Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions.

IMPORTANCE Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious progeny particle production. Importantly, we successfully generated a BKRF4 knockout virus of Akata using CRISPR/Cas9 technology, confirming the phenotype in this separate strain. We further showed that BKRF4 interacted with another virion protein, BGLF2, and demonstrated the importance of this interaction in infectious virion production. These results shed light on the elusive process of EBV progeny maturation in the lytic cycle. Notably, this study describes a successful example of the generation and characterization of an EBV construct with a disrupted lytic gene using CRISPR/Cas9 technology.

人伽马疱疹病毒的成员爱泼斯坦-巴尔病毒（EBV）主要感染B细胞。EBV有两个备选生命周期，即潜伏期和裂解期，偶尔会从潜伏期到裂解期重新激活。为了抗击与EBV相关的疾病，了解EBV裂解复制周期的分子机制也很重要。在这里，我们集中于EBV裂解基因BKRF4。使用我们的抗BKRF4抗体，我们揭示了BKRF4基因产物在裂解周期中具有后期动力学表达。为了表征BKRF4的作用，我们使用细菌人工染色体（BAC）和CRISPR / Cas9系统构建了BKRF4-敲除突变体。尽管BKRF4基因的破坏对病毒蛋白表达和DNA合成几乎没有影响，但它显着降低了HEK293和Akata细胞的子代病毒体水平。此外，我们表明，BKRF4不仅参与子代病毒体的生产，还参与增加病毒颗粒的感染性。免疫沉淀测定表明BKRF4与病毒体蛋白BGLF2相互作用。我们表明，BKRF4的C末端区域对于这种相互作用和有效的后代生产至关重要。免疫荧光分析表明，BKRF4与BGLF2在核和核周区域中部分共定位。最后，我们表明BKRF4是一种磷酸化的可能的外皮蛋白，而EBV蛋白激酶BGLF4对于这种磷酸化可能很重要。两者合计，我们的数据表明BKRF4参与感染性病毒粒子的生产。免疫沉淀测定表明BKRF4与病毒体蛋白BGLF2相互作用。我们表明，BKRF4的C末端区域对于这种相互作用和有效的后代生产至关重要。免疫荧光分析显示，BKRF4与BGLF2在核和核周区域中部分共定位。最后，我们表明BKRF4是一种磷酸化的可能的外皮蛋白，而EBV蛋白激酶BGLF4对于这种磷酸化可能很重要。两者合计，我们的数据表明BKRF4参与感染性病毒粒子的生产。免疫沉淀测定表明BKRF4与病毒体蛋白BGLF2相互作用。我们表明，BKRF4的C末端区域对于这种相互作用和有效的后代生产至关重要。免疫荧光分析显示，BKRF4与BGLF2在核和核周区域中部分共定位。最后，我们表明BKRF4是一种磷酸化的可能的外皮蛋白，而EBV蛋白激酶BGLF4对于这种磷酸化可能很重要。两者合计，我们的数据表明BKRF4参与感染性病毒粒子的生产。免疫荧光分析显示，BKRF4与BGLF2在核和核周区域中部分共定位。最后，我们表明BKRF4是一种磷酸化的可能的外皮蛋白，而EBV蛋白激酶BGLF4对于这种磷酸化可能很重要。两者合计，我们的数据表明BKRF4参与感染性病毒粒子的生产。免疫荧光分析显示，BKRF4与BGLF2在核和核周区域中部分共定位。最后，我们表明BKRF4是一种磷酸化的可能的外皮蛋白，而EBV蛋白激酶BGLF4对于这种磷酸化可能很重要。两者合计，我们的数据表明BKRF4参与感染性病毒粒子的生产。

重要性尽管已经广泛研究了EBV的潜伏基因，但裂解基因的表征却欠佳。这项研究的重点是这样一种裂解基因BKRF4，该基因仅在伽马疱疹病毒（卡波西氏肉瘤相关疱疹病毒或鼠疱疹病毒68的ORF45）中保守。使用B95-8 EBV-BAC制备BKRF4基因敲除病毒后，我们证明BKRF4基因参与了感染性子代颗粒的产生。重要的是，我们使用CRISPR / Cas9技术成功产生了Akata的BKRF4基因敲除病毒，确认了该单独菌株中的表型。我们进一步表明，BKRF4与另一种病毒体蛋白BGLF2相互作用，并证明了这种相互作用在感染性病毒体生产中的重要性。这些结果揭示了EBV后代在裂解周期中难以捉摸的过程。尤其，