Abstract
Herpesvirus subfamilies typically acquire their final envelope in various cytoplasmic compartments such as the trans-Golgi network (TGN), and endosomes prior to their secretion into the extracellular space. However, the sites for the final envelopment of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, are poorly understood. Here, we characterized the sites for the final envelopment of EBV in Burkitt’s lymphoma cell lines induced into the lytic cycle by crosslinking cell surface IgG. Electron microscopy revealed the various stages of maturation and egress of progeny virions including mature EBV in irregular cytoplasmic vesicles. Immunofluorescence staining showed that gp350/220, the major EBV glycoprotein, and the viral capsid antigen, p18, efficiently colocalized with a cis-Golgi marker, GM130. gp350/220 partly colocalized with the TGN, which was distributed in a fragmented and dispersed pattern in the cells induced into the lytic cycle. In contrast, limited colocalization was observed between gp350/220 and endosomal markers, such as a multi-vesicular bodies marker, CD63, a recycling endosome marker, Rab11, and a regulatory secretion vesicles marker, Rab27a. Finally, we observed that treatment of cells with brefeldin A, an inhibitor of vesicle trafficking between the endoplasmic reticulum and Golgi apparatus, resulted in the perinuclear accumulation of gp350/220 and inhibition of its distribution to the plasma membrane. Brefeldin A also inhibited the release of infectious EBV. Taken together, our findings support a model in which EBV acquires its final envelope in intracellular compartments containing markers of Golgi apparatus, providing new insights into how EBV matures.
Highlights
Epstein–Barr virus (EBV), a ubiquitous human gammaherpesvirus, infects a majority of the population worldwide and establishes a persistent lifelong, mostly asymptomatic infection in them
Peng et al (2010) took advantage of electron tomography to characterize the ultrastructural basis of the MHV-68 lifecycle. They showed that tegumented capsids bud into vesicles located adjacent to Golgi apparatus to acquire a final envelope. These findings suggest that EBV shares the mechanism of virion maturation and egress with other herpesvirus subfamilies, the sites for its final envelopment have not been demonstrated because of the lack of a fully permissive culture model for the lytic cycle
To characterize the site for the final envelopment of EBV, the viral lytic cycle was induced by cross linking the cell surface IgG of Akata− EBV-eGFP by adding F(ab’)2 fragments of goat anti-human IgG polyclonal antibody (Takada, 1984)
Summary
Epstein–Barr virus (EBV), a ubiquitous human gammaherpesvirus, infects a majority of the population worldwide and establishes a persistent lifelong, mostly asymptomatic infection in them. EBV infection is associated with various lymphoid and epithelial malignancies such as Burkitt’s lymphoma (BL), Hodgkin’s disease, gastric carcinoma, and nasopharyngeal carcinoma (Longnecker et al, 2013). In latently infected cells the EBV genome is efficiently maintained extrachromosomally (Yates et al, 1996; Nanbo et al, 2007). Either spontaneously or exogenous induction initiates lytic infection, which leads to several 100-fold amplification of the viral genome within 1–2 days (Hammerschmidt and Sugden, 1988). Lytic DNA replication produces long concatemers (Bloss and Sugden, 1994), which eventually are cleaved and packaged as linear genome units in capsids. Replication of EBV DNA and its packaging occur in the nucleus
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