Scherer Julian, Yaffe Zachary A, Vershinin Michael, Enquist Lynn W
Department of Molecular Biology and Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
Department of Physics and Astronomy, University of Utah, Salt Lake City, Utah, USA.
J Virol. 2016 Oct 14;90(21):9997-10006. doi: 10.1128/JVI.01122-16. Print 2016 Nov 1.
Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.
α疱疹病毒，如单纯疱疹病毒和伪狂犬病病毒（PRV），是具有神经侵袭性的双链DNA（dsDNA）病毒，可在其天然宿主的外周神经系统（PNS）神经元中建立终身潜伏感染。重新激活后，感染可扩散回最初的感染黏膜部位，或在极少数情况下扩散至中枢神经系统，通常会导致严重后果。在病毒进出细胞的过程中，病毒衣壳依赖基于微管的分子马达进行高效快速运输。在PNS神经元的轴突中，胞质动力蛋白为向胞体的逆行运动提供动力，而驱动蛋白则沿相反的顺行方向运输货物。通过将荧光蛋白与小衣壳蛋白VP26融合，可对细胞中病毒颗粒的动态特性进行成像，融合后的荧光蛋白会被整合到衣壳中。然而，单色荧光蛋白标签无法区分接种病毒和子代病毒。因此，我们构建了一个双色系统，方法是在表达绿色VP26融合蛋白（PK15-mNG-VP26）的稳定细胞系上培养表达红色荧光VP26融合蛋白的重组PRV（PRV180）。由此产生的双色病毒制剂（PRV180G）的衣壳同时带有红色和绿色荧光蛋白标签，且97%的病毒颗粒含有可检测水平的mNeonGreen（mNG）标记的VP26。在神经元细胞中复制后，所有PRV180G子代病毒只含有单体红色荧光蛋白（mRFP）-VP26标记的衣壳。我们使用PRV180G分析PNS神经元中轴突衣壳的运输动力学。快速双色全内反射荧光（TIRF）显微镜、单颗粒追踪和运动分析显示，在病毒进入细胞过程中，胞质动力蛋白和驱动蛋白介导衣壳进行强大的双向运动，而子代病毒颗粒仅由驱动蛋白运输至远端释放位点。α疱疹病毒是具有神经侵袭性的病毒，在其生命周期中会感染受感染宿主的外周神经系统（PNS）。在PNS神经元中建立静止或潜伏感染是大多数α疱疹病毒的一个标志。在自然宿主中，感染扩散至中枢神经系统的情况极为罕见，但可能是致命的。伪狂犬病病毒（PRV）是一种宿主范围广泛的猪α疱疹病毒，可进入神经元细胞并利用细胞内运输过程来建立感染并在细胞间传播。通过使用一种病毒制剂，其荧光病毒衣壳会根据感染周期的阶段改变颜色，我们发现，在病毒进入细胞过程中，PNS神经元的轴突支持衣壳进行强大的双向运动，类似于细胞货物向细胞体的运输。相比之下，子代病毒颗粒似乎由驱动蛋白单向运输至远端释放位点。
