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Title: Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis}
Article:
Abstract:
Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus( PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence( TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.
Introduction:
Pseudorabies virus( PRV; suid herpesvirus 1) is a veterinary pathogen, widely used as a neuroanatomical tracing tool, and related to the human alpha herpesviruses varicella-zoster virus( VZV) and herpes simplex virus 1 and 2( HSV-1 &-2). Transport and egress of newly assembled alpha herpesvirus particles is a highly dynamic process involving viral components working in concert with host membrane transport systems. After capsid assembly and genome packaging in the nucleus, particles exit the nucleus by budding through the inner and outer nuclear membranes( reviewed in[1]). Viral membrane proteins are produced in the secretory pathway and traffic to the site of secondary envelopment, thought to be trans-Golgi[2]–[4] and/or endosomal membranes[5],[6]. Virus particles acquire their envelopes by budding into these membranes, producing an enveloped virion inside an intracellular vesicle. This virion transport vesicle then traffics to the plasma membrane, where the virion exits the infected cell by exocytosis. While this general description of viral egress is widely accepted, the specific mechanisms involved are not well studied. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final steps in PRV particle transport and exocytosis. This method takes advantage of total internal reflection fluorescence( TIRF) microscopy to selectively image particle dynamics near the plasma membrane, and a pH sensitive fluorescent probe that reveals the precise moment and location of exocytosis. We characterized particle movement by single-particle tracking and mean squared displacement( MSD) analysis. We found that particles are tightly confined at the plasma membrane before and after exocytosis, and undergo a sharp movement during the tens of milliseconds immediately after pHluorin dequenching. Previous studies in the HSV-1 literature sought to identify Rab proteins involved in alpha herpesvirus replication. Rab GTPases regulate essentially all intracellular membrane traffic. In their active GTP-bound form, Rab proteins bind to intracellular membranes, recruit molecular motors, and interact with a wide variety of effector proteins that mediate docking and fusion with target membranes[7]. The Elliott and Lippé laboratories screened siRNAs targeting Rab proteins[5],[6],[8], and Zenner et al. screened a panel of putative Rab GTPase activating proteins( RabGAPs)[9]. Collectively, these studies showed that many different Rab proteins – ER, Golgi, endocytic, and secretory Rabs – are involved in virus replication. However, a fundamental limitation of such loss-of-function screens is that it remains unclear whether these Rab proteins are involved directly in viral egress, or have other indirect or upstream effects. To resolve this ambiguity, we imaged Rab proteins in live cells to identify only those that are spatially and dynamically associated with viral exocytosis. We also investigated the plasma membrane sites of viral exocytosis, and found no evidence for specialized cytoskeleton-depleted exocytosis sites, contrary to a previous report on HSV-1[10]. However, we did observe that viral exocytosis events occur most frequently near plasma membrane patches of LL5β, part of a complex that anchors microtubules to the plasma membrane and regulates exocytosis. Finally, based on previous reports in the cell biological and alpha herpesvirus literature combined with our present findings, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.
Discussion:
Our work contradicts an earlier study that used TIRF microscopy to investigate the distribution of HSV-1 particles on the surface of infected cells[10]. The Brown laboratory imaged fixed cells primarily at 12 hpi, and did not directly observe individual virus exocytosis events. Rather, they observed plasma membrane invaginations containing many virus particles, which they inferred to be sites of virus egress. It is possible that these structures represent sites of preferential particle accumulation late in infection, and may form well after the actual particle exocytosis events. The authors reported that these structures are depleted of cytoskeleton elements, including actin and microtubules. However, the use of TIRF microscopy comes with an important caveat: the TIRF excitation field selectively excites fluorescent molecules within a few hundred nanometers of the coverslip, so only the adherent “footprint” of the cell can be observed. Since the plasma membrane invaginations observed by the Brown lab are up to a micrometer deep, it is likely that cytoskeleton proteins were outside of the TIRF excitation field, rather than biologically depleted. We observe virus particle exocytosis frequently occurs near LL5β complexes, as has also been reported for cell biological cargoes. Thus, our data favor a model in which virus egress sites are specified primarily by cellular mechanisms, rather than induced by viral processes. Rab6 is primarily associated with the Golgi and is present on post-Golgi secretory vesicles[24],[33], whereas Rab11 is typically, but not exclusively, associated with recycling endosomes[34]. Recent studies from the Elliott laboratory showed that siRNA knockdown of either Rab6 or Rab11 caused defects in secondary envelopment, resulting in a significant decrease in infectious titer( >100-fold or>10-fold reduction, respectively)[5],[6]. In their proposed model, viral glycoproteins traffic via the plasma membrane to recycling endosomes, which form the membrane of secondary envelopment. Accordingly, their model suggests two separate exocytosis routes in viral assembly and egress: viral glycoproteins take a Rab6-dependent route from the trans-Golgi to the plasma membrane, and assembled virions take a Rab11-dependent route from recycling endosomes to the plasma membrane. In contrast, we do not observe any such distinction: both Rab6 and Rab11 are associated with exocytosis of particles, and we confirmed that Rab6 is associated with exocytosis of assembled virions containing capsids( Figure 4). While Rab6 and Rab11 are associated with post-Golgi transport and recycling endosomes, respectively, Rab8 functions in both. Rab8 is associated with Golgi-derived secretory vesicles[35],[36] in combination with Rab6[23], but it is also associated with recycling endosomes in combination with Rab11[34]. Knockdown of Rab8 had a small effect, not statistically significant[6], thus it may be a minor player in these processes. Yet, in light of these overlapping functions, whether virus secretory vesicles are derived directly from trans-Golgi or recycling endosomes cannot be ascertained based on Rab proteins alone. Combining recent reports in the literature with our spatial and dynamic data on viral egress, we propose an integrated model of the molecular interactions that link the final transport and exocytosis steps during egress( Figure 7B): First, Rab proteins may promote intracellular transport by recruiting microtubule motors. Rab6a and Rab11a have been reported to interact with kinesins( kinesin-1 and rabkinesin-6) and dynein[7],[24]. Although viral membrane and tegument proteins also interact with microtubule motors[21],[37], siRNA knockdown of several individual motor proteins has little impact on production of infectious alpha herpesvirus[6]. It may be that these Rab proteins contribute to functional redundancy in motor recruitment, ensuring efficient intracellular transport of virus particles during egress( Figure 7B). Second, LL5β complexes anchor stabilized microtubules to the plasma membrane. Naghavi et al. recently reported that HSV-1 infection induces the formation of stabilized microtubules that run from the trans-Golgi to the plasma membrane[38]. These microtubules are regulated by microtubule plus-end tracking proteins( +TIPs), and are anchored to the plasma membrane by cytoplasmic linker associated proteins( CLASPs) through a complex with LL5β[39]. Naghavi et al. showed that siRNA knockdown of CLASPs impairs efficient alpha herpesvirus production and cell-cell spread[38]. Thus, stabilized microtubules, anchored at the plasma membrane by LL5β, may provide an efficient pathway linking the site of secondary envelopment to the site of egress( Figure 7B). Finally, after arrival to the plasma membrane, virus particle exocytosis occurs most frequently near patches of LL5β. The Akhmanova laboratory showed that this preferential exocytosis depends on molecular interactions between Rab6a and Rab8a with LL5β complexes, via ELKS/ERC1 and MICAL3, respectively[23],[24]. Importantly, Johns et al. showed that siRNA knockdown of ELKS/ERC1 impairs HSV-1 production 5–10 fold[6]. Thus, the LL5β complex plays a central role, linking intracellular transport to exocytosis during the final steps in alpha herpesvirus egress( Figure 7B). In this study, we investigated alpha herpesvirus egress in non-polarized epithelial cells that perform constitutive exocytosis. Initial alpha herpesvirus infections typically begin at epithelial cell surfaces, but infection subsequently spreads to polarized cells with highly specialized regulated secretory pathways. VZV infects T cells[40], which perform regulated exocytosis of cytokines and cytotoxic granules at the immunological synapse[41]. All alpha herpesviruses infect sensory and autonomic neurons[42]. Neurons have highly specialized secretory pathways, and exocytosis of neurotransmitters and neuropeptides is highly regulated at neuronal synapses. In addition to their role in constitutive exocytosis, LL5β complexes mediate regulated exocytosis at presynaptic active zones in neurons[43]. Rab3 and Rab27 are associated with regulated exocytosis in many cell types, including neurons and T cells[44], and are reported to be involved in HSV-1 transport and replication in neurons and oligodendrocytes, respectively[45],[46]. Despite these clues, the relationship between constitutive and regulated secretory mechanisms and alpha herpesvirus assembly and egress in these cell types is not known, and is an important topic for future studies.
Pseudorabies virus , an alpha herpesvirus , is an important veterinary pathogen , and related to human varicella-zoster virus and herpes simplex viruses . New alpha herpesvirus particles are assembled inside an infected cell , and must exit from the infected cell by taking advantage of cellular mechanisms . How these virus particles are transported inside the infected cell and secreted at the cell surface is not understood in great detail . In particular , how this process unfolds over time is not easily observed using previous methods . In this study , we developed a new method to observe this egress process . Using this method , we described how virus particles move on their way out: individual virus particles travel to the cell surface , directly to the exit site , where they pause for several seconds before crossing out of the cell . We identified several cellular proteins that are involved in this process . After exiting , virus particles remained stuck to the outer cell surface . Finally , we draw connections between our observations and other recent studies to propose an integrated model of how alpha herpesvirus particles exit from infected cells .