review of this manuscript. This work was supported
in part by the Research and Development Administration at Southern Illinois University, Carbondale,
Illinois, U.S.A.
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