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Journal of Virology, May 2003, p. 6014-6028, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.6014-6028.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Vaccinia Virus I3L Gene Product Is Localized to a Complex Endoplasmic Reticulum-Associated Structure That Contains the Viral Parental DNA

Sonja Welsch, Laura Doglio,{dagger} Sibylle Schleich, and Jacomine Krijnse Locker*

European Molecular Biology Laboratory, 69117 Heidelberg, Germany

Received 18 December 2002/ Accepted 20 February 2003

The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogenous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.

* Corresponding author. Mailing address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Phone: 49 6221 387508. Fax: 49 6221 387306. E-mail: Krijnse{at}EMBL-Heidelberg.DE.

{dagger} Present address: Fondazione Cavalieri Ottolenghi, Universita degli Studi di Torino, A. O. San Luigi Gonzaga, Regione Gonzole 10-10043, Orabssano, Italy.

Journal of Virology, May 2003, p. 6014-6028, Vol. 77, No. 10
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.10.6014-6028.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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