Nuclear Import of HSV-1 DNA Polymerase Processivity Factor UL42 Is Mediated by a C-Terminally Located Bipartite Nuclear Localization Signal

Abstract

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.

Nuclear Transport of Epstein-Barr Virus DNA Polymerase Is Dependent on the BMRF1 Polymerase Processivity Factor and Molecular Chaperone Hsp90

“…Processivity factors other than p41 of HHV-6 have been shown to contain a functional NLS (12,35,(37)(38)(39). Indeed, we observed that BMRF1 protein, when it was expressed alone, localized to the nucleus (Fig.…”

Susceptibility of Human Placenta Derived Mesenchymal Stromal/Stem Cells to Human Herpesviruses Infection

“…Sometimes nuclei were stained by incubating with DAPI (1 µg/mL) for 4 min at RT. Samples were washed with PBS, mounted on coverslips in PBS/glycerol 50% (v/v), and imaged using a Nikon Eclipse E600 microscope equipped with a Nikon DXM1200 digital camera and a Nikon Plan Fluor 40× objective (Nikon, Tokyo, Japan), essentially as previously [68]. Live cells grown on Willcodishes (Willcowells, Amsterdam, The Netherlands) were infected with TOZGFP-HSV1 [51] and analyzed for GFP expression using a Nikon Eclipse TE2000-U inverted microscope equipped with a Nikon DXM1200 digital camera and a Nikon Plan Fluor 40× objective (Nikon, Tokio, Japan), as previously [69]–[71].…”

Regulated Transport into the Nucleus of Herpesviridae DNA Replication Core Proteins

“…Besides ssDNA binding protein, the two main complexes are a multimeric DNA polymerase holoenzyme and helicase/primase complexes. Given the high molecular weight of such proteins, their nuclear transport has been shown to be an active process mediated by a number of different IMPs, which recognize specific NLSs on the cargo proteins [52,53,54,55,56,57,58,59]. In some cases, individual subunits are capable of localizing to the cell nucleus independently of the others, like in the case of HSV and HCMV DNA polymerase holoenzyme subunits [52,53,54,55,56].…”

“…Given the high molecular weight of such proteins, their nuclear transport has been shown to be an active process mediated by a number of different IMPs, which recognize specific NLSs on the cargo proteins [52,53,54,55,56,57,58,59]. In some cases, individual subunits are capable of localizing to the cell nucleus independently of the others, like in the case of HSV and HCMV DNA polymerase holoenzyme subunits [52,53,54,55,56]. However in the case of KSHV and EBV DNA polymerase holoenzymes [59,60] and most of their helicase/primase complex subunits, formation of the holoenzyme appears to be a pre-requisite for nuclear transport [61,62,63,64] (see Table 1).…”

“…Indeed different proteins can be translocated independently, or only as fully assembled holoenzymes, wherein a NLS present on the PAP mediates import of the catalytic subunit via a piggy-back mechanism. In all known herpesviruses, a functional NLS has been characterized on the PAP [52,55,57,58,59], which plays a central role in nuclear targeting of the holoenzyme (Figure 2 and Table 2). In addition, in the case of HCMV a complex phosphorylation-dependent regulation system finely tunes nuclear import of the PAP [51,52,69,70].…”