Honglin Chen - Academia.edu (original) (raw)

Papers by Honglin Chen

Research paper thumbnail of The K526R substitution in viral protein PB2 enhances the effects of E627K on influenza virus replication

Nature Communications, 2014

Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for rep... more Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for replication of avian influenza A viruses in mammalian hosts. Here we show that mutation PB2-K526R is present in some human H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesia and, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970. Polymerase complexes containing PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increased polymerase activity. PB2-526R also enhances viral transcription and replication in cells. In comparison with viruses carrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficiently in mammalian (but not avian) cells and in mouse lung tissues, and cause greater body weight loss and mortality in infected mice. PB2-K526R interacts with nuclear export protein and our results suggest that it contributes to enhance replication for certain influenza virus subtypes, particularly in combination with 627K.

Research paper thumbnail of Low dose inocula of SARS-CoV-2 Alpha variant transmits more efficiently than earlier variants in hamsters

Communications Biology, 2021

Emerging variants of SARS-CoV-2 have been shown to rapidly replace original circulating strains i... more Emerging variants of SARS-CoV-2 have been shown to rapidly replace original circulating strains in humans soon after they emerged. There is a lack of experimental evidence to explain how these natural occurring variants spread more efficiently than existing strains of SARS-CoV-2 in transmission. We found that the Alpha variant (B.1.1.7) increased competitive fitness over earlier parental D614G lineages in in-vitro and in-vivo systems. Using hamster transmission model, we further demonstrated that the Alpha variant is able to replicate and shed more efficiently in the nasal cavity of hamsters than other variants with low dose and short duration of exposure. The capability to initiate effective infection with low inocula may be one of the key factors leading to the rapid transmission of emerging variants of SARS-CoV-2.

Research paper thumbnail of Development and Evaluation of an Epstein-Barr Virus (EBV) Immunoglobulin M Enzyme-Linked Immunosorbent Assay Based on the 18-Kilodalton Matrix Protein for Diagnosis of Primary EBV Infection

Journal of Clinical Microbiology, 1998

A new immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) based on the recombinant E... more A new immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) based on the recombinant Epstein-Barr virus (EBV) matrix protein was developed. Compared to indirect immunofluorescence for the detection of IgM antibody to the EBV capsid antigen on clinical specimens, the sensitivity and specificity of the new IgM ELISA were 96 and 96%, respectively.

Research paper thumbnail of NF-κB Signaling Regulates Epstein-Barr Virus BamHI-Q-Driven EBNA1 Expression

Cancers, Jan 16, 2018

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by ... more Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the HI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, HI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, ...

Research paper thumbnail of Establishment and validation of a two-step screening scheme for improved performance of serological screening of nasopharyngeal carcinoma

Cancer medicine, Jan 25, 2018

Nasopharyngeal carcinoma (NPC), which is closely associated with Epstein-Barr virus (EBV), is one... more Nasopharyngeal carcinoma (NPC), which is closely associated with Epstein-Barr virus (EBV), is one of the most prevalent cancers in southeast China. Most NPC patients are diagnosed at late stage due to inconspicuous symptoms at the early stage, and the prognosis of these patients is poor. The early diagnosis rate of NPC could be significantly increased by serological screening, but the positive predictive value (PPV) is relatively low. A simple two-step serological screening scheme was established to improve the PPV of the screening strategy and was validated by a prospective cohort. Serum antibodies specific for EBNA1, Zta, Thymidine Kinase (TK), EAD, EAR, and VCA were detected by enzyme-linked immunosorbent assay. The combination of EBNA1/IgA and VCA/IgA was used in the first step of screening, and anti-early antigens (EAs) were used in the second step of screening. EAD/IgA was the most prominent marker in the second step of screening, and other anti-EAs were complementary to EAD/I...

Research paper thumbnail of Decreased expression of the NKG2D ligand ULBP4 may be an indicator of poor prognosis in patients with nasopharyngeal carcinoma

Oncotarget, Jan 31, 2017

U16-binding protein 4 (ULBP4), a human ligand for natural killer group 2, member D (NKG2D) recept... more U16-binding protein 4 (ULBP4), a human ligand for natural killer group 2, member D (NKG2D) receptor on NK cells and subsets of T cells, is thought to activate anticancer immune responses. However, the expression pattern and prognostic effect of ULBP4 in nasopharyngeal carcinoma (NPC) has not been investigated. We first compared ULBP4 expression between archival 15 NPC tissues and 8 normal nasopharynx (NP) tissues using qPCR. Then ULBP4 expression among 111 NPC specimens was validated on immunohistochemical examination. In addition, the association of ULBP4 expression with clinical characteristics and survival outcomes was analyzed. Furthermore, the impact of ULBP4 expression in NPC cells on the cytotoxic activity of NK cells was investigated. Both mRNA and protein ULBP4 expressions of NPC tissues were significantly lower than those in normal NP tissues. However, no association of ULBP4 expression with clinical characteristics was observed. Patients with NPC having decreased expressi...

Research paper thumbnail of NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma

Journal of Virology, 2016

Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high ... more Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinas...

Research paper thumbnail of CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells

The Journal of general virology, 2015

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9)... more The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep se...

Research paper thumbnail of Targeted Activation of Human Vγ9Vδ2-T Cells Controls Epstein-Barr Virus-Induced B Cell Lymphoproliferative Disease

Cancer Cell, 2014

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a ... more Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a serious and life-threatening complication. Herein we showed that the aminobisphosphonate pamidronate-expanded human Vg9Vd2-T cells efficiently killed EBV-transformed autologous lymphoblastoid B cell lines (EBV-LCL) through g/d-TCR and NKG2D receptor triggering and Fas and TRAIL engagement. By inoculation of EBV-LCL in Rag2 À/À gc À/À mice and humanized mice, we established lethal EBV-LPD with characteristics close to those of the human disease. Adoptive transfer of pamidronate-expanded Vg9Vd2-T cells alone effectively prevented EBV-LPD in Rag2 À/À gc À/À mice and induced EBV-LPD regression in EBV + tumor-bearing Rag2 À/À gc À/À mice. Pamidronate treatment inhibited EBV-LPD development in humanized mice through selective activation and expansion of Vg9Vd2-T cells. This study provides proof-of-principle for a therapeutic approach using pamidronate to control EBV-LPD through Vg9Vd2-T cell targeting. (C) Typical appearance of subcutaneous tumor in Rag2 À/À gc À/À mice from PBS-treated group at autopsy. (D, E, and I) Representative histological analysis, immunohistological stainings for human CD20, LMP1, Ki-67, and in situ hybridization for EBER-1/2 in formalinfixed paraffin-embedded sections from a tumor (D and I) and liver, kidney and lung in mice treated with PAM-expanded Vg9Vd2-T cells or PBS (E). Scale bar represents 100 mm.

Research paper thumbnail of Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses

Respiratory Research, 2007

Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. Th... more Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of α2,3-linked and α2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAα2,6Gal and Maackia amurensis agglutinin (MAA) for SAα2,3Gal in the respiratory tract of normal adults and children. Methods: We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers. Results: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the α2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs. Conclusion: The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAα2,3 binding viruses, such as influenza or human parainfluenza are to be made.

Research paper thumbnail of The Epstein–Barr virus latencyBamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss ofBamHI-Q promoter activity

Proceedings of the National Academy of Sciences, 1999

In Epstein–Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expressi... more In Epstein–Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expression is highly restricted. EBV-encoded nuclear antigen (EBNA)-1 is expressed, whereas the immunogenic and proliferative EBNAs are not. This pattern of EBNA expression is generated by usage of theBamHI-Q promoter (Qp). We have determined that the JAK/STAT pathway positively regulates Qp activity. In transient-transfection assays, a Qp–CAT reporter was activated by cotransfected JAK-1 and by treatment of cells with the cytokine IL-6. The ability of Qp to bind signal transducer and activator of transcription (STAT) proteins was directly demonstrated by electrophoretic mobility-shift assay, and mutation of potential STAT-binding sites reduced Qp responsiveness to Janus kinase (JAK)-1. Consistent with a role for STATs in Qp function, Qp using Burkitt’s lymphoma Rael cells and cultured nasopharyngeal carcinoma (NPC) cells contained nuclear STAT protein. We investigated whether the inability to m...

Research paper thumbnail of Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21 CIP-1 -mediated G 1 cell cycle arrest through CCAAT/enhancer-binding protein-α

Proceedings of the National Academy of Sciences, 2002

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma... more Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G 1 cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12–24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPα and p21 CIP-1 proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPα in vitro and stimulates C/EBPα-induced expression from both the C/EBPα and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPα and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G 1 to S cell cycle progression in the same cells. All...

Research paper thumbnail of Hypothesis: a novel route for immortalization of epithelial cells by Epstein-Barr virus

Research paper thumbnail of Regulation of Expression of the Epstein-Barr Virus BamHI-A Rightward Transcripts

Journal of Virology, 2005

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expre... more The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expressed in all EBV latency programs, including EBV-infected B cells in healthy carriers. Despite their ubiquitous expression, the regulation and biological function of BARTs are still unclear. In this study, the BART 5′ termini were characterized by using a procedure that selects capped, full-length mRNAs. Two TATA-less promoter regions, designated P1 and P2, were mapped. P1 had relatively high basal activity in both epithelial and B cells, whereas P2 exhibited higher activity in epithelial cells. Upon EBV infection of B cells, transcription from P1 was detected soon after infection, while expression from P2 was delayed. Promoter-reporter assays in transiently transfected cells revealed that P1 and P2 were differentially regulated. Interferon regulatory factor 7 (IRF7) and IRF5 negatively regulated P1 activity. c-Myc and C/EBP family members positively regulated P2. Regulation of P2 by C/EB...

Research paper thumbnail of Lytic Viral Replication as a Contributor to theDetection of Epstein-Barr Virus in BreastCancer

Journal of Virology, 2003

Epstein-Barr virus (EBV) has an accepted association with the epithelial malignancy nasopharyngea... more Epstein-Barr virus (EBV) has an accepted association with the epithelial malignancy nasopharyngeal carcinoma and has also been reported in other more controversial carcinoma settings. Evaluation of EBV association with epithelial carcinomas such as breast cancer would benefit from a better understanding of the outcome of EBV infection of these cells. Cell-free preparations of a green fluorescent protein-expressing virus, BX1, were used to infect breast cancer cell lines, which were then examined for EBV gene expression and viral genome copy number. Reverse transcription-PCR analyses revealed that the cells supported a mixture of latency II and lytic EBV gene expression. Lytic Zta and BMRF1 protein expression was detected by immunohistochemistry, and DNA PCR analyses estimated an EBV copy number of 300 to 600 genomes per infected cell. Evidence for lytic EBV expression was also found in breast tissue, where reverse transcription-PCR analyses detected lytic Zta transcripts in 7 of 10 ...

Research paper thumbnail of CCAAT/Enhancer Binding Protein α Interacts with ZTA and Mediates ZTA-Induced p21 CIP-1 Accumulation and G 1 Cell Cycle Arrest during the Epstein-Barr Virus Lytic Cycle

Journal of Virology, 2003

Cellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has anti... more Cellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G 1 /S through stabilization of p21 CIP-1 /WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G 1 /S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G 1 /S cell cycle arrest occurred in the majority of ZTA-positive cells, but not ...

Research paper thumbnail of Epstein-Barr Virus Bam HI-A Rightward Transcript-Encoded RPMS Protein Interacts with the CBF1-Associated Corepressor CIR To Negatively Regulate the Activity of EBNA2 and NotchIC

Journal of Virology, 2001

The Epstein-Barr virus (EBV) Bam HI-A rightward transcripts (BARTs) are expressed in all EBV-asso... more The Epstein-Barr virus (EBV) Bam HI-A rightward transcripts (BARTs) are expressed in all EBV-associated tumors as well as in latently infected B cells in vivo and cultured B-cell lines. One of the BART family transcripts contains an open reading frame, RPMS1, that encodes a nuclear protein termed RPMS. Reverse transcription-PCR analysis revealed that BART transcripts with the splicing pattern that generates the RPMS1 open reading frame are commonly expressed in EBV-positive lymphoblastoid cell lines and are also detected in Hodgkin's disease tissues. Experiments undertaken to determine the function of RPMS revealed that RPMS interacts with both CBF1 and components of the CBF1-associated corepressor complex. RPMS interaction with CBF1 was demonstrated in a glutathione S -transferase (GST) affinity assay and by the ability of RPMS to alter the intracellular localization of a mutant CBF1. A Gal4-RPMS fusion protein mediated transcriptional repression, suggesting an additional inter...

Research paper thumbnail of Cell Cycle Arrest by Kaposi's Sarcoma-Associated Herpesvirus Replication-Associated Protein Is Mediated at both the Transcriptional and Posttranslational Levels by Binding to CCAAT/Enhancer-Binding Protein α and p21 CIP-1

Journal of Virology, 2003

Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes... more Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G 1 cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPα and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPα and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBPα. Cotransfection of RAP with C/EBPα cooperatively stimulated both the C/EBPα and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPα, but both the N-terminal and C-terminal domains were required for transcript...

Research paper thumbnail of Contribution of C/EBP Proteins to Epstein-Barr Virus Lytic Gene Expression and Replication in Epithelial Cells

Journal of Virology, 2006

The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replicat... more The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPβ and had limited C/EBPα expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPα and C/EBPβ. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPβ expression and a prolonged increase in C/EBPα expression. In AGS/BX1 cells, endogenous C/EBPα and C/EBPβ proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPα and C/EBPβ proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs f...

Research paper thumbnail of A Positive Autoregulatory Loop of LMP1 Expression and STAT Activation in Epithelial Cells Latently Infected with Epstein-Barr Virus

Journal of Virology, 2003

ABSTRACTSTAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC... more ABSTRACTSTAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protei...

Research paper thumbnail of The K526R substitution in viral protein PB2 enhances the effects of E627K on influenza virus replication

Nature Communications, 2014

Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for rep... more Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for replication of avian influenza A viruses in mammalian hosts. Here we show that mutation PB2-K526R is present in some human H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesia and, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970. Polymerase complexes containing PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increased polymerase activity. PB2-526R also enhances viral transcription and replication in cells. In comparison with viruses carrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficiently in mammalian (but not avian) cells and in mouse lung tissues, and cause greater body weight loss and mortality in infected mice. PB2-K526R interacts with nuclear export protein and our results suggest that it contributes to enhance replication for certain influenza virus subtypes, particularly in combination with 627K.

Research paper thumbnail of Low dose inocula of SARS-CoV-2 Alpha variant transmits more efficiently than earlier variants in hamsters

Communications Biology, 2021

Emerging variants of SARS-CoV-2 have been shown to rapidly replace original circulating strains i... more Emerging variants of SARS-CoV-2 have been shown to rapidly replace original circulating strains in humans soon after they emerged. There is a lack of experimental evidence to explain how these natural occurring variants spread more efficiently than existing strains of SARS-CoV-2 in transmission. We found that the Alpha variant (B.1.1.7) increased competitive fitness over earlier parental D614G lineages in in-vitro and in-vivo systems. Using hamster transmission model, we further demonstrated that the Alpha variant is able to replicate and shed more efficiently in the nasal cavity of hamsters than other variants with low dose and short duration of exposure. The capability to initiate effective infection with low inocula may be one of the key factors leading to the rapid transmission of emerging variants of SARS-CoV-2.

Research paper thumbnail of Development and Evaluation of an Epstein-Barr Virus (EBV) Immunoglobulin M Enzyme-Linked Immunosorbent Assay Based on the 18-Kilodalton Matrix Protein for Diagnosis of Primary EBV Infection

Journal of Clinical Microbiology, 1998

A new immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) based on the recombinant E... more A new immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) based on the recombinant Epstein-Barr virus (EBV) matrix protein was developed. Compared to indirect immunofluorescence for the detection of IgM antibody to the EBV capsid antigen on clinical specimens, the sensitivity and specificity of the new IgM ELISA were 96 and 96%, respectively.

Research paper thumbnail of NF-κB Signaling Regulates Epstein-Barr Virus BamHI-Q-Driven EBNA1 Expression

Cancers, Jan 16, 2018

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by ... more Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the HI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, HI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, ...

Research paper thumbnail of Establishment and validation of a two-step screening scheme for improved performance of serological screening of nasopharyngeal carcinoma

Cancer medicine, Jan 25, 2018

Nasopharyngeal carcinoma (NPC), which is closely associated with Epstein-Barr virus (EBV), is one... more Nasopharyngeal carcinoma (NPC), which is closely associated with Epstein-Barr virus (EBV), is one of the most prevalent cancers in southeast China. Most NPC patients are diagnosed at late stage due to inconspicuous symptoms at the early stage, and the prognosis of these patients is poor. The early diagnosis rate of NPC could be significantly increased by serological screening, but the positive predictive value (PPV) is relatively low. A simple two-step serological screening scheme was established to improve the PPV of the screening strategy and was validated by a prospective cohort. Serum antibodies specific for EBNA1, Zta, Thymidine Kinase (TK), EAD, EAR, and VCA were detected by enzyme-linked immunosorbent assay. The combination of EBNA1/IgA and VCA/IgA was used in the first step of screening, and anti-early antigens (EAs) were used in the second step of screening. EAD/IgA was the most prominent marker in the second step of screening, and other anti-EAs were complementary to EAD/I...

Research paper thumbnail of Decreased expression of the NKG2D ligand ULBP4 may be an indicator of poor prognosis in patients with nasopharyngeal carcinoma

Oncotarget, Jan 31, 2017

U16-binding protein 4 (ULBP4), a human ligand for natural killer group 2, member D (NKG2D) recept... more U16-binding protein 4 (ULBP4), a human ligand for natural killer group 2, member D (NKG2D) receptor on NK cells and subsets of T cells, is thought to activate anticancer immune responses. However, the expression pattern and prognostic effect of ULBP4 in nasopharyngeal carcinoma (NPC) has not been investigated. We first compared ULBP4 expression between archival 15 NPC tissues and 8 normal nasopharynx (NP) tissues using qPCR. Then ULBP4 expression among 111 NPC specimens was validated on immunohistochemical examination. In addition, the association of ULBP4 expression with clinical characteristics and survival outcomes was analyzed. Furthermore, the impact of ULBP4 expression in NPC cells on the cytotoxic activity of NK cells was investigated. Both mRNA and protein ULBP4 expressions of NPC tissues were significantly lower than those in normal NP tissues. However, no association of ULBP4 expression with clinical characteristics was observed. Patients with NPC having decreased expressi...

Research paper thumbnail of NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma

Journal of Virology, 2016

Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high ... more Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinas...

Research paper thumbnail of CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells

The Journal of general virology, 2015

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9)... more The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep se...

Research paper thumbnail of Targeted Activation of Human Vγ9Vδ2-T Cells Controls Epstein-Barr Virus-Induced B Cell Lymphoproliferative Disease

Cancer Cell, 2014

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a ... more Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a serious and life-threatening complication. Herein we showed that the aminobisphosphonate pamidronate-expanded human Vg9Vd2-T cells efficiently killed EBV-transformed autologous lymphoblastoid B cell lines (EBV-LCL) through g/d-TCR and NKG2D receptor triggering and Fas and TRAIL engagement. By inoculation of EBV-LCL in Rag2 À/À gc À/À mice and humanized mice, we established lethal EBV-LPD with characteristics close to those of the human disease. Adoptive transfer of pamidronate-expanded Vg9Vd2-T cells alone effectively prevented EBV-LPD in Rag2 À/À gc À/À mice and induced EBV-LPD regression in EBV + tumor-bearing Rag2 À/À gc À/À mice. Pamidronate treatment inhibited EBV-LPD development in humanized mice through selective activation and expansion of Vg9Vd2-T cells. This study provides proof-of-principle for a therapeutic approach using pamidronate to control EBV-LPD through Vg9Vd2-T cell targeting. (C) Typical appearance of subcutaneous tumor in Rag2 À/À gc À/À mice from PBS-treated group at autopsy. (D, E, and I) Representative histological analysis, immunohistological stainings for human CD20, LMP1, Ki-67, and in situ hybridization for EBER-1/2 in formalinfixed paraffin-embedded sections from a tumor (D and I) and liver, kidney and lung in mice treated with PAM-expanded Vg9Vd2-T cells or PBS (E). Scale bar represents 100 mm.

Research paper thumbnail of Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses

Respiratory Research, 2007

Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. Th... more Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of α2,3-linked and α2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAα2,6Gal and Maackia amurensis agglutinin (MAA) for SAα2,3Gal in the respiratory tract of normal adults and children. Methods: We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers. Results: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the α2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs. Conclusion: The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAα2,3 binding viruses, such as influenza or human parainfluenza are to be made.

Research paper thumbnail of The Epstein–Barr virus latencyBamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss ofBamHI-Q promoter activity

Proceedings of the National Academy of Sciences, 1999

In Epstein–Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expressi... more In Epstein–Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expression is highly restricted. EBV-encoded nuclear antigen (EBNA)-1 is expressed, whereas the immunogenic and proliferative EBNAs are not. This pattern of EBNA expression is generated by usage of theBamHI-Q promoter (Qp). We have determined that the JAK/STAT pathway positively regulates Qp activity. In transient-transfection assays, a Qp–CAT reporter was activated by cotransfected JAK-1 and by treatment of cells with the cytokine IL-6. The ability of Qp to bind signal transducer and activator of transcription (STAT) proteins was directly demonstrated by electrophoretic mobility-shift assay, and mutation of potential STAT-binding sites reduced Qp responsiveness to Janus kinase (JAK)-1. Consistent with a role for STATs in Qp function, Qp using Burkitt’s lymphoma Rael cells and cultured nasopharyngeal carcinoma (NPC) cells contained nuclear STAT protein. We investigated whether the inability to m...

Research paper thumbnail of Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21 CIP-1 -mediated G 1 cell cycle arrest through CCAAT/enhancer-binding protein-α

Proceedings of the National Academy of Sciences, 2002

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma... more Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G 1 cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12–24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPα and p21 CIP-1 proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPα in vitro and stimulates C/EBPα-induced expression from both the C/EBPα and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPα and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G 1 to S cell cycle progression in the same cells. All...

Research paper thumbnail of Hypothesis: a novel route for immortalization of epithelial cells by Epstein-Barr virus

Research paper thumbnail of Regulation of Expression of the Epstein-Barr Virus BamHI-A Rightward Transcripts

Journal of Virology, 2005

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expre... more The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expressed in all EBV latency programs, including EBV-infected B cells in healthy carriers. Despite their ubiquitous expression, the regulation and biological function of BARTs are still unclear. In this study, the BART 5′ termini were characterized by using a procedure that selects capped, full-length mRNAs. Two TATA-less promoter regions, designated P1 and P2, were mapped. P1 had relatively high basal activity in both epithelial and B cells, whereas P2 exhibited higher activity in epithelial cells. Upon EBV infection of B cells, transcription from P1 was detected soon after infection, while expression from P2 was delayed. Promoter-reporter assays in transiently transfected cells revealed that P1 and P2 were differentially regulated. Interferon regulatory factor 7 (IRF7) and IRF5 negatively regulated P1 activity. c-Myc and C/EBP family members positively regulated P2. Regulation of P2 by C/EB...

Research paper thumbnail of Lytic Viral Replication as a Contributor to theDetection of Epstein-Barr Virus in BreastCancer

Journal of Virology, 2003

Epstein-Barr virus (EBV) has an accepted association with the epithelial malignancy nasopharyngea... more Epstein-Barr virus (EBV) has an accepted association with the epithelial malignancy nasopharyngeal carcinoma and has also been reported in other more controversial carcinoma settings. Evaluation of EBV association with epithelial carcinomas such as breast cancer would benefit from a better understanding of the outcome of EBV infection of these cells. Cell-free preparations of a green fluorescent protein-expressing virus, BX1, were used to infect breast cancer cell lines, which were then examined for EBV gene expression and viral genome copy number. Reverse transcription-PCR analyses revealed that the cells supported a mixture of latency II and lytic EBV gene expression. Lytic Zta and BMRF1 protein expression was detected by immunohistochemistry, and DNA PCR analyses estimated an EBV copy number of 300 to 600 genomes per infected cell. Evidence for lytic EBV expression was also found in breast tissue, where reverse transcription-PCR analyses detected lytic Zta transcripts in 7 of 10 ...

Research paper thumbnail of CCAAT/Enhancer Binding Protein α Interacts with ZTA and Mediates ZTA-Induced p21 CIP-1 Accumulation and G 1 Cell Cycle Arrest during the Epstein-Barr Virus Lytic Cycle

Journal of Virology, 2003

Cellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has anti... more Cellular CCAAT/enhancer binding protein α (C/EBPα) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G 1 /S through stabilization of p21 CIP-1 /WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G 1 /S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G 1 /S cell cycle arrest occurred in the majority of ZTA-positive cells, but not ...

Research paper thumbnail of Epstein-Barr Virus Bam HI-A Rightward Transcript-Encoded RPMS Protein Interacts with the CBF1-Associated Corepressor CIR To Negatively Regulate the Activity of EBNA2 and NotchIC

Journal of Virology, 2001

The Epstein-Barr virus (EBV) Bam HI-A rightward transcripts (BARTs) are expressed in all EBV-asso... more The Epstein-Barr virus (EBV) Bam HI-A rightward transcripts (BARTs) are expressed in all EBV-associated tumors as well as in latently infected B cells in vivo and cultured B-cell lines. One of the BART family transcripts contains an open reading frame, RPMS1, that encodes a nuclear protein termed RPMS. Reverse transcription-PCR analysis revealed that BART transcripts with the splicing pattern that generates the RPMS1 open reading frame are commonly expressed in EBV-positive lymphoblastoid cell lines and are also detected in Hodgkin's disease tissues. Experiments undertaken to determine the function of RPMS revealed that RPMS interacts with both CBF1 and components of the CBF1-associated corepressor complex. RPMS interaction with CBF1 was demonstrated in a glutathione S -transferase (GST) affinity assay and by the ability of RPMS to alter the intracellular localization of a mutant CBF1. A Gal4-RPMS fusion protein mediated transcriptional repression, suggesting an additional inter...

Research paper thumbnail of Cell Cycle Arrest by Kaposi's Sarcoma-Associated Herpesvirus Replication-Associated Protein Is Mediated at both the Transcriptional and Posttranslational Levels by Binding to CCAAT/Enhancer-Binding Protein α and p21 CIP-1

Journal of Virology, 2003

Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes... more Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G 1 cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPα and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPα and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBPα. Cotransfection of RAP with C/EBPα cooperatively stimulated both the C/EBPα and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPα, but both the N-terminal and C-terminal domains were required for transcript...

Research paper thumbnail of Contribution of C/EBP Proteins to Epstein-Barr Virus Lytic Gene Expression and Replication in Epithelial Cells

Journal of Virology, 2006

The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replicat... more The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPβ and had limited C/EBPα expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPα and C/EBPβ. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPβ expression and a prolonged increase in C/EBPα expression. In AGS/BX1 cells, endogenous C/EBPα and C/EBPβ proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPα and C/EBPβ proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs f...

Research paper thumbnail of A Positive Autoregulatory Loop of LMP1 Expression and STAT Activation in Epithelial Cells Latently Infected with Epstein-Barr Virus

Journal of Virology, 2003

ABSTRACTSTAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC... more ABSTRACTSTAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protei...