Epstein-Barr virus (EBV) is the pathogenic factor of numerous human tumors, yet certain of its encoded proteins have not been studied. As a first step for functional identification, we presented the construction of a library of expression constructs for most of the EBV encoded proteins and an explicit subcellular localization map of 81 proteins encoded by EBV in mammalian cells. Viral open reading frames were fused with enhanced yellow fluorescent protein (EYFP) tag in eukaryotic expression plasmid then expressed in COS-7 live cells, and protein localizations were observed by fluorescence microscopy. As results, 34.57% (28 proteins) of all proteins showed pan-nuclear or subnuclear localization, 39.51% (32 proteins) exhibitted pan-cytoplasmic or subcytoplasmic localization, and 25.93% (21 proteins) were found in both the nucleus and cytoplasm. Interestingly, most envelope proteins presented pan-cytoplasmic or membranous localization, and most capsid proteins displayed enriched or complete localization in the nucleus, indicating that the subcellular localization of specific proteins are associated with their roles during viral replication. Taken together, the subcellular localization map of EBV proteins in live cells may lay the foundation for further illustrating the functions of EBV-encoded genes in human diseases especially in its relevant tumors.

Epstein‐Barr virus (EBV), a ubiquitous gammaherpesvirus, can regulate the antiviral response of NF‐κB signaling, which is critical for cell survival, growth transformation, and virus latency. Here, we showed that tegument protein BGLF2 could inhibit TNF‐α‐induced NF‐κB activity. BGLF2 was shown to interplay with the NF‐κB subunits p65 and p50, and the Rel homology domain of p65 was the pivotal region to interact with BGLF2. Nonetheless, BGLF2 did not influence the development of p65‐p50 dimerization. Yet, overexpression of BGLF2 inhibited the phosphorylation of p65 Ser536 (but not Ser276) and blocked the nuclear translocation of p65. In addition, knockdown of BGLF2 during EBV lytic replication elevated NF‐κB activity and the phosphorylation of p65 Ser536. Taken together, these results suggest that the inhibition of NF‐κB activation may serve as a strategy to escape the host's antiviral innate immunity to EBV during its lytic infection.—Chen, T., Wang, Y., Xu, Z., Zou, X., Wang, P., Ou, X., Li, Y., Peng, T., Chen, D., Li, M., Cai, M. Epstein‐Barr virus tegument protein BGLF2 inhibits NF‐κB activity by preventing p65 Ser536 phosphorylation. FASEB J. 33, 10563–10576 (2019). http://www.fasebj.org

Background/Aims: Epstein-Barr virus (EBV) BFLF2, the homologue of herpes simplex virus 1 (HSV-1) UL31, is crucial for the efficient viral DNA packaging and primary egress across the nuclear membrane. However, we still do not know its subcellular transport mechanisms. Methods: Interspecies heterokaryon assays were utilized to detect the nucleocytoplasmic shuttling of BFLF2, and mutation analysis, plasmid transfection and fluorescence microscopy assays were performed to identify the functional nuclear localization sequence (NLS) and nuclear export sequence (NES) of BFLF2 in live cells. Furthermore, the nuclear import and export of BFLF2 were assessed by confocal microscopy, co-immunoprecipitation and immunoblot assays. Results: BFLF2 was confirmed to shuttle between the nucleus and cytoplasm. Two predicted NESs were shown to be nonfunctional, yet we proved that the nuclear export of BFLF2 was mediated through transporter associated with antigen processing (TAP), but not chromosomal region maintenance 1 (CRM1) dependent pathway. Furthermore, one functional NLS, ²²RRLMHPHHRNYTASKASAH⁴⁰, was identified, and the aa22-23, aa22-25, aa28-30 and aa37-40 had an important role in the nuclear localization of BFLF2. Besides, the nuclear import of BFLF2 was demonstrated through Ran-, importin α7-, importin β1- and transportin-1-dependent mechanism that does not require importin α1, α3 and α5. Conclusion: These works are of significance for the further study of the functions of BFLF2 during EBV infection, as well as for further insights into the design of new antiviral drug target and vaccine development against EBV.

Viperin is an interferon-inducible protein that responsible for a variety of antiviral responses to different viruses. Our previous study has shown that the ribonuclease UL41 of herpes simplex virus 1 (HSV-1) can degrade the mRNA of viperin to promote HSV-1 replication. However, it is not clear whether other HSV-1 encoded proteins can regulate the function of viperin. Here, one novel viperin associated protein, glycoprotein D (gD), was identified. To verify the interaction between gD and viperin, gD and viperin expression plasmids were firstly co-transfected into COS-7 cells, and fluorescence microscope showed they co-localized at the perinuclear region, then this potential interaction was confirmed by co-immunoprecipitation (Co-IP) assays. Moreover, confocal microscopy demonstrated that gD and viperin co-localized at the Golgi body and lipid droplets. Furthermore, dual-luciferase reporter and Co-IP assays showed gD and viperin interaction leaded to the increase of IRF7-mediated IFN-β expression through promoting viperin and IRAK1 interaction and facilitating K63-linked IRAK1 polyubiquitination. Nevertheless, gD inhibited TRAF6-induced NF-κB activity by decreasing the interaction of viperin and TRAF6. In addition, gD restrained viperin-mediated interaction between IRAK1 and TRAF6. Eventually, gD and viperin interaction was corroborated to significantly inhibit the proliferation of HSV-1. Taken together, this study would open up new avenues toward delineating the function and physiological significance of gD and viperin during HSV-1 replication cycle.