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Journal of Virology, April 2000, p. 3525-3536, Vol. 74, No. 8
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

Ketil Pedersen,1,2 Eric J. Snijder,3 Sibylle Schleich,1 Norbert Roos,2 Gareth Griffiths,1 and Jacomine Krijnse Locker1,*

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.

* 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.

Journal of Virology, April 2000, p. 3525-3536, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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Copyright © 2000 by the American Society for Microbiology. All rights reserved.