E pstein-Barr virus (EBV), first discovered in association withBurkitt's lymphoma (27), is linked to a variety of human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, gastric carcinoma, and posttransplant lymphoproliferative disease (105). EBV infection results in either lytic replication or the establishment of viral latency. Both latent and lytic EBV gene products have been implicated in the development of cancer (28,51,72,105). EBV can be reactivated from latency by various reagents, such as 5-bromodeoxyuridine (39, 46), phorbol esters (110), anti-Ig antibodies (23,92,97), sodium butyrate (71), methotrexate (28), bortezomib (33, 34), thapsigargin (88, 95), and arsenic trioxide (89). The transition from latency to lytic replication is mediated by two EBV immediate-early genes, BZLF1 and BRLF1. The encoded proteins, ZTA and RTA, function as transcriptional activators that regulate the expression of EBV lytic cycle genes and lytic viral DNA replication (16,21,22,31,36,69,70,83,86,96). The lytic induction of EBV has been postulated as a therapeutic strategy for the treatment of virus-associated tumors (29,30,33,77).The small ubiquitin-related modifier (SUMO) was first identified as a posttranslational modifier of RanGAP1 (73,75). Similarly to the ubiquitination pathway, SUMOylation involves a series of sequential enzymatic reactions. The SUMO precursor protein is first cleaved by sentrin-specific proteases (SENPs) to generate a C-terminal diglycine motif. This then forms an E1ϳSUMO thioester, which is transferred to the E2-conjugating enzyme UBC9. E2ϳSUMO directly transfers SUMO to the substrate at lysine residues to form an isopeptide bond. E3 SUMO protein ligases facilitate this process by recruiting E2ϳSUMO to specific substrates and by enhancing the transfer process. SUMOylated targets can be de-SUMOylated by the SENP removal of SUMO (37). SUMOylation has been implicated in a variety of cellular processes, including transcriptional regulation, cell cycle regulation, signal transduction, the DNA damage response (DDR), and the regulation of protein-protein interactions (38). Both latent and lytic EBV proteins interact with the SUMO system. EBNA3C is SUMOylated (84), while LMP1 modulates the SUMOylation processes by interaction with UBC9 (6). SUMOylation regulates the transcriptional activity of ZTA and RTA (10,13,14,45,47,80). Noncovalent SUMO-protein interactions can also occur through a SUMO interaction motif (SIM) in the target proteins (3,57,90,91,93). EBNA3C contains a SIM motif and upregulates EBNA2-mediated gene activation by binding to a SUMOylated protein (84).In this study, we used an EBV protein microarray to identify additional EBV proteins that bind to SUMO. One of the identified proteins was the conserved protein kinase BGLF4. BGLF4 is present in the virion and expressed at an early stage of the lytic cycle (40,41,99). BGLF4 phosphorylates multiple EBV proteins, including BMRF1 (18, 42), EBNA2 (106), EBNA-LP (55), ZTA (4), EBNA1, and virion proteins (108). BGLF4 also phosp...

The protein array provided a sensitive platform for global screening; identified new, frequently recognized viral antigens; and revealed a broader humoral response to EBV compared with KSHV in the same patients.

1747 Individuals with HIV infection have an increased susceptibility to EBV- and KSHV-associated malignancies. Evaluating a possible role for specific humoral responses to individual viral proteins in the control of viral load and prevention of EBV or KSHV related disease development in this population has been hampered by the limited number of viral antigens currently used for screening serum samples. We have developed a protein array platform that can be utilized for EBV and KSHV serological screening. The array consists of EBV and KSHV proteins printed onto glass slides. All eighty four EBV open reading frames and eighty six KSHV open reading frames were cloned into bacterial vectors and the inserts validated by DNA sequencing in both directions. Authenticated inserts were transferred to a yeast vector that expresses proteins as N-terminal GST-fusions. Seventy nine EBV proteins and eighty one KSHV proteins were successfully purified from yeast. First generation EBV/KSHV protein arrays have been printed with these proteins plus a variety of control proteins that include GST, and human IgG, IgM and IgA. Initial serological assays compared IgG antibody responses in HIV positive individuals, cancer patients and healthy adults. The HIV positive group (i) had a broader EBV antigenic response than the cancer patients or healthy normal's and (ii) recognized a wider range of EBV antigens compared to KSHV. This may reflect more extensive exposure to replicating EBV versus KSHV in these patients. In addition to the previously described dominant antigenic responses to EBV and KSHV, the assays identified multiple additional immunogenic viral proteins. The ability to compare serological responses to the complete repertoire of individual EBV and KSHV encoded proteins should provide new insight into B cell mediated immune regulation of these viruses. Disclosures: No relevant conflicts of interest to declare.

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