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Vol. 12, Issue 12, 3875-3891, December 2001
*EMBL, Cell Biology and Biophysics Programme, 69117 Heidelberg,
Germany
Vaccinia virus (vv) early transcription can be reconstituted in
vitro from purified virions; in this assay mRNAs are made inside the
viral core and subsequently extruded. Although the in vitro process has
been extensively characterized, relatively little is known about vv
early transcription in vivo. In the present study the fate of vv early
mRNAs in infected HeLa cells was followed by BrUTP transfection and
confocal and electron microscopy. The extruded vv early mRNAs were
found to be organized into unique granular cytoplasmic structures that
reached a size up to 1 µm. By EM these structures appeared as
amorphous electron-dense cytoplasmic aggregates that were surrounded by
ribosomes. Confocal images showed that the RNA structures were located
some distance away from intracellular cores and that both structures
appeared to be aligned on microtubules (MTs), implying that MT tracks
connected mRNAs and cores. Accordingly, intact MTs were found to be
required for the typical punctate organization of viral mRNAs.
Biochemical evidence supported the notion that vv mRNAs were MT
associated and that MT depletion severely affected viral (but not
cellular) mRNA synthesis and stability. By confocal microscopy the
viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting
away from intracellular cores to their sites of cytoplasmic accumulation.
Corresponding author. E-mail
address: krijnse{at}embl-heidelberg.de.
Present address: Max Planck Institute for
Developmental Biology, Spemannstrasse 35, 72076 Tuebingen, Germany.
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