BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex To Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins

Abstract

BGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin ␤ was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha-and betaherpesviruses. IMPORTANCEDuring lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

“…(iii) ORF36 might regulate the nuclear translocation of virions. Recently, it was reported that BGLF4 induces the nuclear redistribution of several EBV lytic proteins, including the major capsid protein, VCA (ORF25) (53). Interestingly, the same study found that KSHV ORF36 was also capable of inducing the nuclear translocation of VCA.…”

The human cytomegalovirus nuclear egress complex unites multiple functions: Recruitment of effectors, nuclear envelope rearrangement, and docking to nuclear capsids

“…Moreover, alternative mechanisms of NEC inhibition could be similarly effective, eg, the inhibitory targeting of NEC phosphorylation. Considering the fact that most if not all of the NEC‐associated proteins undergo phosphorylation, inhibitors of NEC phosphorylation appear likewise attractive for use in antiviral applications . In addition, considering the various structural changes predicted or already proven as a prerequisite for NEC assembly and down‐stream functions, small molecules interfering with conformational switches might be attractive for further development.…”

“…113,114 During herpesviral replication, the nuclear lamina is locally distorted, mainly on the basis of a disassembly of lamins A/C induced by the site-specific phosphorylation at Ser22 (in part also Ser392). A number of laminphosphorylating protein kinase activities have been described, including cyclin-dependent kinase (CDK) 1, protein kinase C (PKC), protein TABLE 1 Herpesviral core NEC proteins and NEC-associated proteins Alpha-herpesviruses Beta-herpesviruses Gamma-herpesviruses [78][79][80][81][82][83][84][85][86][87] HSV kinase B, and CDK-like enzymes, including herpesviral protein kinases. 111,113 In addition, the association of further proteins leads to a remodeling and functional alteration of the NE through fine-regulated processes.…”

The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-Mediated Nuclear Envelope Modification for Epstein-Barr Virus Maturation

“…In addition, EBV-encoded BGLF4 is a Ser/Thr kinase that can mimic cyclin-dependent kinase 1 to induce several prophase-like phenomena, such as chromosome condensation and partial disassembly of the nuclear lamina, for the nuclear egress of viral nu-cleocapsids (1). BGLF4 also modulates the transport preference of NPCs for the nuclear import of viral components (2).…”