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. 2003 Jun;77(11):6351-8.
doi: 10.1128/jvi.77.11.6351-6358.2003.

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28

Colleen A White  1 , Nigel D Stow, Arvind H Patel, Michelle Hughes, Valerie G Preston
Affiliations

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28

Colleen A White et al. J Virol. 2003 Jun.

Abstract

The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.

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Figures

FIG. 1.
FIG. 1.
Coimmunoprecipitation of UL6 and UL28. Sf cells were infected with AcUL6, AcUL28, or the two viruses together, and the virus-infected cell polypeptides were labeled with [35S]methionine. Cell extracts were incubated with anti-UL6 antibody (YE583), and the immunoprecipitates, along with samples of total cell proteins, were analyzed by SDS-PAGE. The radioactive protein bands were detected in the dried gel by phosphorimager analysis (A). The positions of UL6 and UL28 are indicated. In a separate polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane and the UL28 protein was detected with antibody R123 by Western blot analysis (B). MI, mock-infected cell extract.
FIG. 2.
FIG. 2.
Coimmunoprecipitation of UL6 and UL15. Sf cells were infected with AcUL6, AcUL15pp65, or the two viruses together, and the virus-infected cell polypeptides were labeled with [35S]methionine. The cell extracts were incubated with anti-UL6 antibody (YE583), and the immunoprecipitates, along with the total extracts, were analyzed by SDS-PAGE. The radioactive protein bands were detected in the dried gel with a phosphorimager (A). In a separate gel, the proteins were transferred to a nitrocellulose membrane and the UL15pp65 protein was detected with anti-pp65 antibody by Western blot analysis (B). The complexes precipitated from extracts of cells coinfected with AcUL6 and AcUL15pp65 or AcUL6 and AcUL28, as indicated, with UL6 immune or preimmune serum are shown in panel C.
FIG. 3.
FIG. 3.
Intracellular colocalization of UL6AU1 and UL28. Vero cells were transfected with pAS30AU1 expressing UL6AU1 (panels a to c), pUL28 expressing UL28 (panels d to f), or both plasmids together (panels g to i). The proteins were detected by an indirect immunofluorescence assay with the AU1 mouse monoclonal antibody, specific for UL6AU1, and the R123 polyclonal rabbit antibody, specific for UL28, as primary antibodies and Cy5-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG as secondary antibodies. The cells were examined by confocal microscopy. Each set of three digital confocal images (from left to right) shows the same field of cells, with FITC fluorescence in green, Cy5 fluorescence in red, and a merged image of the two.
FIG. 4.
FIG. 4.
Intracellular colocalization of UL6 and UL28-c-myc. The digital confocal images show transfected Vero cells expressing UL6 (panels a to c), UL28-c-myc (panels d to f), and UL6 and UL28-c-myc together (panels g to i). The HSV-1 proteins were detected by treating cells with YE583 polyclonal rabbit antibody (specific for UL6) and c-myc mouse monoclonal antibody (specific for UL28-c-myc) and then incubating them with FITC-conjugated anti-rabbit IgG and Cy5-conjugated anti-mouse IgG. Each set of three panels shows FITC staining of UL6 (left), Cy5 staining of UL28-c-myc (middle), and a merged image of the two for the same field of transfected cells (right). Note that in panels g to i, only one of the two UL6-expressing cells contains UL28-c-myc.
FIG. 5.
FIG. 5.
Intracellular distribution of wt UL6, mutant UL6, and UL15pp65. (A). Digital images of transfected Vero cells expressing wt UL6 (a to c), UL15pp65 (d to f), or a mixture of the two proteins (g to i). (B). Images of transfected Vero cells expressing mutant protein UL6in161 alone (a to c) or with UL15pp65 (d to f), UL6in269 alone (g to i) or with UL15pp65 (j to l), and UL6in368 alone (m to o) or withUL15pp65 (p to r). The expressed proteins were detected by confocal microscopy with polyclonal rabbit antibody YE583 (specific for UL6) and mouse monoclonal antibody pp65 (specific for UL15pp65) as primary antibodies, followed by FITC-conjugated anti-rabbit IgG and Cy5-conjugated anti-mouse IgG. The sets of three panels show FITC staining of wt or mutant UL6 protein, Cy5 staining of UL15pp65, and a merged image of the same field of cells.
FIG. 5.
FIG. 5.
Intracellular distribution of wt UL6, mutant UL6, and UL15pp65. (A). Digital images of transfected Vero cells expressing wt UL6 (a to c), UL15pp65 (d to f), or a mixture of the two proteins (g to i). (B). Images of transfected Vero cells expressing mutant protein UL6in161 alone (a to c) or with UL15pp65 (d to f), UL6in269 alone (g to i) or with UL15pp65 (j to l), and UL6in368 alone (m to o) or withUL15pp65 (p to r). The expressed proteins were detected by confocal microscopy with polyclonal rabbit antibody YE583 (specific for UL6) and mouse monoclonal antibody pp65 (specific for UL15pp65) as primary antibodies, followed by FITC-conjugated anti-rabbit IgG and Cy5-conjugated anti-mouse IgG. The sets of three panels show FITC staining of wt or mutant UL6 protein, Cy5 staining of UL15pp65, and a merged image of the same field of cells.
FIG. 6.
FIG. 6.
Intracellular localization of UL6 mutant proteins and UL28. Digital confocal images of transfected Vero cells expressing UL6in161, UL6in269, or UL6in368 alone or with UL28-c-myc. The HSV proteins were detected by confocal microscopy with YE583, which is specific for UL6, and anti-c-myc, which is specific for UL28-c-myc, as primary antibodies and FITC-conjugated anti-rabbit IgG and Cy5-conjugated anti-mouse IgG as secondary antibodies. The top row (a to d) shows merged FITC and Cy5 fluorescent images of the HSV protein expressed on its own. The remainder of the figure shows individual FITC or Cy5 staining and a merged image for UL6in161 and UL28-c-myc (e to g), UL6in269 and UL28-c-myc (h to j), and UL6in368 and UL28-c-myc (k to m). Note that in panels k to m, only one of the UL6-expressing cells contains UL28-c-myc.
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