title: An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus.
abstract: Although the 1235 amino acids human herpes simplex virus type 1 (HSV-1) DNA polymerase catalytic subunit, pUL30, is essential for HSV-1 replication in the nucleus of host cells, little information is available regarding its nuclear import mechanism. The present study addresses this issue directly, characterizing pUL30's nuclear import pathway for the first time using quantitative confocal laser scanning microscopy (CLSM) on living cells, and fluorescent binding assays. In addition to a previously described nuclear localization signal (NLS) located within the pUL30 binding site for the polymerase accessory protein (PAP) pUL42, that appears to be dispensable for nuclear targeting, pUL30 possesses three putative basic NLSs. Intriguingly, the core of pUL30-NLS2 (residues 1114-1120) is highly homologous to that of the recently described NLS, similarly located upstream of the PAP binding site, of the human cytomegalovirus (HCMV) DNA polymerase catalytic subunit, pUL54. Here we show for the first time that pUL30-NLS2 itself is only partially functional in terms of nuclear import due to residue P1118 present in position 3 of the NLS core. Intriguingly, pUL30-NLS2 together with pUL30-NLS3 (residues 1133-1136) represents a fully functional bipartite NLS (pUL30-NLSbip), required for nuclear targeting of pUL30, and able to confer nuclear localization on heterologous proteins by conferring high-affinity interaction with the importin (IMP) alpha/beta heterodimer. Since nuclear targeting of HSV-1 proteins forming the replication fork is crucial for viral replication, the pUL30-NLSbip emerges for the first time as a viable therapeutic target.
authors.affiliation: Dipartimento di Medicina Clinica Specialistica e Sperimentale, Divisione di Microbiologia, Universtià degli Studi di Bologna, Bologna, Italia. gualtiero.alvisi3@unibo.it
authors.forename: Gualtiero
authors.initials: G
authors.lastname: Alvisi
authors.forename: Daniele
authors.initials: D
authors.lastname: Musiani
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ripalti
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics*; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D014764:Viral Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; D011392:Proline; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus