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Journal of Virology, April 2000, p. 3525-3536, Vol. 74, No. 8
European Molecular Biology Laboratory, Cell
Biology Programme, 69117 Heidelberg, Germany1;
EM Unit for Biological Sciences, University of Oslo, Blindern,
N-0316 Oslo, Norway2; and Department of
Virology, Institute of Medical Microbiology, Leiden University,
2300 RC Leiden, The Netherlands3
Received 29 November 1999/Accepted 13 January 2000
The entry of vaccinia virus (VV) into the host cell results in the
delivery of the double-stranded DNA genome-containing core into the
cytoplasm. The core is disassembled, releasing the viral DNA in order
to initiate VV cytoplasmic transcription and DNA replication. Core
disassembly can be prevented using the VV early transcription
inhibitor actinomycin D (actD), since early VV protein synthesis is
required for core uncoating. In this study, VV intracellular cores were
accumulated in the presence of actD and isolated from infected cells.
The content of these cores was analyzed by negative staining EM and by
Western blotting using a collection of antibodies to VV core and
membrane proteins. By Western blot analyses, intracellular actD cores,
as well as cores prepared by NP-40-dithiothreitol treatment of
purified virions (NP-40/DTT cores), contained the core proteins p25
(encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small
amounts of the VV membrane proteins p32 (D8L) and p35
(H3L). While NP-40/DTT cores contained the major putative DNA-binding
protein p11 (F17R), actD cores entirely lacked this protein. Labeled
cryosections of cells infected for different periods of time in the
presence or absence of actD were subsequently used to follow the fate
of VV core proteins by EM. These EM images confirmed that p11 was lost
at the plasma membrane upon core penetration. The cores that
accumulated in the presence of actD were labeled with antibodies to 4a,
p39, p25, and DNA at all times examined. In the absence of the drug the
cores gradually lost their electron-dense inner part, concomitant
with the loss of p25 and DNA labeling. The remaining core shell still
labeled with antibodies to p39 and 4a/4b, implying that these proteins
are part of this structure. These combined data are discussed with
respect to the structure of VV as well as core disassembly.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of Vaccinia Virus Intracellular Cores:
Implications for Viral Uncoating and Core Structure
*
Corresponding author. Mailing address: European
Molecular Biology Laboratory, Cell Biology Programme, Meyerhofstrasse
1, 69117 Heidelberg, Germany. Phone: 49 6221 387244. Fax: 49 6221 387306. E-mail: Krijnse{at}EMBL-Heidelberg.DE.
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