Chunfu Zheng - Academia.edu (original) (raw)
Papers by Chunfu Zheng
The Journal of Eukaryotic Microbiology, 2003
Strain bacillus Calmette-Guerin (BCG) of Mycobacterium hovis has been used as a live bacterial va... more Strain bacillus Calmette-Guerin (BCG) of Mycobacterium hovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-l/HN Plasmodium falciparum genome, sequenced, and expressed in M . bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.
BMC Proceedings, 2011
Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen of skin and mucous membranes w... more Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen of skin and mucous membranes which associates with the infections of the mucocutaneous membranes, brain, and internal organs of infected neonates. As a member of the human herpesvirus family, HSV-1 contains a large DNA genome, encoding 84 unique open reading frames (ORFs), but the majority of its function is still elusive. In the present study, the genome of HSV-1 F strain was cloned as a stable and infectious BAC without any deletions of the viral genes. The BAC backbone sequences flanked by loxP sites were inserted into the intergenic region between UL37 and UL38. Cotransfection of the recombinant virus with a Cre recombinase plasmid resulted in the excision of the BAC sequences. Additionally, a firefly luciferase cassette was inserted to generate a novel luciferase HSV-1 BAC. Importantly, the resulting recombinant HSV-1 BAC Luc behaved indistinguishably from the wild-type virus in vero cells, and the resulting luciferase activity could be quantified in vitro expediently. The recombinant HSV-1 BAC Luc will facilitate HSV-1 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.
BMC Proceedings, 2011
The ORF9 protein, a VZV-encoded late protein consisting of 302 amino acid (aa) residues, is a mem... more The ORF9 protein, a VZV-encoded late protein consisting of 302 amino acid (aa) residues, is a member of the highly conserved alphaherpesvirus UL49 gene family but shares limited identity with the UL49 prototype, HSV-1 VP22. As the orthologue of HSV-1 VP22, ORF9 is believed to be a major constituent of the VZV virion tegument. HSV-1 VP22 has been extensively studied; however, the functional properties of ORF9 are less well understood, despite the fact that its transcript is the most abundant viral message expressed during VZV lytic infection. As an important step toward understanding the detailed functions of ORF9 in vivo is to determine its precise subcellular localization, in the work presented here, to avoid the flaw of fixation protocol, living cells fluorescence microscopy technique, which is widely applied and developed in our group, was applied to deeply analyze the intracellular distribution of ORF9 protein and, to characterize its functional nuclear localization signal (NLS) and nuclear export signal (NES), as well as its transport mechanism in living cells.
Journal of virology, Jan 11, 2015
Receptor-interacting kinase3(RIP3) and its substrate MLKL (mixed lineage kinase domain like prote... more Receptor-interacting kinase3(RIP3) and its substrate MLKL (mixed lineage kinase domain like protein) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defensive mechanism. Here, we report that human herpes simplex virus type 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of a RIP1/RIP3 necrosome. HSV R1 was sufficient to suppress TNF-induced necrosis and its RHIM domain was required to disrupt RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defensive mechanism of...
Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of... more Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of two copies of VP23 and one copy of VP19C. Here, we identified a non-classical NLS of VP19C between aa 50 and 61, and the nuclear import of VP19C was mediated by RanGTP and importin b1-, but not importin a5-, dependent pathway. Additionally, recombinant virus harbouring this NLS mutation (NLSm) replicates less efficiently as wild-type. These data strongly suggested that the nuclear import of VP19C is required for efficient HSV-1 production.
Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
BMC Proceedings, 2011
Herpes simplex virus type 1 (HSV-1) is a common and widely studied human pathogen that can replic... more 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. Cite this article as: Xing et al.: Screening and identification of host cellular factors interaction with immediate-early protein ICP22 of herpes simplex virus type 1.
Virologica Sinica, 2011
The function of the herpes simplex virus type 1 (HSV-1) UL4 protein is still elusive. Our objecti... more The function of the herpes simplex virus type 1 (HSV-1) UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP), the nuclear export signals (NES) of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattern of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 (CRM1)-dependent manner involving RanGTP hydrolysis.
Virus research, 2010
The herpes simplex virus type I (HSV-1) US11 protein is an RNA-binding multifunctional regulator ... more The herpes simplex virus type I (HSV-1) US11 protein is an RNA-binding multifunctional regulator that specifically and stably associates with nucleoli. Although the C-terminal part of US11 was responsible for its nucleolar localization, the precise nucleolar localization signal (NoLS) and nuclear export signal (NES) of US11 and its nuclear import and export mechanisms are still elusive. In this study, fluorescence microscopy was employed to investigate the subcellular localization of US11 and characterize its transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP), three novel NoLSs of US11 were for the first time mapped to amino acids 84-125, 126-152, and 89-146, respectively. Additionally, the NES was identified to locate between amino acids 89 and 119. Furthermore, the US11 protein was demonstrated to target to the cytoplasm through the NES by chromosomal region maintenance 1 (CRM1)-independent pathwa...
Virus research, 2010
In previous study, we have identified a nuclear localization signal (NLS) and a nucleolar localiz... more 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 lept...
Virologica Sinica, 2008
The herpes simplex virus type 1 (HSV-1) infected-cell protein 27 (ICP27) is an essential, highly ... more The herpes simplex virus type 1 (HSV-1) infected-cell protein 27 (ICP27) is an essential, highly conserved protein involved in various steps of HSV-1 gene regulation as well as in the shut-off of host gene expression during infection. It functions primarily at the post-transcriptional level in inhibiting precursor mRNA splicing and in promoting nuclear export of viral transcripts. Recently, many novel functions performed by the HSV-1 ICP27 protein were shown, including leptomycin B resistance, inhibition of the typeⅠinterferon signaling, regulation of the viral mRNA translation and determining the composition of HSV-1 virions.
It has been reported that herpes simplex virus type 1 UL3, UL4, and UL20.5 proteins are localized... more 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.
BMC Proceedings, 2011
Pseudorabies virus (PRV) UL54 protein localized almost exclusively to the nucleolus. By construct... more Pseudorabies virus (PRV) UL54 protein localized almost exclusively to the nucleolus. By constructing a series of mutants, the putative nuclear localization signal (NLS), nucleolar localization signal (NoLS) and nuclear export signal (NES) of UL54 were for the first time mapped to amino acids 45 RRRRGGRGGRAAR 57 , 61 RQRRR 65 and 240 LQNLRLKLGPFL 251 , respectively. In addition, nuclear localization of UL54 was important for its transcriptional regulation of glycoprotein C promoter. The nuclear import of UL54 was abrogated by dominant negative RanGTP and importin β1, respectively, indicating that UL54 targeted to the nucleus by means of a classic Ran-and importin β-dependent nuclear import mechanism. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1). However, ectopic expression of the mRNA export receptor TAP(NXF1) promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP(NXF1), but not CRM1, dependent nuclear export pathway. The present study demonstrated that UL54 is a nucleolar protein, adding UL54 to the growing list of transactivators which localize to the nucleolus and shuttle between the nucleus and the cytoplasm.
Virus research, Jan 9, 2010
This article has been withdrawn at the request of the authors. The Publisher apologizes for any i... more This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
Virology, 2011
The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV... more The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein and essential for HSV-1 infection. To determine if UL54 might shuttle between the nucleus and cytoplasm, as has been shown for its homologues in human herpesviruses, the molecular determinants for its nucleocytoplasmic shuttling were investigated. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of chromosome region maintenance 1 (CRM1). However, TAP/NXF1 promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP/NXF1, but not CRM1, dependent nuclear export pathway. Furthermore, UL54 was demonstrated to target to the nucleus through a classic Ran-, importin β1- and α5-dependent nuclear import mechanism.
Protein & cell, 2012
Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more s... more Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.
Virologica Sinica, 2010
As one of the immediate-early (IE) proteins of herpes simplex virus type 1 (HSV-1), ICP22 is a mu... more 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.
Virologica Sinica, 2010
The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only parti... more The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.
Virology, 2010
The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion... more The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 131 PRPR 134 NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.
Virologica Sinica, 2010
Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious d... more Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.
The Journal of Eukaryotic Microbiology, 2003
Strain bacillus Calmette-Guerin (BCG) of Mycobacterium hovis has been used as a live bacterial va... more Strain bacillus Calmette-Guerin (BCG) of Mycobacterium hovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-l/HN Plasmodium falciparum genome, sequenced, and expressed in M . bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.
BMC Proceedings, 2011
Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen of skin and mucous membranes w... more Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen of skin and mucous membranes which associates with the infections of the mucocutaneous membranes, brain, and internal organs of infected neonates. As a member of the human herpesvirus family, HSV-1 contains a large DNA genome, encoding 84 unique open reading frames (ORFs), but the majority of its function is still elusive. In the present study, the genome of HSV-1 F strain was cloned as a stable and infectious BAC without any deletions of the viral genes. The BAC backbone sequences flanked by loxP sites were inserted into the intergenic region between UL37 and UL38. Cotransfection of the recombinant virus with a Cre recombinase plasmid resulted in the excision of the BAC sequences. Additionally, a firefly luciferase cassette was inserted to generate a novel luciferase HSV-1 BAC. Importantly, the resulting recombinant HSV-1 BAC Luc behaved indistinguishably from the wild-type virus in vero cells, and the resulting luciferase activity could be quantified in vitro expediently. The recombinant HSV-1 BAC Luc will facilitate HSV-1 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.
BMC Proceedings, 2011
The ORF9 protein, a VZV-encoded late protein consisting of 302 amino acid (aa) residues, is a mem... more The ORF9 protein, a VZV-encoded late protein consisting of 302 amino acid (aa) residues, is a member of the highly conserved alphaherpesvirus UL49 gene family but shares limited identity with the UL49 prototype, HSV-1 VP22. As the orthologue of HSV-1 VP22, ORF9 is believed to be a major constituent of the VZV virion tegument. HSV-1 VP22 has been extensively studied; however, the functional properties of ORF9 are less well understood, despite the fact that its transcript is the most abundant viral message expressed during VZV lytic infection. As an important step toward understanding the detailed functions of ORF9 in vivo is to determine its precise subcellular localization, in the work presented here, to avoid the flaw of fixation protocol, living cells fluorescence microscopy technique, which is widely applied and developed in our group, was applied to deeply analyze the intracellular distribution of ORF9 protein and, to characterize its functional nuclear localization signal (NLS) and nuclear export signal (NES), as well as its transport mechanism in living cells.
Journal of virology, Jan 11, 2015
Receptor-interacting kinase3(RIP3) and its substrate MLKL (mixed lineage kinase domain like prote... more Receptor-interacting kinase3(RIP3) and its substrate MLKL (mixed lineage kinase domain like protein) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defensive mechanism. Here, we report that human herpes simplex virus type 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of a RIP1/RIP3 necrosome. HSV R1 was sufficient to suppress TNF-induced necrosis and its RHIM domain was required to disrupt RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defensive mechanism of...
Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of... more Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of two copies of VP23 and one copy of VP19C. Here, we identified a non-classical NLS of VP19C between aa 50 and 61, and the nuclear import of VP19C was mediated by RanGTP and importin b1-, but not importin a5-, dependent pathway. Additionally, recombinant virus harbouring this NLS mutation (NLSm) replicates less efficiently as wild-type. These data strongly suggested that the nuclear import of VP19C is required for efficient HSV-1 production.
Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
BMC Proceedings, 2011
Herpes simplex virus type 1 (HSV-1) is a common and widely studied human pathogen that can replic... more 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. Cite this article as: Xing et al.: Screening and identification of host cellular factors interaction with immediate-early protein ICP22 of herpes simplex virus type 1.
Virologica Sinica, 2011
The function of the herpes simplex virus type 1 (HSV-1) UL4 protein is still elusive. Our objecti... more The function of the herpes simplex virus type 1 (HSV-1) UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP), the nuclear export signals (NES) of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattern of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 (CRM1)-dependent manner involving RanGTP hydrolysis.
Virus research, 2010
The herpes simplex virus type I (HSV-1) US11 protein is an RNA-binding multifunctional regulator ... more The herpes simplex virus type I (HSV-1) US11 protein is an RNA-binding multifunctional regulator that specifically and stably associates with nucleoli. Although the C-terminal part of US11 was responsible for its nucleolar localization, the precise nucleolar localization signal (NoLS) and nuclear export signal (NES) of US11 and its nuclear import and export mechanisms are still elusive. In this study, fluorescence microscopy was employed to investigate the subcellular localization of US11 and characterize its transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP), three novel NoLSs of US11 were for the first time mapped to amino acids 84-125, 126-152, and 89-146, respectively. Additionally, the NES was identified to locate between amino acids 89 and 119. Furthermore, the US11 protein was demonstrated to target to the cytoplasm through the NES by chromosomal region maintenance 1 (CRM1)-independent pathwa...
Virus research, 2010
In previous study, we have identified a nuclear localization signal (NLS) and a nucleolar localiz... more 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 lept...
Virologica Sinica, 2008
The herpes simplex virus type 1 (HSV-1) infected-cell protein 27 (ICP27) is an essential, highly ... more The herpes simplex virus type 1 (HSV-1) infected-cell protein 27 (ICP27) is an essential, highly conserved protein involved in various steps of HSV-1 gene regulation as well as in the shut-off of host gene expression during infection. It functions primarily at the post-transcriptional level in inhibiting precursor mRNA splicing and in promoting nuclear export of viral transcripts. Recently, many novel functions performed by the HSV-1 ICP27 protein were shown, including leptomycin B resistance, inhibition of the typeⅠinterferon signaling, regulation of the viral mRNA translation and determining the composition of HSV-1 virions.
It has been reported that herpes simplex virus type 1 UL3, UL4, and UL20.5 proteins are localized... more 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.
BMC Proceedings, 2011
Pseudorabies virus (PRV) UL54 protein localized almost exclusively to the nucleolus. By construct... more Pseudorabies virus (PRV) UL54 protein localized almost exclusively to the nucleolus. By constructing a series of mutants, the putative nuclear localization signal (NLS), nucleolar localization signal (NoLS) and nuclear export signal (NES) of UL54 were for the first time mapped to amino acids 45 RRRRGGRGGRAAR 57 , 61 RQRRR 65 and 240 LQNLRLKLGPFL 251 , respectively. In addition, nuclear localization of UL54 was important for its transcriptional regulation of glycoprotein C promoter. The nuclear import of UL54 was abrogated by dominant negative RanGTP and importin β1, respectively, indicating that UL54 targeted to the nucleus by means of a classic Ran-and importin β-dependent nuclear import mechanism. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1). However, ectopic expression of the mRNA export receptor TAP(NXF1) promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP(NXF1), but not CRM1, dependent nuclear export pathway. The present study demonstrated that UL54 is a nucleolar protein, adding UL54 to the growing list of transactivators which localize to the nucleolus and shuttle between the nucleus and the cytoplasm.
Virus research, Jan 9, 2010
This article has been withdrawn at the request of the authors. The Publisher apologizes for any i... more This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
Virology, 2011
The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV... more The pseudorabies virus (PRV) early protein UL54 is a homologue of the herpes simplex virus 1 (HSV-1) immediate-early protein ICP27, which is a multifunctional protein and essential for HSV-1 infection. To determine if UL54 might shuttle between the nucleus and cytoplasm, as has been shown for its homologues in human herpesviruses, the molecular determinants for its nucleocytoplasmic shuttling were investigated. Heterokaryon assays demonstrated that UL54 was a nucleocytoplasmic shuttling protein and this property could not be blocked by leptomycin B, an inhibitor of chromosome region maintenance 1 (CRM1). However, TAP/NXF1 promoted the nuclear export of UL54 and interacted with UL54, suggesting that UL54 shuttles between the nucleus and the cytoplasm via a TAP/NXF1, but not CRM1, dependent nuclear export pathway. Furthermore, UL54 was demonstrated to target to the nucleus through a classic Ran-, importin β1- and α5-dependent nuclear import mechanism.
Protein & cell, 2012
Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more s... more Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.
Virologica Sinica, 2010
As one of the immediate-early (IE) proteins of herpes simplex virus type 1 (HSV-1), ICP22 is a mu... more 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.
Virologica Sinica, 2010
The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only parti... more The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.
Virology, 2010
The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion... more The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 131 PRPR 134 NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.
Virologica Sinica, 2010
Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious d... more Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.