England Matthew R, Purdy John G, Ropson Ira J, Dalessio Paula M, Craven Rebecca C
Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA.
J Virol. 2014 Jul;88(13):7170-7. doi: 10.1128/JVI.00309-14. Epub 2014 Apr 9.
During virion maturation, the Rous sarcoma virus (RSV) capsid protein is cleaved from the Gag protein as the proteolytic intermediate CA-SP. Further trimming at two C-terminal sites removes the spacer peptide (SP), producing the mature capsid proteins CA and CA-S. Abundant genetic and structural evidence shows that the SP plays a critical role in stabilizing hexameric Gag interactions that form immature particles. Freeing of CA-SP from Gag breaks immature interfaces and initiates the formation of mature capsids. The transient persistence of CA-SP in maturing virions and the identification of second-site mutations in SP that restore infectivity to maturation-defective mutant viruses led us to hypothesize that SP may play an important role in promoting the assembly of mature capsids. This study presents a biophysical and biochemical characterization of CA-SP and its assembly behavior. Our results confirm cryo-electron microscopy (cryo-EM) structures reported previously by Keller et al. (J. Virol. 87:13655-13664, 2013, doi:10.1128/JVI.01408-13) showing that monomeric CA-SP is fully capable of assembling into capsid-like structures identical to those formed by CA. Furthermore, SP confers aggressive assembly kinetics, which is suggestive of higher-affinity CA-SP interactions than observed with either of the mature capsid proteins. This aggressive assembly is largely independent of the SP amino acid sequence, but the formation of well-ordered particles is sensitive to the presence of the N-terminal β-hairpin. Additionally, CA-SP can nucleate the assembly of CA and CA-S. These results suggest a model in which CA-SP, once separated from the Gag lattice, can actively promote the interactions that form mature capsids and provide a nucleation point for mature capsid assembly.
The spacer peptide is a documented target for antiretroviral therapy. This study examines the biochemical and biophysical properties of CA-SP, an intermediate form of the retrovirus capsid protein. The results demonstrate a previously unrecognized activity of SP in promoting capsid assembly during maturation.
在病毒粒子成熟过程中，劳氏肉瘤病毒（RSV）衣壳蛋白作为蛋白水解中间体CA-SP从Gag蛋白上裂解下来。在两个C末端位点的进一步修剪去除了间隔肽（SP），产生成熟的衣壳蛋白CA和CA-S。大量的遗传和结构证据表明，SP在稳定形成未成熟颗粒的六聚体Gag相互作用中起关键作用。CA-SP从Gag中释放会破坏未成熟界面并启动成熟衣壳的形成。CA-SP在成熟病毒粒子中的短暂存在以及在SP中恢复成熟缺陷突变病毒感染性的第二位点突变的鉴定使我们推测SP可能在促进成熟衣壳的组装中起重要作用。本研究展示了CA-SP及其组装行为的生物物理和生化特征。我们的结果证实了Keller等人（《病毒学杂志》87:13655 - 13664，2013，doi:10.1128/JVI.01408 - 13）之前报道的冷冻电子显微镜（cryo-EM）结构，表明单体CA-SP完全能够组装成与CA形成的衣壳样结构相同的结构。此外，SP赋予了快速的组装动力学，这表明CA-SP相互作用的亲和力高于两种成熟衣壳蛋白中的任何一种。这种快速组装在很大程度上与SP氨基酸序列无关，但有序颗粒的形成对N末端β-发夹的存在敏感。此外，CA-SP可以引发CA和CA-S的组装。这些结果提出了一个模型，其中CA-SP一旦与Gag晶格分离，就可以积极促进形成成熟衣壳的相互作用，并为成熟衣壳组装提供成核点。
间隔肽是抗逆转录病毒疗法的一个已记录靶点。本研究研究了逆转录病毒衣壳蛋白的中间形式CA-SP的生化和生物物理性质。结果证明了SP在成熟过程中促进衣壳组装方面具有先前未被认识的活性。
