Nuclear import of HSV-1 DNA polymerase processivity factor UL42 is mediated by a C-terminally located bipartite nuclear localization signal.
The polymerase accessory protein of the human herpes simplex virus type 1 (HSV-1) DNA polymerase UL42 plays an essential role in viral replication, conferring processivity to the catalytic subunit UL30. We show here that UL42 is imported to the nucleus of living cells in a Ran- and energy-dependent fashion, through a process that requires a C-terminally located bipartite nuclear localization signal (UL42-NLSbip; PTTKRGRSGGEDARADALKKPK(413)). Moreover cytoplasmic mutant derivatives of UL42 lacking UL42-NLSbip are partially relocalized into the cell nucleus upon HSV-1 infection or coexpression with UL30, implying that the HSV-1 DNA polymerase holoenzyme can assemble in the cytoplasm before nuclear translocation occurs, thus explaining why the UL42 C-terminal domain is not strictly required for viral replication in cultured cells. However, mutation of both UL30 and UL42 NLS results in retention of the DNA polymerase holoenzyme in the cytoplasm, suggesting that simultaneous inhibition of both NLSs could represent a viable strategy to hinder HSV-1 replication. Intriguingly, UL42-NLSbip is composed of two stretches of basic amino acids matching the consensus for classical monopartite NLSs (NLSA, PTTKRGR(397); NLSB, KKPK(413)), neither of which are capable of targeting GFP to the nucleus on their own, consistent with the hypothesis that P and G residues in position +3 of monopartite NLSs are not compatible with nuclear transport in the absence of additional basic sequences located in close proximity. Our results showing that substitution of G or P of the NLS with an A residue partially confers NLS function will help to redefine the consensus for monopartite NLSs.
Alvisi
Gualtiero
G
Dipartimento di Ematologia e Scienze Oncologiche L.A. Seragnoli, Universita degli Studi di Bologna, Bologna, Italia. gualtiero.alvisi3@unibo.it
Avanzi
Simone
S
Musiani
Daniele
D
Camozzi
Daria
D
Leoni
Valerio
V
Ly-Huynh
Jennifer D
JD
Ripalti
Alessandro
A
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D016384:Consensus Sequence; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D009154:Mutation; D019913:Nuclear Localization Signals; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D019913:Nuclear Localization Signals; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus