Interaction of the putative human cytomegalovirus portal protein pUL104 with the large terminase subunit pUL56 and its inhibition by benzimidazole-D-ribonucleosides.
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.
Dittmer
Alexandra
A
Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.
Drach
John C
JC
Townsend
Leroy B
LB
Fischer
Anke
A
Bogner
Elke
E
D001562:Benzimidazoles / Q000494:pharmacology*; D036022:Capsid Proteins / Q000378:metabolism*; D018929:Cell Culture Techniques; D003587:Cytomegalovirus / Q000187:drug effects* / Q000502:physiology; D004706:Endodeoxyribonucleases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D012263:Ribonucleosides / Q000494:pharmacology*; D014764:Viral Proteins / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*
D001562:Benzimidazoles; D036022:Capsid Proteins; D004791:Enzyme Inhibitors; D012263:Ribonucleosides; C481341:UL104 protein, human cytomegalovirus; C090238:UL56 protein, cytomegalovirus; D014764:Viral Proteins; D015678:Viral Structural Proteins; C031000:benzimidazole; D004706:Endodeoxyribonucleases; C030894:terminase