Characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27

Abstract

Bovine herpesvirus-1 infected cell protein 27 (BICP27) was detected predominantly in the nucleolus. The open reading frame of BICP27 was fused with the enhanced yellow fluorescent protein (EYFP) gene to investigate its subcellular localization in live cells and BICP27 was able to direct monomeric, dimeric or trimeric EYFP exclusively to the nucleolus. By constructing a series of deletion mutants, the putative nuclear localization signal (NLS) and nucleolar localization signal (NoLS) were mapped to (81)RRAR(84) and (86)RPRRPRRRPRRR(97) respectively. Specific deletion of the putative NLS, NoLS or both abrogated nuclear localization, nucleolar localization or both respectively. Furthermore, NLS was able to direct trimeric EYFP predominantly to the nucleus but excluded from the nucleolus, whereas NoLS targeted trimeric EYFP primarily to the nucleus, and enriched in the nucleolus with faint staining in the cytoplasm. NLS+NoLS directed trimeric EYFP predominantly to the nucleolus with faint staining in the nucleus. Moreover, deletion of NLS+NoLS abolished the transactivating activity of BICP27 on gC promoter, whereas deletion of either NLS or NoLS did not. The study demonstrated that BICP27 is a nucleolar protein, adding BICP27 to the growing list of transactivators which localize to the nucleolus.

Identification of Nuclear and Nucleolar Localization Signals of Pseudorabies Virus (PRV) Early Protein UL54 Reveals that Its Nuclear Targeting Is Required for Efficient Production of PRV

“…In addition, it is well known that some fixation protocols may alter the subcellular localization of proteins, resulting in misleading conclusions in the analysis of the intracellular distribution of a specific protein (26). Therefore, two different IFA fixation methods were applied, namely, methanol-acetic acid based (25) or formaldehyde based (19,32). Untagged UL54 or HAtagged UL54 localized in the nucleolus (Fig.…”

“…Immunofluorescence assay (IFA) was applied to analyze the subcellular localization of PRV UL54 in transfected or infected cells. To investigate the subcellular localization of UL54 in transfected cells, two different fixation methods were performed, either methanol-acetic acid-based (25) or formaldehydebased (19,32). For the former, the transfected cells were fixed with 95% methanol and 5% glacial acetic acid, permeabilized with 0.25% Triton X-100 and 5% dimethyl sulfoxide (DMSO), and incubated with the anti-UL54 pAb and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Zymed Laboratories).…”

“…For the latter, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with the anti-UL54 or anti-EP0 pAb, respectively. To detect the subcellular localization of UL54 in infected cells, mock-infected PK-15 cells and cells infected with PRV at a multiplicity of infection (MOI) of 1 or 0.1 for several hours were subjected to formaldehydebased (19), fixation-based IFA using the anti-UL54 or anti-EP0 pAb. After each incubation step, the cells were washed extensively with PBS.…”

Characterization of the nuclear import and export signals, and subcellular transport mechanism of varicella-zoster virus ORF9

“…However, it is well known that some fixation protocols may alter the subcellular localization of proteins. As an alternative method to investigate the intracellular distribution of ORF9, a living-cell fluorescence microscopy technique and enhanced-yellow-fluorescent protein (EYFP)-tagged variants were used (Ding et al, 2010;Guo et al, 2009;Xing et al, 2010). To study the subcellular localization of ORF9 in the absence of other viral proteins, plasmid expressing EYFP monomer, dimer (dEYFP) or trimer (tEYFP) fused to the C terminus of ORF9 or EYFP fused to the N terminus of ORF9 were constructed (Fig.…”

“…1a) using VZV BAC DNA as a template (Zhang et al, 2008). COS-7 cells (ATCC CRL1651), a useful tool for studying subcellular localization of proteins, were transfected with plasmids pEYFP-N1, pORF9-EYFP, pORF9-dEYFP, pORF9-tEYFP or pEYFP-ORF9 as described previously (Ding et al, 2010;Guo et al, 2009;Xing et al, 2010). The subcellular localization of these fusion proteins was imaged in live cells.…”