Butler-Cole Christine, Wagner Mary J, Da Silva Melissa, Brown Gordon D, Burke Robert D, Upton Chris
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.
Virol J. 2007 Jul 24;4:76. doi: 10.1186/1743-422X-4-76.
Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins.
Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed.
These results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton.
肌动蛋白结合蛋白对真核生物的细胞骨架动力学至关重要；然而，对其病毒对应物却知之甚少。在本研究中，通过多种实验和生物信息学技术对痘病毒肌动蛋白结合蛋白同源物，即埃可病毒莫斯科株基因141（ECTV-PH）进行了研究，以表征其与细胞和病毒蛋白的相互作用。
迄今为止，所有已测序的正痘病毒均编码肌动蛋白结合蛋白样蛋白，且氨基酸（aa）同源性超过90%。序列比较显示与哺乳动物1型肌动蛋白结合蛋白相似度最高；然而，哺乳动物3型肌动蛋白结合蛋白和痘病毒肌动蛋白结合蛋白中一个保守的3个氨基酸缺失表明，这些同源物可能关系更为密切。结构分析表明，ECTV-PH可以成功地模拟到肌动蛋白结合蛋白1晶体结构和肌动蛋白结合蛋白3同源模型上，尽管被认为是结合肌动蛋白、聚（L-脯氨酸）和磷脂酰肌醇-4,5-二磷酸（PIP2）所必需的表面残基很少保守。免疫沉淀和质谱鉴定出两种在感染细胞内与ECTV-PH相互作用的蛋白：α-原肌球蛋白，一种38 kDa的细胞肌动蛋白结合蛋白，以及痘苗病毒西储株（VACV-WR）148的84 kDa产物，它是正痘病毒A型包涵体（ATI）蛋白的截短型VACV对应物。蛋白质免疫印迹和Far-Western印迹表明与α-原肌球蛋白的相互作用是直接的，免疫荧光实验表明ECTV-PH和α-原肌球蛋白可能共定位于类似于肌动蛋白尾和细胞突起的结构。痘病毒ATI蛋白的序列比较表明，虽然全长直系同源物仅存在于牛痘病毒和埃可病毒中，但约700个氨基酸的截短型ATI蛋白在超过90%的已测序正痘病毒中保守。免疫荧光研究表明，ECTV-PH定位于由病毒ATI蛋白的截短型和全长型形成的细胞质包涵体。此外，可以观察到ECTV-PH和截短型ATI蛋白共定位于细胞表面的突起。
这些结果表明ECTV-PH在病毒蛋白的细胞内运输或病毒的细胞间传播中起作用。更广泛的意义包括更好地理解病毒-宿主关系以及细胞组织和控制肌动蛋白细胞骨架的机制。
