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The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARYPolypeptides synthesized in Vero cells infected with African swine fever virus (ASFV) can be divided into early and late classes on the basis of their sensitivity to cytosine arabinoside. As ASFV does not inhibit cell protein synthesis until late in infection, immunoprecipitation was used to identify virus-specific polypeptides. Eighteen early and 15 late polypeptides were detected by polyacrylamide gel electrophoresis. Early polypeptides can be further divided into those which are transiently expressed at early times and the majority which are synthesized throughout infection. In vitro translation products of RNAs from infected cells at different stages of infection were compared with in vivo products after immunoprecipitation. A good correspondence was observed between the in vivo and in vitro patterns. All early RNAs translated in vitro were synthesized in the presence of cytosine arabinoside and cycloheximide and can therefore be classified as immediate early RNAs. Only two polypeptides transcribed from early RNA were not present among late RNA products. No evidence was obtained for a discrete class of delayed early RNAs.

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