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Abstract：
Kaposi\'s sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta- and gammaherpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gammaherpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the four cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components.
OBJECTIVE: The initiation of late gene transcription is universally conserved across the beta- and gammaherpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the four conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production to demonstrate for the first time that these cysteines are pivotal for such functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.
OBJECTIVE: The initiation of late gene transcription is universally conserved across the beta- and gammaherpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the four conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production to demonstrate for the first time that these cysteines are pivotal for such functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.
摘要：
卡波西肉瘤疱疹病毒（KSHV）ORF34作为病毒前起始复合物（vPIC）的组成部分发挥重要作用，这对于跨β-和γ-疱疹病毒的晚期基因表达是必不可少的。尽管已经认识到ORF34在vPIC中的关键作用及其作为hub蛋白的功能，需要进一步澄清其对vPIC功能的具体贡献以及与其他组件的交互。这项研究采用了ORF34的深度学习算法辅助结构模型，揭示了位于结构化域中的人类β-和γ疱疹病毒的高度保守的氨基酸残基。因此，我们通过用丙氨酸取代保守残基改造ORF34丙氨酸扫描突变体.评估这些突变体与其他vPIC因子相互作用并恢复携带ORF34缺陷型KSHV-BAC的细胞中的病毒产生的能力。我们的实验结果强调了ORF34中保守的四个半胱氨酸残基的关键作用：由一对C-Xn-C共有基序组成的四面体排列。这表明金属阳离子在与ORF24和ORF66vPIC组分相互作用中的潜在掺入，促进晚期基因转录，并通过捕获金属阳离子来促进整体病毒生产。总之,我们的发现强调了KSHVORF34中保守的半胱氨酸对于有效的vPIC组装和病毒复制的重要作用，从而增强我们对vPIC组件之间复杂相互作用的理解。
目的：晚期基因转录的起始在β-和γ-疱疹病毒家族中普遍保守。该过程采用病毒预起始复合物（vPIC），这类似于细胞PIC。尽管KSHVORF34是病毒复制的关键因素，并且是vPIC的组成部分，vPIC形成的细节和对其功能至关重要的基本结构域仍不清楚.结构预测表明,四个保守的半胱氨酸(C170、C175、C256和C259)形成与金属阳离子配位的四面体。我们研究了这些保守氨基酸在与其他vPIC成分相互作用中的作用，晚期基因表达，和病毒生产首次证明这些半胱氨酸对于这些功能是关键的。这一发现不仅加深了我们对ORF34和vPIC动力学的全面理解，而且为今后进一步研究疱疹病毒复制机制奠定了基础。
目的：晚期基因转录的起始在β-和γ-疱疹病毒家族中普遍保守。该过程采用病毒预起始复合物（vPIC），这类似于细胞PIC。尽管KSHVORF34是病毒复制的关键因素，并且是vPIC的组成部分，vPIC形成的细节和对其功能至关重要的基本结构域仍不清楚.结构预测表明,四个保守的半胱氨酸(C170、C175、C256和C259)形成与金属阳离子配位的四面体。我们研究了这些保守氨基酸在与其他vPIC成分相互作用中的作用，晚期基因表达，和病毒生产首次证明这些半胱氨酸对于这些功能是关键的。这一发现不仅加深了我们对ORF34和vPIC动力学的全面理解，而且为今后进一步研究疱疹病毒复制机制奠定了基础。
