State Key Lab of Biocontrol, Sun Yat-sen University, Guangzhou, China.
State Key Lab of Biocontrol, Sun Yat-sen University, Guangzhou, China
J Virol. 2018 Apr 13;92(9). doi: 10.1128/JVI.01989-17. Print 2018 May 1.
Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene () interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene () and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in -deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly.
病毒基因组的包裹，导致核衣壳的组装形成感染性的子代病毒粒子，是许多病毒生命周期中的关键步骤。杆状病毒核衣壳的组装是一个复杂的过程，涉及许多蛋白质。我们之前的研究表明，核心基因 () 的缺失通过产生缺乏病毒基因组的衣壳鞘来中断核衣壳的组装，其机制尚不清楚。所有 38K 的同源物都含有参与磷酸转移的卤代酸脱卤酶超家族的保守基序。本研究证实了这些基序对核衣壳组装的要求，表明 38K 可能是一种功能性的卤代酸脱卤酶。P6.9 也是由核心基因 () 编码的，是病毒基因组包裹所必需的。据报道，在病毒感染的细胞中存在多种磷酸化形式的 P6.9，而在芽生病毒中仅检测到未磷酸化的形式。因此，研究了 38K 是否介导 P6.9 的去磷酸化。在 -缺失或 -突变病毒转染的细胞中检测到 P6.9 的另一种磷酸化形式，并通过质谱确定了 38K 介导的去磷酸化位点。为了评估 38K 介导的 P6.9 去磷酸化对病毒复制的影响，这些位点被突变为谷氨酸（磷酸化模拟突变体）或丙氨酸（磷酸化缺陷突变体）。研究表明，在磷酸化模拟突变体病毒转染的细胞中，核衣壳的组装被中断。总之，我们的研究结果表明，38K 介导 P6.9 的 C 末端特定位点的去磷酸化，这对于病毒基因组的包裹是必不可少的。基因组包装是病毒生命周期中的一个基本过程，病毒有不同的策略来完成这一步骤。对于一些双链 DNA (dsDNA) 病毒，在基因组包裹之前形成原衣壳，这可能需要碱性蛋白来帮助中和核酸电荷排斥，以促进基因组在有限的衣壳空间内的压缩。杆状病毒编码一种小的碱性蛋白 P6.9，它是病毒感染周期中多种过程所必需的。P6.9 的磷酸化被认为导致核衣壳脱壳，而 P6.9 的去磷酸化则参与核衣壳组装过程中的病毒 DNA 包裹。在这里，我们证明杆状病毒核心基因编码的卤代酸脱卤酶同源物通过介导 P6.9 的 C 末端 5 个特定位点的去磷酸化参与核衣壳组装。这一发现有助于理解病毒核衣壳组装的机制。
