Epstein-Barr Virus Transforming Protein LMP1 Plays a Critical Role in Virus Production

Abstract

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.

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“…In a previous study, lack of LMP1 expression severely impaired virus release into culture supernatants, which resulted in poor infection efficiency. These results have suggested that LMP1 serves an important role in EBV particle release from cells during the lytic cycle and in the infection of new host cells (46). Furthermore, a different study identified that low expression of LMP1 suppressed the activity of the EBV latent replication origin oriP, and that the LMP1 binding site for tumor necrosis factor receptor-associated factor was essential for this suppressive effect (49).…”

“…A number of studies have demonstrated that LMP1 serves an important role in the EBV lytic cycle (24,45,46). While LMP1 may be expressed in some states of EBV latency, significant induction of full-length LMP1 is frequently observed during virus reactivation into the lytic cycle (13,47,48).…”

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“…Culture supernatants were prepared from cell clone harboring d.LMP1-EBV [16] and used to infect peripheral B cells. The culture supernatant containing AK-BAC-GFP virus [15] was also used for another infection as a control.…”

“…We previously constructed a recombinant Akata strain EBV as AK-BAC-GFP that had a GFP gene in their genome 15 and d.LMP1-EBV by using AK-BAC-GFP as the starting material for mutagenesis [16] . This d.LMP1-EBV also expressed all viral genes except LMP1.…”

“…Previously established AK-BAC-GFP (recombinant EBV containing Bacterial artificial chromosome (BAC) and Green fluorescent protein (GFP) gene in their genome) [15] and d.LMP1-EBV (LMP1 knockout AK-BAC-GFP) [16] were maintained in RPMI 1640 medium (Sigma-Aldrich Fine Chemicals, St. Louis, Mo.) containing 10% fetal bovine serum at 37°C in a 5% humidified CO 2 atmosphere.…”