Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs.
The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.
Alvisi
Gualtiero
G
Dipartimento di Medicina Clinica Specialistica e Sperimentale, Sezione di Microbiologia, Università degli Studi di Bologna, via Massarenti 9, 40138 Bologna, Italy. gualtiero.alvisi@med.monash.edu.au
Ripalti
Alessandro
A
Ngankeu
Apollinaire
A
Giannandrea
Maila
M
Caraffi
Stefano G
SG
Dias
Manisha M
MM
Jans
David A
DA
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003587:Cytomegalovirus / Q000201:enzymology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019913:Nuclear Localization Signals; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
D019913:Nuclear Localization Signals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; D004259:DNA-Directed DNA Polymerase