Nikhil Sharma - Academia.edu (original) (raw)

Papers by Nikhil Sharma

Research paper thumbnail of Epstein-Barr Virus Latent Antigen 3C Can Mediate the Degradation of the Retinoblastoma Protein through an SCF Cellular Ubiquitin Ligase

Proceedings of The National Academy of Sciences, 2005

Transfection. In general, 10 million human embryonic kidney (HEK) 293T, BJAB, or U2OS cells were ... more Transfection. In general, 10 million human embryonic kidney (HEK) 293T, BJAB, or U2OS cells were transfected by electroporation with a Bio-Rad Gene Pulser in 0.4-cm-gap cuvettes at 210-220 V and 975 microfarads. Unless otherwise indicated, transfected samples were harvested at 24 h, and SDS͞PAGE was performed with 5% of the total normalized protein lysate.

Research paper thumbnail of A Cyclin-Binding Motif within the Amino-Terminal Homology Domain of EBNA3C Binds Cyclin A and Modulates Cyclin A-Dependent Kinase Activity in Epstein-Barr Virus-Infected Cells

Journal of Virology, 2004

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essent... more The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the ␣1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. . It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.

Research paper thumbnail of SCFSkp2 Complex Targeted by Epstein-Barr Virus Essential Nuclear Antigen

Molecular and Cellular Biology, 2005

The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-pro... more The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCF Skp2 , known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCF Skp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCF Skp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCF Skp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCF Skp2 -dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCF Skp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.

Research paper thumbnail of Induction of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen by the Lytic Transactivator RTA: a Novel Mechanism for Establishment of Latency

Journal of Virology, 2005

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to developme... more Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein J (RBP-J), as a RBP-J-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-J. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.

Research paper thumbnail of A peptide-based inhibitor for prevention of B cell hyperproliferation induced by Epstein–Barr virus

Virology, 2006

The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-t... more The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-transplant lymphoproliferative disease causes significant complications in transplant recipients, including loss of the transplanted organ and even death. The limited treatment options include, nonspecific targeting of B cell surface antigens with monoclonal antibodies or withdrawal of immunosuppression. These therapies fail in ∼50% of patients. Clearly, treatments that specifically target EBV-infected cells are desirable. The EBV antigen EBNA3C regulates cell cycle by targeting critical cellular complexes such as cyclin A/cdk2, SCF Skp2 , and Rb. Here, we use a 20-amino-acid EBNA3C-derived peptide, fused to an HIV TAT tag for efficient delivery, to disrupt cell cycle regulation by EBNA3C. The peptide inhibited hyperproliferation of EBV-infected B cell lines and reduced in vitro immortalization of primary B lymphocytes by EBV. Importantly, the peptide inhibited lymphoblastoid outgrowth from the blood of an EBVpositive transplant patient in vitro.

Research paper thumbnail of Epstein-Barr Virus Latent Antigen 3C Can Mediate the Degradation of the Retinoblastoma Protein through an SCF Cellular Ubiquitin Ligase

Proceedings of The National Academy of Sciences, 2005

Transfection. In general, 10 million human embryonic kidney (HEK) 293T, BJAB, or U2OS cells were ... more Transfection. In general, 10 million human embryonic kidney (HEK) 293T, BJAB, or U2OS cells were transfected by electroporation with a Bio-Rad Gene Pulser in 0.4-cm-gap cuvettes at 210-220 V and 975 microfarads. Unless otherwise indicated, transfected samples were harvested at 24 h, and SDS͞PAGE was performed with 5% of the total normalized protein lysate.

Research paper thumbnail of A Cyclin-Binding Motif within the Amino-Terminal Homology Domain of EBNA3C Binds Cyclin A and Modulates Cyclin A-Dependent Kinase Activity in Epstein-Barr Virus-Infected Cells

Journal of Virology, 2004

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essent... more The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the ␣1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. . It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.

Research paper thumbnail of SCFSkp2 Complex Targeted by Epstein-Barr Virus Essential Nuclear Antigen

Molecular and Cellular Biology, 2005

The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-pro... more The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCF Skp2 , known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCF Skp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCF Skp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCF Skp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCF Skp2 -dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCF Skp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.

Research paper thumbnail of Induction of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen by the Lytic Transactivator RTA: a Novel Mechanism for Establishment of Latency

Journal of Virology, 2005

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to developme... more Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein J (RBP-J), as a RBP-J-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-J. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.

Research paper thumbnail of A peptide-based inhibitor for prevention of B cell hyperproliferation induced by Epstein–Barr virus

Virology, 2006

The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-t... more The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-transplant lymphoproliferative disease causes significant complications in transplant recipients, including loss of the transplanted organ and even death. The limited treatment options include, nonspecific targeting of B cell surface antigens with monoclonal antibodies or withdrawal of immunosuppression. These therapies fail in ∼50% of patients. Clearly, treatments that specifically target EBV-infected cells are desirable. The EBV antigen EBNA3C regulates cell cycle by targeting critical cellular complexes such as cyclin A/cdk2, SCF Skp2 , and Rb. Here, we use a 20-amino-acid EBNA3C-derived peptide, fused to an HIV TAT tag for efficient delivery, to disrupt cell cycle regulation by EBNA3C. The peptide inhibited hyperproliferation of EBV-infected B cell lines and reduced in vitro immortalization of primary B lymphocytes by EBV. Importantly, the peptide inhibited lymphoblastoid outgrowth from the blood of an EBVpositive transplant patient in vitro.