The gene encoding the African swine fever virus protein of Mr 11500, present in the virus particle, has been mapped and sequenced in the genome of the Vero cell-adapted virus strain BA71V. A serum raised against virion proteins of Mr 12000 to 13000 isolated from polyacrylamide gels was used to screen a plasmid expression library, containing viral DNA random fragments, that expresses viral polypeptides fused to β-galactosidase. Using this method, we have identified and sequenced the open reading frame (ORF) A137R, which initiates at the right end of the EcoRI A restriction fragment and extends into the EcoRI F fragment. Expression of the protein in Escherichia coli has confirmed that ORF A137R encodes a protein with an Mr of about 12000. A specific serum was raised against the E. coli-expressed protein, and has been used to identify the protein encoded by the ORF, which is translated at late times of infection and incorporated into the virus particle. Immunofluorescence experiments have shown that the protein localizes in virus factories.
AlcamíA.,
AnguloA.,
López-OtínC.,
MuñozM.,
FreijeJ. M. P.,
CarrascosaA. L.,
ViñuelaE.1992; Amino acid sequence and structural properties of protein pi2, an African swine fever virus attachment protein. Journal of Virology 66:3860–3868
AlmazánF.,
RodríguezJ. M.,
AndrésG.,
PérezR.,
ViñuelaE.,
RodríguezJ. F.1992; Transcriptional analysis of multigene family 110 of African swine fever virus. Journal of Virology 66:6655–6667
AlmazánF.,
RodríguezJ. M.,
AnguloA.,
ViñuelaE.,
RodríguezJ. F.1993; Transcriptional mapping of a late gene coding for the p12 attachment protein of African swine fever virus. Journal of Virology 67:553–556
AnguloA.,
ViñuelaE.,
AlcamíA.1993; Inhibition of African swine fever virus binding and infectivity by purified, recombinant virus attachment protein pl2. Journal of Virology 67:5463–5476
BonnerW. M.,
LaskeyR. A.1974; A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46:83–88
CamachoA.,
ViñuelaE.1991; Protein p22 of African swine fever virus: an early structural protein that is incorporated into the membrane of infected cells. Virology 181:251–257
CarrascosaJ. L.,
CarazoJ. M.,
CarrascosaA. L.,
GarcíaN.,
SantistebanA.,
ViñuelaE.1984; General morphology and capsid fine structure of African swine fever virus particles. Virology 132:160–172
De BoerC. J.1967; Studies to determine neutralizing antibodies in sera from animals recovered from African swine fever and laboratory animals inoculated with African virus with adjuvants. Archiv fur die gesamte Virusforschung 20:164–179
DixonL. K.,
WilkinsonP. J.,
SumptionK. J.,
EkueF.1990; Diversity of the African swine fever virus genome. In Molecular Biology of Iridoviruses pp 271–295 Edited by
DaraiG.
Boston: Kluwer Academic Publishers;
EstevesA.,
MarquesM. I.,
CostaJ. V.1986; Two-dimensional analysis of African swine fever proteins and proteins induced in infected cells. Virology 152:181–191
García-BarrenoB.,
SanzA.,
NogalM. L.,
ViñuelaE.,
EnjuanesL.1986; Monoclonal antibodies of African swine fever virus: antigenic differences among field virus isolates and viruses passaged in cell culture. Journal of Virology 58:385–392
López-OtínC.,
SimónC.,
MéndezE.,
ViñuelaE.1988; Mapping and sequence of the gene encoding protein p37, a major structural protein of African swine fever virus. Virus Genes 1:291–303
López-OtínC.,
Simón-MateoC.,
MartínezL.,
ViñuelaE.1989; Gly-Gly-X, a novel consensus sequence for the proteolytic processing of viral and cellular proteins. Journal of Biological Chemistry 264:9107–9110
López-OtínC.,
FreijeJ. M. P.,
ParraF.,
MéndezE.,
ViñuelaE.1990; Mapping and sequence of the gene coding for protein p72, the major capsid protein of African swine fever virus. Virology 175:477–484
RodríguezJ. M.,
AlmazánF.,
ViñuelaE.,
RodríguezJ. F.1992; Genetic manipulation of African swine fever virus: construction of recombinant viruses expressing the β-galactosidase gene. Virology 188:67–76
SalasM. L.,
KuznarJ.,
ViñuelaE.1981; Polyadenylation, methylation, and capping of the RNA synthesized in vitro by African swine fever virus. Virology 113:484–491
SambrookJ.,
FritschE. F.,
ManiatisT.1989Molecular Cloning: A Laboratory Manual 2nd edn New York: Cold Spring Harbor Laboratory;
SangerF.,
NicklenS.,
CoulsonA. R.1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U,. S,. A 74:5463–5467
Simón-MateoC.,
AndrésG.,
ViñuelaE.1993; Polyprotein processing in African swine fever virus: a novel strategy of gene expression for a DNA virus. EMBO Journal 12:2977–2987
StudierF. W.,
RosenbergA. H.,
DunnJ. J.,
DubendorffJ. W.1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185:60–89
VieiraJ.,
MessingJ.1982; The pUC plasmids, an M13mp7-derived system for insertion, mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268