Protein Array Identification of Substrates of the Epstein-Barr Virus Protein Kinase BGLF4

A conserved family of herpesvirus protein kinases plays a crucial role in herpesvirus DNA replication and virion production. However, despite the fact that these kinases are potential therapeutic targets, no systematic studies have been performed to identify their substrates. We generated an Epstein-Barr virus (EBV) protein array to evaluate the targets of the EBV protein kinase BGLF4. Multiple proteins involved in EBV lytic DNA replication and virion assembly were identified as previously unrecognized substrates for BGLF4, illustrating the broad role played by this protein kinase. Approximately half of the BGLF4 targets were also in vitro substrates for the cellular kinase CDK1/cyclin B. Unexpectedly, EBNA1 was identified as a substrate and binding partner of BGLF4. EBNA1 is essential for replication and maintenance of the episomal EBV genome during latency. BGLF4 did not prevent EBNA1 binding to sites in the EBV latency origin of replication, oriP. Rather, we found that BGLF4 was recruited by EBNA1 to oriP in cells transfected with an oriP vector and BGLF4 and in lytically induced EBV-positive Akata cells. In cells transfected with an oriP vector, the presence of BGLF4 led to more rapid loss of the episomal DNA, and this was dependent on BGLF4 kinase activity. Similarly, expression of doxycycline-inducible BGLF4 in Akata cells led to a reduction in episomal EBV genomes. We propose that BGLF4 contributes to effective EBV lytic cycle progression, not only through phosphorylation of EBV lytic DNA replication and virion proteins, but also by interfering with the EBNA1 replication function.Herpesviruses encode two families of serine/threonine protein kinases, one of which, the BGLF4 (Epstein-Barr virus [EBV])/UL97 (human cytomegalovirus)/UL13 (herpes simplex virus)/ORF36 (Kaposi''s sarcoma-associated herpesvirus)/ORF47 (varicella-zoster virus) family, is the sole protein kinase encoded by beta and gamma herpesviruses. The protein kinases phosphorylate both viral and host proteins (16, 21, 42) and are necessary for efficient virus lytic replication. Consequently, these kinases have been of interest as potential targets for antiviral drug development (37), and the compound 1263W94 (maribavir), which inhibits the cytomegalovirus UL97 protein (3), has been used in phase I clinical trials (27, 31, 47).EBV infection is prevalent worldwide, and primary infection in adolescence or early adulthood is associated in 30 to 40% of cases with infectious mononucleosis. EBV efficiently infects B cells in the lymphoid tissues of the Waldeyer ring (43). EBV infection of B cells is biased toward establishment of latency with limited viral-gene expression (49). During latent infection, EBV genomes are maintained as extrachromosomal episomes. Replication of episomal genomes utilizes the latency origin of replication, oriP. The only EBV-encoded protein required is the origin binding protein EBNA1. All other essential replication factors are provided by the cell. Expression of the EBV replicative cycle and production of progeny virus take place in terminally differentiated plasma B cells (11, 29), and epithelial cells may also contribute to the cycle of virus replication and spread that is an important component of both persistent infection of the individual and transmission of virus from one individual to the next (4, 22). Lytic DNA replication initiates at separate origins, oriLyt. EBV encodes a set of six core lytic replication proteins, along with ancillary proteins, such as thymidine kinase (TK), that are involved in nucleotide metabolism (13, 44).Several substrates have been described for the EBV BGLF4 protein kinase, including the core lytic EBV replication protein BMRF1, the polymerase processivity factor (8, 17). BGLF4 has also been found to locate to sites of lytic viral replication (46), to be required for efficient lytic DNA replication and release of nucleocapsids from the nucleus (18), and to contribute to the compaction of cell chromatin seen in cells undergoing lytic replication (32). Protein chip technology provides a new tool for global analysis of activities for biologically important enzymes, such as ubiquitin ligases, DNA repair enzymes, and kinases (7, 19, 36, 38, 52). Using an EBV protein array for unbiased screening, we identified multiple new BGLF4 substrates involved in lytic DNA replication, capsid assembly, and DNA packaging. Unexpectedly, we also identified EBNA1 as a substrate and binding partner for BGLF4. The data suggest that the contribution of BGLF4 to the EBV lytic cycle extends beyond the previously recognized contributions to lytic DNA replication and virion production and includes facilitating the switch from latent to lytic DNA replication by downregulating the EBNA1 replication function.     

Open Reading Frame 33 of a Gammaherpesvirus Encodes a Tegument Protein Essential for Virion Morphogenesis and Egress

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi''s sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.Gammaherpesviruses are associated with tumorigenesis. Like other herpesviruses, they are characterized as having two distinct stages in their life cycle: lytic replication and latency (15, 16, 18, 21, 54). Latency provides the viruses with advantages to escape host immune surveillance and to establish lifelong persistent infection and contributes to transformation and development of malignancies. However, it is through lytic replication that viruses propagate and transmit among hosts to maintain viral reservoirs. Both viral latency and lytic replication play important roles in tumorigenesis. The gammaherpesvirus subfamily includes Epstein-Barr virus (EBV), Kaposi''s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 and murine gammaherpesvirus 68 (MHV-68), among others. EBV is associated with Burkitt''s lymphoma, nasopharyngeal carcinoma, Hodgkin''s disease, and lymphoproliferative diseases in immunodeficient patients (28). KSHV is etiologically linked with Kaposi''s sarcoma, primary effusion lymphoma, and multicentric Castleman''s disease (11-13, 22, 52). Neither in vivo nor in vitro studies of EBV and KSHV are convenient due to their propensity to establish latency in cell culture and their limited host ranges.MHV-68 is genetically related to these two human gammaherpesviruses, especially to KSHV, based on the alignment of their genomic sequences and other biological properties (55). As a natural pathogen of wild rodents, MHV-68 also infects laboratory mice (6, 40, 46) and replicates to a high titer in a variety of fibroblast and epithelial cell lines. These advantages make MHV-68 an excellent model for studying the lytic replication of gammaherpesviruses in vitro and certain aspects of virus-host interactions in vivo. In addition, the MHV-68 genome has been cloned as a bacterial artificial chromosome (BAC) that can propagate in Escherichia coli (1, 2, 36, 51), making it convenient to study the function of each open reading frame (ORF) by genetic methods. Exploring the functions of MHV-68 ORFs will likely shed light on the functions of their homologues in human gammaherpesviruses.Gammaherpesviral particles have a characteristic multilayered architecture. An infectious virion contains a double-stranded DNA genome, an icosahedral capsid shell, a thick, proteinaceous tegument compartment, and a lipid bilayer envelope spiked with glycoproteins (14, 30, 47, 49). As a unique structure of herpesviruses, the tegument plays important roles in multiple aspects of the viral life cycle, including virion assembly and egress (38, 48, 53), translocation of nucleocapsids into the nucleus, transactivation of viral immediate-early genes, and modulation of host cell gene expression, innate immunity, and signal transduction (9, 10, 23, 60). Some components of MHV-68 tegument have been identified by a mass spectrometric study (8), and the functions of some tegument proteins have been revealed, such as ORF45, ORF52, and ORF75c (7, 24, 29).MHV-68 ORF33 is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. Its homologues include human herpes simplex virus type 1 (HSV-1) UL16, human herpes simplex virus type 2 (HSV-2) UL16, human cytomegalovirus (HCMV) UL94, EBV BGLF2, KSHV ORF33, and rhesus monkey rhadinovirus (RRV) ORF33. HSV-1 UL16 has been identified as a tegument protein and may function in viral DNA packaging, virion assembly, budding, and egress (5, 32, 35, 41, 44). HCMV UL94 is a virion associated protein and might function in virion assembly and budding (31, 57). EBV BGLF2, KSHV ORF33, and RRV ORF33 are also virion-associated proteins, but their functions are not clear (26, 43, 59). The mass spectrometric study of MHV-68 did not identify ORF33 as a virion component (8), although ORF33 is found to be essential for viral lytic replication by transposon mutagenesis of the MHV-68 genome cloned as a BAC (51). However, insertion of the 1.2-kbp Mu transposon in that study may influence the expression of ORFs approximate to ORF33. Consequently, the role ORF33 plays in viral replication needs to be confirmed, preferably through site-directed mutagenesis. Whether ORF33 is a tegument protein and the exact viral replication stage in which it functions also need to be investigated.We determined that MHV-68 ORF33 encodes a tegument protein and is expressed with true late kinetics. To explore the function of ORF33 in viral lytic phase, we used site-directed mutagenesis and generated an ORF33-null mutant, taking advantage of the MHV-68 BAC system. We showed that the ORF33-null mutant is capable of viral DNA replication, early and late gene expression, capsid assembly, and DNA packaging, but incapable of virion release. The defect of ORF33-null mutant can be rescued in trans by an ORF33 expression plasmid.