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Journal of Virology, August 2008, p. 7551-7566, Vol. 82, No. 15
0022-538X/08/$08.00+0     doi:10.1128/JVI.00016-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Atomic Force Microscopy Investigation of Vaccinia Virus Structure{triangledown}

Y. Kuznetsov, P. D. Gershon,{dagger} and A. McPherson{dagger}*

Department of Molecular Biology and Biochemistry, University of California—Irvine, Irvine, California 92697

Received 3 January 2008/ Accepted 21 May 2008

Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be "soldered" by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome.

* Corresponding author. Mailing address: University of California, Irvine, Department of Molecular Biology and Biochemistry, 560 Steinhaus Hall, Irvine, CA 92697-3900. Phone: (949) 824-1931. Fax: (949) 824-1954. E-mail: amcphers{at}uci.edu

{triangledown} Published ahead of print on 28 May 2008.

{dagger} These two authors contributed equally to this work.

Journal of Virology, August 2008, p. 7551-7566, Vol. 82, No. 15
0022-538X/08/$08.00+0     doi:10.1128/JVI.00016-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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