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Journal of Virology, June 2007, p. 6419-6433, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.00047-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Putative Terminase Subunits of Herpes Simplex Virus 1 Form a Complex in the Cytoplasm and Interact with Portal Protein in the Nucleus{triangledown}

Kui Yang,1 Fred Homa,2 and Joel D. Baines1*

Department of Microbiology and Immunology, Cornell University, New York School of Veterinary Medicine, Ithaca, New York,1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania2

Received 8 January 2007/ Accepted 19 March 2007

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocates DNA through the portal vertex in the capsid during DNA packaging. The HSV terminase is believed to consist of the UL15, UL28, and UL33 gene products (pUL15, pUL28, and pUL33, respectively), whereas the HSV type 1 portal vertex is encoded by UL6. Immunoprecipitation reactions revealed that pUL15, pUL28, and pUL33 interact in cytoplasmic and nuclear lysates. Deletion of a canonical nuclear localization signal (NLS) from pUL15 generated a dominant-negative protein that, when expressed in an engineered cell line, decreased the replication of wild-type virus up to 80-fold. When engineered into the genome of recombinant HSV, this mutation did not interfere with the coimmunoprecipitation of pUL15, pUL28, and pUL33 from cytoplasmic lysates of infected cells but prevented viral replication, most nuclear import of both pUL15 and pUL28, and coimmunoprecipitation of pUL15, pUL28, and pUL33 from nuclear lysates. When the pUL15/pUL28 interaction was reduced in infected cells by the truncation of the C terminus of pUL28, pUL28 remained in the cytoplasm. Whether putative terminase components localized in the nucleus or cytoplasm, pUL6 localized in infected cell nuclei, as viewed by indirect immunofluorescence. The finding that the portal and terminase do eventually interact was supported by the observation that pUL6 coimmunoprecipitated strongly with pUL15 and weakly with pUL28 from extracts of infected cells in 1.0 M NaCl. These data are consistent with the hypothesis that the pUL15/pUL28/pUL33 complex forms in the cytoplasm and that an NLS in pUL15 is used to import the complex into the nucleus where at least pUL15 and pUL28 interact with the portal to mediate DNA packaging.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Cornell University, New York School of Veterinary Medicine, Ithaca, NY 14853. Phone: (607) 253-3391. Fax: (607) 253-3384. E-mail: jdb11{at}cornell.edu

{triangledown} Published ahead of print on 28 March 2007.

Journal of Virology, June 2007, p. 6419-6433, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.00047-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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