Christopher B. Whitehurst

The Epstein-Barr Virus (EBV) Deubiquitinating Enzyme BPLF1 Reduces EBV Ribonucleotide Reductase Activity

ABSTRACT A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first 246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1 requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect. These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.

Epstein-Barr Virus BPLF1 Deubiquitinates PCNA and Attenuates Polymerase η Recruitment to DNA Damage Sites

ABSTRACT PCNA is monoubiquitinated in response to DNA damage and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway, translesion synthesis (TLS). Since PCNA is reported to associate with Epstein-Barr virus (EBV) DNA during its replication, we investigated whether the EBV deubiquitinating (DUB) enzyme encoded by BPLF1 targets ubiquitinated PCNA and disrupts TLS. An N-terminal BPLF1 fragment (a BPLF1 construct containing the first 246 amino acids [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured cells. Moreover, monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 in vitro. BPLF1 1-246 dysregulated TLS by reducing recruitment of the specialized repair polymerase polymerase η (Polη) to the detergent-resistant chromatin compartment and virtually abolished localization of Polη to nuclear repair foci, both hallmarks of TLS. Expression of BPLF1 1-246 decreased viability of UV-treated cells and led to cell death, presumably through deubiquitination of PCNA and the inability to repair damaged DNA. Importantly, deubiquitination of PCNA could be detected endogenously in EBV-infected cells in comparison with samples expressing short hairpin RNA (shRNA) against BPLF1. Further, the specificity of the interaction between BPLF1 and PCNA was dependent upon a PCNA-interacting peptide (PIP) domain within the N-terminal region of BPLF1. Both DUB activity and PIP sequence are conserved in the members of the family Herpesviridae. Thus, deubiquitination of PCNA, normally deubiquitinated by cellular USP1, by the viral DUB can disrupt repair of DNA damage by compromising recruitment of TLS polymerase to stalled replication forks. PCNA is the first cellular target identified for BPLF1 and its deubiquitinating activity.

Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) C-Terminal-Activating Region 3 Contributes to LMP1-Mediated Cellular Migration via Its Interaction with Ubc9

ABSTRACT Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), the principal viral oncoprotein and a member of the tumor necrosis factor receptor superfamily, is a constitutively active membrane signaling protein that regulates multiple signal transduction pathways via its C-terminal-activating region 1 (CTAR1) and CTAR2, and also the less-studied CTAR3. Because protein sumoylation among other posttranslational modifications may regulate many signaling pathways induced by LMP1, we investigated whether during EBV latency LMP1 regulates sumoylation processes that control cellular activation and cellular responses. By immunoprecipitation experiments, we show that LMP1 interacts with Ubc9, the single reported SUMO-conjugating enzyme. Requirements for LMP1-Ubc9 interactions include enzymatically active Ubc9: expression of inactive Ubc9 (Ubc9 C93S) inhibited the LMP1-Ubc9 interaction. LMP1 CTAR3, but not CTAR1 and CTAR2, participated in the LMP1-Ubc9 interaction, and amino acid sequences found in CTAR3, including the JAK-interacting motif, contributed to this interaction. Furthermore, LMP1 expression coincided with increased sumoylation of cellular proteins, and disruption of the Ubc9-LMP1 CTAR3 interaction almost completely abrogated LMP1-induced protein sumoylation, suggesting that this interaction promotes the sumoylation of downstream targets. Additional consequences of the disruption of the LMP1 CTAR3-Ubc9 interaction revealed effects on cellular migration, a hallmark of oncogenesis. Together, these data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling and leads to biological effects. We propose that LMP1, by interaction with Ubc9, modulates sumoylation processes, which regulate signal transduction pathways that affect phenotypic changes associated with oncogenesis.

Maribavir Inhibits Epstein-Barr Virus Transcription in Addition to Viral DNA Replication

ABSTRACT Although many drugs inhibit the replication of Epstein-Barr virus (EBV) in cell culture systems, there is still no drug that is effective and approved for use in primary EBV infection. More recently, maribavir (MBV), an l-ribofuranoside benzimidazole, has been shown to be a potent and nontoxic inhibitor of EBV replication and to have a mode of action quite distinct from that of acyclic nucleoside analogs such as acyclovir (ACV) that is based primarily on MBVs ability to block the phosphorylation of target proteins by EBV and human cytomegalovirus protein kinases. However, since the antiviral mechanisms of the drug are complex, we have carried out a comprehensive analysis of the effects of MBV on the RNA expression levels of all EBV genes with a quantitative real-time reverse transcription-PCR-based array. We show that in comparisons with ACV, the RNA expression profiles produced by the two drugs are entirely different, with MBV causing a pronounced inhibition of multiple viral mRNAs and with ACV causing virtually none. The results emphasize the different modes of action of the two drugs and suggest that the action of MBV may be linked to indirect effects on the transcription of EBV genes through the interaction of BGLF4 with multiple viral proteins.

Location and Role of Free Cysteinyl Residues in the Sindbis Virus E1 and E2 Glycoproteins

ABSTRACT Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.

Knockout of Epstein-Barr Virus BPLF1 Retards B-Cell Transformation and Lymphoma Formation in Humanized Mice

ABSTRACT BPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 function in vivo. Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice. IMPORTANCE Epstein-Barr virus infects approximately 90% of the worlds population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBVs BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV. Epstein-Barr virus infects approximately 90% of the worlds population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBVs BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV.

The Rad6/18 Ubiquitin Complex Interacts with the Epstein-Barr Virus Deubiquitinating Enzyme, BPLF1, and Contributes to Virus Infectivity

ABSTRACT Epstein-Barr virus (EBV) encodes BPLF1, a lytic cycle protein with deubiquitinating activity that is contained in its N-terminal domain and conserved across the Herpesviridae. EBV replication is associated with cellular DNA replication and repair factors, and initiation of EBV lytic replication induces a DNA damage response, which can be regulated at least in part by BPLF1. The cellular DNA repair pathway, translesion synthesis (TLS), is disrupted by BPLF1, which deubiquitinates the DNA processivity factor, PCNA, and inhibits the recruitment of the TLS polymerase, polymerase eta (Pol eta), after damage to DNA by UV irradiation. Here we showed that the E3 ubiquitin ligase, which activates TLS repair by monoubiquitination of PCNA, is also affected by BPLF1 deubiquitinating activity. First, BPLF1 interacts directly with Rad18, and overexpression of BPLF1 results in increased levels of the Rad18 protein, suggesting that it stabilizes Rad18. Next, expression of functionally active BPLF1 caused relocalization of Rad18 into nuclear foci, which is consistent with sites of cellular DNA replication that occur during S phase. Also, levels of Rad18 remain constant during lytic reactivation of wild-type virus, but reactivation of BPLF1 knockout virus resulted in decreased levels of Rad18. Finally, the contribution of Rad18 levels to infectious virus production was examined with small interfering RNA (siRNA) targeting Rad18. Results demonstrated that reducing levels of Rad18 decreased production of infectious virus, and infectious titers of BPLF1 knockout virus were partially restored by overexpression of Rad18. Thus, BPLF1 interacts with and maintains Rad18 at high levels during lytic replication, which assists in production of infectious virus. IMPORTANCE Characterization of EBV BPLF1s deubiquitinating activity and identification of its targets and subsequent functional effects remain little studied. All members of the Herpesviridae contain BPLF1 homologs with conserved enzymatic activity, and findings discovered with EBV BPLF1 are likely applicable to other members of the family. Discovery of new targets of BPLF1 will point to cellular pathways and viral processes regulated by the enzymatic activity of the EBV-encoded deubiquitinating enzyme. Here we determined the importance of the cellular ubiquitin ligase Rad18 in these processes and how it is affected by BPLF1. Our findings demonstrate that EBV can co-opt Rad18 as a novel accessory factor in the production of infectious virus.

Maribavir Inhibits Epstein-Barr Virus Transcription through the EBV Protein Kinase

ABSTRACT Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus.

LMP1-Induced Sumoylation Influences the Maintenance of Epstein-Barr Virus Latency through KAP1

ABSTRACT As a herpesvirus, Epstein-Barr virus (EBV) establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme Ubc9 via its C-terminal activating region-3 (CTAR3) and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or leakier in EBV-transformed lymphoblastoid cell lines. The cells are also more sensitive to viral reactivation induced by irradiation, which results in the increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR3-mediated sumoylation regulates the function of KAP1. KAP1 also binds to EBV OriLyt and immediate early promoters in a CTAR3-dependent manner, and inhibition of sumoylation processes abrogates the binding of KAP1 to these promoters. These data provide an additional line of evidence that supports our findings that CTAR3 is a distinct functioning regulatory region of LMP1 and confirm that LMP1-induced sumoylation may help stabilize the maintenance of EBV latency. IMPORTANCE Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) plays an important role in the maintenance of viral latency. Previously, we documented that LMP1 targets cellular proteins to be modified by a ubiquitin-like protein (SUMO). We have now identified a function for this LMP1-induced modification of cellular proteins in the maintenance of EBV latency. Because latently infected cells have to undergo viral reactivation in order to be vulnerable to antiviral drugs, these findings identify a new way to increase the rate of EBV reactivation, which increases cell susceptibility to antiviral therapies.

The translesion polymerase pol η is required for efficient epstein-barr virus infectivity and is regulated by the viral deubiquitinating enzyme BPLF1

ABSTRACT Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the worlds population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkins lymphoma, Burkitts lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBVs BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production.