de Oliveira Anna Paula, Glauser Daniel L, Laimbacher Andrea S, Strasser Regina, Schraner Elisabeth M, Wild Peter, Ziegler Urs, Breakefield Xandra O, Ackermann Mathias, Fraefel Cornel
Institute of Virology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland.
J Virol. 2008 May;82(10):4974-90. doi: 10.1128/JVI.02431-07. Epub 2008 Mar 12.
We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.
我们构建了一种重组1型单纯疱疹病毒（HSV-1），它同时编码来自病毒体三个区室（衣壳、被膜和包膜）的选定结构蛋白，并与自发荧光蛋白融合。这种三荧光重组体rHSV-RYC具有复制能力，尽管动力学延迟，它将融合蛋白整合到病毒体的所有三个区室中，并且在超微结构水平上与野生型HSV-1相当。衣壳融合蛋白VP26（单体红色荧光蛋白[mRFP]-VP26）最初在整个细胞核中被观察到，随后在病毒复制区室中积累。在感染过程中，mRFP-VP26在复制区室的周边形成小焦点，随着时间的推移，这些小焦点扩大并合并成大得多的焦点。包膜糖蛋白H（gH）融合蛋白（增强型黄色荧光蛋白[EYFP]-gH）最初在细胞质中以囊泡模式积累，然后主要整合到核膜中。被膜融合蛋白VP16（VP16-增强型青色荧光蛋白[ECFP]）最初以弥漫性核模式被观察到，然后在病毒复制区室中积累。此外，它还在复制区室的周边形成小焦点，然而，这些小焦点与小的mRFP-VP26焦点不共定位。后来，VP16-ECFP从细胞核重新分布到细胞质中，在那里它积累在囊泡焦点和核周簇中，类似于高尔基体。在感染后期，发现mRFP-VP26、EYFP-gH和VP16-ECFP在质膜上的点中共定位，可能代表成熟的子代病毒。总之，这项研究为衣壳、被膜和包膜蛋白之间的区室化和相互作用动力学提供了新的见解。类似的策略也可用于评估病毒生命周期中的其他动态事件，如进入和运输。
