Gretchen L. Bentz

Human Cytomegalovirus Induces Monocyte Differentiation and Migration as a Strategy for Dissemination and Persistence

ABSTRACT Human cytomegalovirus (HCMV) pathogenesis is characterized by multiple organ system involvement due to viral spread to host organs after a cell-associated viremia. The cell type responsible for HCMV dissemination is unknown. Monocytes are the most likely candidate since they are the predominant cell type infected in the blood. However, monocytes are not productive for viral replication and are abortively infected. The results presented here provide a potential answer to this conundrum. We report that primary HCMV infection of monocytes induces transendothelial migration and monocyte-to-macrophage differentiation and that these HCMV-differentiated macrophages are productive for viral replication. Together, our data suggest a novel mechanism for HCMV pathogenesis; HCMV induces cellular changes in monocytes to promote viral replication and spread to host organs.

Human Cytomegalovirus (HCMV) Infection of Endothelial Cells Promotes Naïve Monocyte Extravasation and Transfer of Productive Virus To Enhance Hematogenous Dissemination of HCMV

ABSTRACT Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of naïve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased naïve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.

Human CMV infection of endothelial cells induces an angiogenic response through viral binding to EGF receptor and β1 and β3 integrins

Human cytomegalovirus (HCMV) infection is associated with atherosclerosis, transplant vascular sclerosis, and coronary restenosis. A common theme in these vascular diseases is an increased rate of angiogenesis. Angiogenesis is a complex biological process mediated by endothelial cell (EC) proliferation, migration, and morphogenesis. Although angiogenesis is a normal process in the host, its dysregulation, after viral infection or injury to the vessel wall, is associated with plaque development in atherosclerotic patients. We now document that HCMV infection results in increased EC proliferation, motility, and capillary tube formation. The observed HCMV-induced angiogenic response depended on viral binding to and signaling through the β1 and β3 integrins and the epidermal growth factor receptor, via their ability to activate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase signaling pathways. Because a proangiogenic response drives the neovascularization observed in atherosclerotic disease, our findings identify a possible mechanism for how HCMV infection contributes to vascular disease.

TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1

ABSTRACT We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6−/− mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1s ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IκB kinase ε. In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor.

PI3K-Dependent Upregulation of Mcl-1 by Human Cytomegalovirus Is Mediated by Epidermal Growth Factor Receptor and Inhibits Apoptosis in Short-Lived Monocytes

Monocytes are a primary target for human CMV (HCMV) infection and are a key cell type responsible for hematogenous dissemination of the virus. Biologically, these cells have a short lifespan of 1–3 d in the circulation, yet infected cells remain viable for weeks despite the lack of viral antiapoptotic gene expression during this period. To understand the mechanism by which HCMV inhibits the initial phase of monocyte apoptosis, we focused on the viral modulation of early prosurvival cell signaling events after infection. We demonstrate in this study that the viral upregulation of the PI3K pathway promotes an early block in apoptosis after infection. Temporal transcriptome and protein analyses revealed Mcl-1, a member of the Bcl-2 family, was transiently induced in a PI3K-dependent manner during the early stages of HCMV infection. In accord with the survival studies, virally induced levels of Mcl-1 expression dissipated to mock levels by 72 h postinfection. Through the use of Mcl-1–specific small interfering RNA, we confirmed the functional role that Mcl-1 plays as a key early regulator of apoptosis in monocytes. Lastly, we showed that HCMV engagement and activation of the epidermal growth factor receptor during viral binding triggered the upregulation of Mcl-1. Overall, our data indicates that activation of the epidermal growth factor receptor/PI3K signaling pathway, via the PI3K-dependent upregulation of Mcl-1, is required to circumvent apoptosis in naturally short-lived monocytes during the early stages of HCMV infection, thus ensuring the early steps in the viral persistence strategy.

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 BRLF1 Inhibits Transcription of IRF3 and IRF7 and Suppresses Induction of Interferon-β

Activation of interferon regulatory factors (IRFs) 3 and 7 is essential for the induction of Type I interferons (IFN) and innate antiviral responses, and herpesviruses have evolved mechanisms to evade such responses. We previously reported that Epstein-Barr virus BZLF1, an immediate-early (IE) protein, inhibits the function of IRF7, but the role of BRLF1, the other IE transactivator, in IRF regulation has not been examined. We now show that BRLF1 expression decreased induction of IFN-beta, and reduced expression of IRF3 and IRF7; effects were dependent on N- and C-terminal regions of BRLF1 and its nuclear localization signal. Endogenous IRF3 and IRF7 RNA and protein levels were also decreased during cytolytic EBV infection. Finally, production of IFN-beta was decreased during lytic EBV infection and was associated with increased susceptibility to superinfection with Sendai virus. These data suggest a new role for BRLF1 with the ability to evade host innate immune responses.

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.

Epstein-Barr Virus Latent Membrane Protein 1 Regulates the Function of Interferon Regulatory Factor 7 by Inducing Its Sumoylation

ABSTRACT Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) induces multiple signal transduction pathways during latent EBV infection via its C-terminal activating region 1 (CTAR1), CTAR2, and the less-studied CTAR3. One mechanism by which LMP1 regulates cellular activation is through the induction of protein posttranslational modifications, including phosphorylation and ubiquitination. We recently documented that LMP1 induces a third major protein modification by physically interacting with the SUMO-conjugating enzyme Ubc9 through CTAR3 and inducing the sumoylation of cellular proteins in latently infected cells. We have now identified a specific target of LMP1-induced sumoylation, interferon regulatory factor 7 (IRF7). We hypothesize that during EBV latency, LMP1 induces the sumoylation of IRF7, limiting its transcriptional activity and modulating the activation of innate immune responses. Our data show that endogenously sumoylated IRF7 is detected in latently infected EBV lymphoblastoid cell lines. LMP1 expression coincided with increased sumoylation of IRF7 in a CTAR3-dependent manner. Additional experiments show that LMP1 CTAR3-induced sumoylation regulates the expression and function of IRF7 by decreasing its turnover, increasing its nuclear retention, decreasing its DNA binding, and limiting its transcriptional activation. Finally, we identified that IRF7 is sumoylated at lysine 452. These data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling, leading to biologic effects. We propose that CTAR3 is an important signaling region of LMP1 that regulates protein function by sumoylation. We have shown specifically that LMP1 CTAR3, in cooperation with CTAR2, can limit the ability of IRF7 to induce innate immune responses by inducing the sumoylation of IRF7.

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.