Human cytomegalovirus (HCMV) DNA polymerase processivity factor ppUL44 dimerizes in the cytosol before translocation to the nucleus.
Replication of the human cytomegalovirus genome takes place in the nuclei of infected cells and is mediated by a viral-encoded DNA polymerase complex formed by the catalytic subunit pUL54 and the processivity factor ppUL44. Although it has recently been shown that the dimerization ability of recombinant pUL44 appears to be crucial for effective DNA binding in vitro, whether ppUL44 can dimerize or not in a cellular context is unknown. Here, we show, by using co-immunoprecipitation and dual-color live imaging approaches on cells expressing fluorescent and differently tagged ppUL44 fusion proteins, that ppUL44 dimerizes in the cytoplasm via its N-terminal domain, before translocating to the nucleus. Furthermore, we show that nuclear translocation of differently tagged ppUL44 heterodimers can occur even when one subunit carries a nonfunctional nuclear localization signal. Importantly, the latter cotransfection assay represents a system to test small-molecule inhibitors for their ability to impair ppUL44 dimerization.
Alvisi
Gualtiero
G
Dipartimento di Medicina Clinica Specialistica e Sperimentale, Sezione di Microbiologia, Università degli Studi di Bologna, Bologna, Italy. gualtiero.alvisi@med.monash.edu.au
Jans
David A
DA
Ripalti
Alessandro
A
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins