Rev. sci. tech. Off. int. Epiz.,
1986, 5 (2), 469-475.
The structure and function of the African
swine fever virus genome
L. K. DIXON*
Summary: African swine fever (ASF) virus resembles the Iridoviridae in its mor­
phology, assembly of virions in the cytoplasm and complexity of its doublestranded DNA genome. However, recent analysis of the structure of the genome
and enzyme composition of virus particles has revealed that, in several important
aspects, ASF virus differs from Iridoviridae but resembles Poxviridae. Thus, the
genomes of ASF and Poxviridae have terminal cross-links and inverted terminal
repeats, whereas the genomes of at least three Iridoviridae are circularly permuted
and terminally redundant. In addition, ASF virus particles, like Poxviruses, con­
tain several enzymes related to DNA transcription and mRNA modification which
are lacking from Iridoviruses.
KEYWORDS : African swine fever
Iridoviridae - Morphology - Poxviridae.
virus - Antigen
structure - Enzymes -
T H E STRUCTURE A N D COMPOSITION OF A F R I C A N
S W I N E F E V E R VIRUS P A R T I C L E S
Structure
African swine fever (ASF) virions are icosahedral in morphology and are assembled
in the cytoplasm of infected cells. The virus particles consist of a nucleoprotein core
about 80 nm in diameter surrounded by a lipid membrane covered by a capsid. Extra­
cellular virions have an external envelope, derived by budding through the cell mem­
brane, and are about 200 nm in diameter (5, 6, 17, 24, 35). The external envelope does
not seem to be essential for infectivity (24). The capsid has a periodic structure and
consists of a hexagonal arrangement of capsomers which appear as hexagonal prisms
13 nm long ( 1 , 6 , 25).
Protein composition
Estimates of the number and molecular weights of ASF virion proteins vary consi­
derably. Earlier reports estimated that there were 5 or 14 proteins (4, 43), whereas more
recently 24 intracellular virion proteins (35, 39, 41) and 34 extracellular virion proteins
(7) have been identified. Of the 34 extracellular virus proteins, 12 are probably of host
cell origin since they incorporate S-methionine when cells are pulse labelled prior to
infection. These may be incorporated into the virion as it buds through the cell mem­
brane (7). Radioactive fucose and glucosamine are incorporated into 3 and 3 Pi into 5
intracellular virion proteins. In contrast, extracellular virus contains no glycoproteins
and no proteins above 150,000 daltons molecular weight (7). The discrepancy between
these reports may reflect differences between intracellular and extracellular virus or bet­
ween the purity of the preparations (44). These studies have been carried out using virus
35
2
* Animal Virus Research Institute, Pirbright, Surrey GU24 0NF, United Kingdom.
— 470 —
adapted to grow in various established cell lines. As yet, purification of virus and
analysis of structural proteins from virus grown in the porcine target cells for field
virus isolates of A S F (monocytes and macrophages) has not been carried out.
The distribution of viral proteins within the virion has been studied by treat­
ment with Nonidet P-40 and 2-mercaptoethanol (39, 4). In the absence of 0.5 M
N a C l this treatment gives rise t o non-infectious subviral particles (core I) of increa­
sed density in CsCl (1.31 g / c m compared with 1.23 g / c m ) and lacking some viral
proteins. In the presence of 0.5 M NaCl, some additional viral proteins are lost
(including the major structural protein with molecular weight 73,000), resulting in
core II structures containing at least 14 proteins (39).
3
3
Enzyme activities
A S F virus particles contain a number of enzyme activities including a DNAdependent R N A polymerase which can transcribe A S F viral D N A in vitro (19) and
resembles vaccinia virus R N A polymerase kinetically (18) and in its pattern of
enzyme inhibition (37, 34). In addition, A S F virions contain enzymes involved in
the 5' and 3 ' end modifications of m R N A s (33), a coumermycin A l sensitive topoisomerase (34), a protein kinase (32) and one or two nucleoside triphosphate phosphohydrolases (20). Similar enzymatic activities are present in vaccinia virus (23, 8).
In contrast, the virions of frog virus 3 (the best studied of the Iridoviridae) do not
contain an R N A polymerase or other enzymes involved in m R N A modification (for
review see 48).
Genome
The genome of A S F virus was first established as consisting of D N A in the
1960's (16, 30). Later studies estimated that the molar mass of A S F D N A is about
100 X 1 - gmol - with a G C content of about 41 % (12). Sedimentation analysis
of A S F D N A in neutral and alkaline sucrose gradients and analysis of the renaturation rate of intact D N A suggested the presence of one or two inter-strand cross­
links in the A S F virus D N A (28). In addition, when A S F D N A cleaved by restric­
tion endonucleases is denatured and rapidly re-annealed, only the terminal frag­
ments become SI resistant duplexes identifiable by gel electrophoresis (2). This evi­
dence indicates that A S F D N A contains terminal cross-links similar to those found
in poxvirus D N A (14, 3).
6
Electron microscopic analysis of heteroduplexes, formed by re-annealing dena­
tured terminal fragments after removal of the terminal cross-links, identified termi­
nal inverted repetitions 2.7 kb long in D N A from a Spanish isolate of the virus
adapted to grow in Vero cells. Shorter internal inverted repetitions were also identi­
fied (36). Vaccinia virus contains terminal inverted repetitions 10 kb long (13, 49).
However, the presence of long terminal inverted repeats in pox and A S F viruses
may not be essential since variola virus (11) and two African isolates of A S F virus
(D'Souza and Black, in preparation) apparently lack them. In contrast, the D N A of
iridoviruses such as frog virus 3 (15), fish lymphocystis disease (9) and Chilo irides­
cent virus (10) is circularly permuted and terminally redundant. These differences in
the structure of the genome reflect the mechanism of D N A replication. It is, there­
fore, likely that A S F virus D N A replicates by a mechanism similar to that proposed
for vaccinia virus D N A (26, 3) rather than as proposed for iridoviruses such as frog
virus 3 (for review see 48).
— 471 —
Molecular cloning of D N A from a Spanish isolate of A S F virus adapted to grow
in Vero cells (21), a Malta and a Malawi isolate (Dixon, unpublished results) has
been carried out, allowing preparation of restriction endonuclease site maps of the
virus (2, 3, Dixon, unpublished results). Genetic variation of A S F isolates has been
studied by restriction enzyme analysis, using virus grown in porcine macrophages
or isolated from pig red blood cell fractions. Analysis of DNA from early European
isolates of A S F virus revealed that the central 125 kb of the genome was conserved
between isolates but considerable variation existed at the termini. The variation
could be accounted for by deletions of varying size of D N A from several locations
near the termini (44). Comparison of restriction enzyme digestion patterns of seve­
ral African, European and Caribbean isolates of A S F virus showed that the Euro­
pean isolates were similar to those from the Cameroon and Caribbean, indicating a
possible c o m m o n origin for infection (47). Other African isolates in general show
considerable variation ; however, similarities are evident between some isolates (47,
D'Souza and Black, in preparation ; Dixon, Wilkinson, Hutchings and Kurnock,
unpublished results). The adaptation process of A S F virus to established cell lines
leads to large deletions in the D N A (42, 45, 46). The coding capacity of ASF virus is
obviously large ; as yet, however, no detailed mapping or studies on gene structure
have been reported.
VIRAL GENE EXPRESSION
Viral D N A synthesis requires a phosphonoacetic acid sensitive, virus induced
DNA polymerase ( 3 1 , 22) as well as a functional nucleus in the infected cell (27).
Pulse chase experiments using infected porcine monocytes indicated that D N A
replication occurs in the cytoplasm (29), in contrast to an earlier report which sug­
gested that viral D N A is synthesised in the nucleus and transported to the cyto­
plasm (38).
Isolated A S F virions can transcribe virus D N A . T h e R N A product synthesised
in vitro hybridises to the same D N A regions as the RNAs synthesised in infected
cells in the presence of either cycloheximide or cytosine arabinoside. This suggests
that, before D N A replication, A S F D N A is transcribed by R N A polymerase present
in the virion (see 44). After D N A replication, new R N A species arise which hybri­
dise with D N A regions not transcribed in infected cells in the presence of protein or
DNA synthesis inhibitors. Both early and late RNAs are transcribed independently
of host cell R N A polymerase II, since cells infected in the presence of 5,6-dichloroß-D-ribofuranosylbenzimidazole synthesise RNAs and proteins which are indistin­
guishable from those made in the absence of the drug (see 4).
Virus-induced protein synthesis reflects R N A transcription, appearing as two
populations early and late after infection. By one-dimensional Polyacrylamide gel
electrophoresis, about 50 virus-induced polypeptides have been detected (40, 41).
The distribution of these proteins in the cytoplasm and nucleus has been studied;
some are restricted to the nuclear fraction and some to the cytoplasmic fraction.
*
* *
— 472 —
STRUCTURE ET FONCTION DU GÉNOME DU VIRUS DE LA PESTE PORCINE
AFRICAINE. — L.K. Dixon.
Résumé : Le virus de la peste porcine africaine (PPA) ressemble aux Iridoviridae par sa morphologie, la disposition des virions dans le cytoplasme et la
complexité de son génome à ADN bicaténaire. Cependant, une analyse récente
de la structure du génome et de la composition enzymatique des particules
virales a révélé que le virus de la PPA diffère par plusieurs aspects importants
des Iridoviridae mais ressemble aux Poxviridae. Ainsi, les génomes du virus
PPA et des Poxviridae ont des croisements terminaux et des répétitions terminales inversées, tandis que les génomes d'au moins trois Iridoviridae sont permutés de manière circulaire, et plus nombreux en position terminale. De plus,
les particules virales de PPA, comme les Poxvirus, contiennent plusieurs enzymes capables de la transcription de l'ADN et de la modification de l'ARNm
alors que ces enzymes n'existent pas chez les Iridovirus.
MOTS-CLÉS : Enzymes - Iridoviridae - Morphologie - Poxviridae - Structure
antigénique - Virus de la peste porcine africaine.
*
ESTRUCTURA Y FUNCIÓN DEL GENOMA DEL VIRUS DE LA PESTE PORCINA
AFRICANA. — L.K. Dixon.
Resumen : El virus de la peste porcina africana (PPA) se parece a los Iridoviridae por su morfología, disposición de los viriones en el citoplasma y complejidad de su genoma con ADN bicatenario. No obstante, un reciente análisis de
la estructura del genoma y de la composición enzimática de las partículas víricas reveló que el virus de la PPA difiere de los Iridoviridae por varios e importantes aspectos, aunque se parece a los Poxviridae. Así, los genomas del virus
PPA y de los Poxviridae tienen cruces terminales y repeticiones terminales
invertidas, mientras que los genomas de por lo menos tres Iridoviridae están
permutados de manera circular, siendo más numerosos en posición terminal.
Además, las partículas víricas de PPA, como los Poxvirus, contienen varias
enzimas con capacidad para la transcripción del ADN y la modificación del
ARNm, mientras que no existen estas enzimas en los Iridovirus.
PALABRAS CLAVE : Enzimas - Estructura antigénica - Iridoviridae - Morfología - Poxviridae - Virus de la peste porcina africana.
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