Interaction of the putative human cytomegalovirus portal protein pUL104 with the large terminase subunit pUL56 and its inhibition by benzimidazole-D-ribonucleosides

Abstract

Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-D-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.

FIG. 1.

Chemical structures of BDCRB, Cl₄RB, and CDMRB.

FIG. 2.

Analysis of intracellular distribution of HCMV pUL104. (A) HFF cells were mock-infected or infected with HCMV strain AD169 for 12, 24, 48, and 72 h at an MOI of 1. After paraformaldehyde fixation, a consecutive immunofluorescence staining with antibody against pUL104, PAbUL104, and antibody against IE1, MAb63-27, was performed. (B) HFF cells infected with HCMV AD169 in the presence of 200 μg/ml PAA were 72 h p.i. fixed with 4% paraformaldehyde. Double-staining was performed with antibodies against pUL104 and IE1, respectively. As a control, phase contrast images were taken to visualize nuclei (phase-c).

FIG. 3.

Confocal microscopy analysis of HCMV pUL104 co-localization with packaging proteins. HFF cells mock infected or infected for 24, 48, and 72 h with HCMV AD169 at an MOI of 1 were double stained with PAbUL104 and MAbM23 against the pUL112-113 gene products (A), MAb28-4 against MCP (B) or PAbUL56 against the terminase subunit pUL56 (C). After paraformaldehyde fixation consecutive immunofluorescence staining was performed. The images represent the central section of the analyzed cells by laser scanning confocal microscopy. A merge of the Cy3- and Cy5-stained cells is shown in addition.

FIG. 4.

Binding of pUL104 to an immobilized carboxy-terminal portion of pUL56 (aa 404 through 850). Glutathione-Sepharose 4B beads loaded with GST, GST-UL56C or GST-UL89 were incubated with in vitro translated UL104 (in vitro UL104) or luciferase (in vitro luciferase). The bound material was subjected to SDS-10% PAGE prior to autoradiography. The arrows indicate the positions of pUL104 and luciferase, the molecular weight markers (M) are shown on the left.

FIG. 5.

Direct interaction of pUL104 with the terminase subunit pUL56 and inhibition by benzimidazole ribonucleosides. (A) Mock-infected (lane 1) or HCMV AD169 infected at an MOI of 1 in the absence (lane 2) or in the presence of 100 μM CDMRB (lane 3), 100 μM BDCRB (lane 4), or 100 μM Cl₄RB (lane 5). HFF cells were coimmunoprecipitated with PAbUL56 at 72 h p.i. and subjected to SDS-8% PAGE. The separated probes were electrotransferred onto nitrocellulose prior to immunoblot analysis using the antibody PAbUL104. (B) Cell extracts from the identical probes of A were separated on an SDS-8% PAGE gel. After transfer onto nitrocellulose immunoblot analysis were performed by using the antibody PAbUL104. (C) As a loading control the immunoblot was consecutively stained with an antibody against actin. The arrows indicate the position of pUL104, the molecular weight markers (M) are shown on the left.

FIG. 6.

Electron microscopy analysis of ultrathin section of HCMV infected cells in the absence (A) or presence of 100 μM Cl₄RB (B). After 3 days, cells were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde with 1% tannin and prepared for electron microscopy. B capsids are indicated by black arrows, A capsids by white arrows, C capsids by bold arrows. N indicates the nucleus, C the cytoplasm.

FIG. 7.

Pulsed-field electrophoresis analysis of infected cells in the presence of Cl₄RB. HFF cells were infected with an MOI of 3 in the absence (w/o) or presence of 100 μM Cl₄RB. 72 h p.i. cells were embedded in agarose and digested with proteinase K and incubated with or without 50 U restriction endonuclease NotI. DNA was resolved by pulsed-field gel electrophoreses, stained with ethidium bromide, and photographed. Concatemers and unit length monomers are indicated.