Settlage Sharon B, See Renee G, Hanley-Bowdoin Linda
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, 27695-7622, USA.
J Virol. 2005 Aug;79(15):9885-95. doi: 10.1128/JVI.79.15.9885-9895.2005.
Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants.
大多数感染双子叶植物的双生病毒编码一种复制增强蛋白（C3、AL3或REn），其小的单链DNA基因组的最佳复制需要该蛋白。C3与C1相互作用，C1是启动滚环复制的必需病毒复制蛋白。C3还能形成同型寡聚体，并与至少两种宿主编码蛋白相互作用，即增殖细胞核抗原（PCNA）和视网膜母细胞瘤相关蛋白（pRBR）。有人提出，蛋白质相互作用有助于C3发挥功能。利用番茄黄化曲叶病毒的C3蛋白，我们研究了C3蛋白家族中保守氨基酸的突变对复制增强和蛋白质相互作用的影响。令人惊讶的是，许多突变并不影响C3在烟草原生质体中的复制增强活性。其他突变要么增强了C3的复制活性，要么对其有害。在酵母双杂交试验中对突变蛋白的分析表明，使C3复制增强活性失活的突变也会降低或使C3寡聚化以及与C1和PCNA的相互作用失活。相比之下，与pRBR结合受损的突变C3蛋白在复制试验中功能完全正常。C3蛋白中部的疏水残基与C3自身、C1和PCNA的相互作用有关，而该蛋白N端和C端的极性残基对C3与pRBR的相互作用很重要。这些实验证实了C3-C3、C3-C1和C3-PCNA相互作用在双生病毒复制中的重要性。虽然C3与pRBR的相互作用在循环细胞中的病毒复制中不是必需的，但它可能在完整植物中分化细胞的感染过程中发挥作用。
