Fusen Lin

Comprehensive Characterization of Interaction Complexes of Herpes Simplex Virus Type 1 ICP22, UL3, UL4, and UL20.5

ABSTRACT It has been reported that herpes simplex virus type 1 UL3, UL4, and UL20.5 proteins are localized to small, dense nuclear bodies together with ICP22 in infected cells. In the present study, we comprehensively characterized these interactions by subcellular colocalization, coimmunoprecipitation, and bimolecular fluorescence complementation assays. For the first time, it was demonstrated that both UL3 and UL20.5 are targeted to small, dense nuclear bodies by a direct interaction with ICP22, whereas UL4 colocalizes with ICP22 through its interaction with UL3 but not UL20.5 or ICP22. There was no detectable interaction between UL3 and UL20.5.

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 81RRAR84 and 86RPRRPRRRPRRR97 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.

Characterization of the subcellular localization of herpes simplex virus type 1 proteins in living cells

In this study, we presented the construction of a library of expression clones for the herpes simplex virus type 1 (HSV-1) proteome and subcellular localization map of HSV-1 proteins in living cells using yellow fluorescent protein (YFP) fusion proteins. As a result, 21 proteins showed cytoplasmic or subcytoplasmic localization, 16 proteins showed nuclear or subnuclear localization, and others were present both in the nucleus and cytoplasm. Interestingly, most capsid proteins showed enriched or exclusive localization in the nucleus, and most of the envelope proteins showed cytoplasmic localization, suggesting that subcellular localization of the proteins correlated with their functions during virus replication. These results present a subcellular localization map of HSV-1 proteins in living cells, which provide useful information to further characterize the functions of these proteins.

Characterization of the nuclear import and export mechanisms of bovine herpesvirus-1 infected cell protein 27

In previous study, we have identified a nuclear localization signal (NLS) and a nucleolar localization signal (NoLS) in bovine herpesvirus-1 (BHV-1) infected cell protein 27 (BICP27), which targets predominantly to the nucleolus. Furthermore, the C-terminal 300 amino acid residues targets exclusively to the cytoplasm, suggesting that BICP27 might contain a nuclear export signal (NES). Amino acid sequence analysis revealed that there is a cluster of leucine-rich residues resembling a NES. Heterokaryon assays demonstrated that BICP27 is capable of shuttling between the nucleus and the cytoplasm of the BHV-1 infected, BICP27 and BICP27-EYFP transfected cells. Deletion mutant analysis revealed that this property is attributed to the leucine-rich NES 299LEELCAARRLSL310. Moreover, the functional NES could mediate transport of a monomer EYFP and a dimer EYFP to the cytoplasm. The nucleocytoplasmic shuttling of BICP27 and the nuclear export of NES-EYFP and NES-dEYFP could be blocked by leptomycin LMB, an inhibitor of the chromosomal region maintenance 1 (CRM1), which is the receptor for exportin-1-dependent nuclear export. In addition, the nuclear import of BICP27 was inhibited by a dominant negative Ran-GTP, namely Ran-GTP Q69L, indicating that BICP27 localized to the nucleus by means of a classic Ran dependent nuclear import mechanism. In conclusion, these results demonstrate that BICP27 shuttles between the nucleus and the cytoplasm by the functional NES and NLS through a CRM1-dependent nuclear export pathway and a Ran dependent nuclear import pathway.

Expression, purification of the UL3 protein of herpes simplex virus type 1, and production of UL3 polyclonal antibody

Herpes simplex virus type 1 (HSV-1) is a common pathogen which causes infections of the mucocutaneous membranes. The UL3 protein belongs to a group of HSV-1 late proteins. To date, the function of the UL3 protein in cell culture, animal models, and natural infection is unknown. To investigate further the function of the UL3 protein, this study was undertaken to express the UL3 protein and raise a polyclonal antibody. The UL3 gene was cloned in the prokaryotic expression vector pET-28a (+) to yield pET-28a (+)-UL3. The His6-tagged UL3 protein was expressed in Escherichia coli (E. coli) BL21 (DE3) cells and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After purification by nickel affinity chromatography and refolding, the recombinant protein was used to raise the anti-UL3 polyclonal antibody. Western blot analysis demonstrated that the UL3 protein was recognized by the polyclonal antibody, and immunofluorescent assay also showed that the antibody was able to recognize the UL3 protein in the cells infected with HSV-1.

A novel virus-encoded nucleocytoplasmic shuttling protein: The UL3 protein of herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) UL3 protein is a nuclear protein. In this study, the molecular mechanism of the subcellular localization of UL3 was characterized by fluorescence microscopy in living cells. A nuclear localization signal (NLS) and a nuclear export signal (NES) were also identified. UL3 was demonstrated to target to the cytoplasm through the NES via chromosomal region maintenance 1 (CRM-1) dependent pathway, and to the nucleus through RanGTP-dependent mechanism. Heterokaryon assays confirmed that UL3 was capable of shuttling between the nucleus and the cytoplasm. These results demonstrate that the UL3 protein is a novel HSV-1 encoded nucleocytoplasmic shuttling protein.

The herpes simplex virus type 1 infected cell protein 22

As one of the immediate-early (IE) proteins of herpes simplex virus type 1 (HSV-1), ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells. It is required in experimental animal systems and some nonhuman cell lines, but not in Vero or HEp-2 cells. ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase II. It has been shown to be required for efficient expression of early (E) genes and a subset of late (L) genes. ICP22, in conjunction with the UL13 kinase, mediates the phosphorylation of RNA polymerase II. Both ICP22 and UL13 are required for the activation of cdc2, the degradation of cyclins A and B and the acquisition of a new cdc2 partner, the UL42 DNA polymerase processivity factor. The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase IIα in an ICP22-dependent manner to promote L gene expression. In addition, ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase II.

Screening and identification of host cellular factors interaction with immediate-early protein ICP22 of herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is a common and widely studied human pathogen that can replicate in epithelial cells and other cells of the host or alternatively can remain latent in peripheral neurons. ICP22 consists of 420 residues and is encoded by a spliced mRNA transcribed from the US1 gene. It is necessary for efficient HSV-1 growth in animal models of infection as well as for efficient in vitro growth in some, but not all, cultured cells. For example, ICP22 mutants grow well in African green monkey kidney (Vero) cells, but not in human embryonic lung (HEL) cells. ICP22 is extensively phosphorylated during infection, primarily by UL13 and another viral protein kinase, US3. In addition to inducing the modification of the host cell RNA Pol II, several other functions have been attributed to ICP22; these functions include the induction of certain viral L genes, the alteration of cell cycle-related proteins, and the determination of virion composition. It is clear that ICP22 is a multifunctional protein localized to the nucleus of infected cells, however, the host cellular factors of ICP22 as well as the biological functions of their interactions are still little known. In the present study, an yeast two-hybrid system was applied to identify the host cellular factors of ICP22 and five target candidates were yielded: (1) TATA box binding protein -associated factor (TAF1); (2) TAO kinase 3 (TAOK3); (3) Alpha thalassemia/mental retardation syndrome X-linked (ATRX); (4) Cyclin-dependent kinase 9 (CDK9); (5) Ras association domain family member 9 (RASSF9); (6) occludin/ELL domain containing 1 (OCEL1). To confirm some of the interactions by co-localization in living cells, ICP22 and two candidate targets were tagged with enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), respectively. Upon cotransfection of COS-7 cells, RASSF9-EYFP and OCEL1-EYFP both co-localized with ICP22-ECFP in distinct nuclear domains, indicating they are host cellular factors interaction with viral ICP22 under physiological conditions.