Acid Trip: Zika Virus Goes Off-Pathway during pH-Triggered Membrane Fusion

Abstract

Viral entry is a major target of efforts to find inhibitors against viral pathogens. Target-based approaches require detailed understanding of the entry mechanism on a molecular level. However, the lack of critical knowledge, such as the identity of the host membrane receptor, can significantly hamper progress. Such is the case for Zika virus (Figure 1 ), a human pathogen that received limited attention clinically and scientifically until a recent epidemic and whose host receptor is poorly defined. In this issue of ACS Central Science, Boxer et al. address this limitation using their distinctive in vitro model system, which enables quantitative analysis of viral membrane fusion events even in the absence of host receptor. 1 Complementary DNA–lipid conjugates bypass the need for viral–receptor interactions by bringing apposing membranes into stable close proximity (Figure 2 ). A pH drop mimics endosome acidification and induces an active, fusion-competent state of the virus. Thus, the system recapitulates crucial aspects of viral membrane fusion while permitting single-particle analysis and experimental control over components and conditions. Figure 1 Zika virus. Illustration by David S. Goodsell. 2 The team of Boxer and Kasson previously established the so-called “receptor-free” system using influenza and validated many crucial factors and assumptions. 3 In particular, at least for influenza, the rate-limiting step in pH-induced membrane fusion is independent of host receptor binding, confirming that long-lived proximity mediated by DNA oligonucleotide annealing is sufficient to capture relevant parameters of the membrane fusion process. In the current study, the authors build on this assumption to provide mechanistic insights into Zika virus membrane fusion. Importantly, low pH and membrane proximity were also sufficient to induce Zika virus fusion, allowing detailed investigation of the process as a function of pH. Single-particle analysis enabled the authors to draw a distinction between fusion rate versus fusion efficiency, defined as the extent of virus–vesicle fusion across the entire population of bound virus. Although fusion efficiencies correlated strongly with pH, fusion rates were largely pH-independent. Studies with related viruses have shown that activation of E protein, a viral surface protein and crucial mediator of membrane fusion, is pH-dependent; this is mirrored in pH-dependent conformations of Zika E protein. 4 Thus, the current observations indicate that E protein activation is not rate-limiting for Zika membrane fusion. Another novel finding was a low but observable level of fusion at neutral pH, which suggests that Zika virus could fuse in the early endosome. This would contrast with fusion in related viruses, such as Dengue, which are primarily active in the late endosome. 5 Using chemical kinetic modeling, the authors confirmed that a reversible, pH-dependent activation step followed by an irreversible pH-independent fusion step accurately described the observed rates of fusion. However, this model was not consistent with the observed fusion efficiencies. Accurate modeling required, at minimum, the inclusion of a pH-independent, off-pathway state that competes with the equilibrium between bound and activated states (Figure 2 ). More sophisticated cellular automaton modeling, which includes factors such as protein distribution and activation, led to the same conclusion. While little experimental information is available for Zika virus, previous studies on the related West Nile virus suggest that an off-pathway state could be related to inactivation of the viral E protein following extension and insertion into the host membrane. 6 Figure 2 Key features of viral–host membrane fusion are captured by a “receptor-free” in vitro model system. Target vesicles (bottom) represent host cell membranes. The virus and target vesicle bear complementary oligonucleotide DNA–lipid conjugates that mediate stable close proximity between membranes in the absence of host receptors. Unbound virus is washed away, so the only initial state is bound (B). A subsequent pH drop mimics endosome acidification that induces an active, fusion-competent state of the virus (A). An increase in fluorescence indicates membrane fusion (F) as dye–lipid conjugates at high concentration in the virus dequench. A microfluidics setup and tethering to the solid support enable rapid reagent and buffer exchange and quantitative imaging of single particles. Unexpectedly, analysis of Zika virus fusion by Rawle et al. revealed fusion rates and efficiencies that require, at minimum, a pH-independent, off-pathway state (O). (Full membrane fusion is depicted, but is not distinguishable from hemifusion in this system.) The success of this and the earlier influenza study encourages the application of the DNA tethering approach to other biological processes that depend on bringing membranes into close proximity, but have eluded mechanistic study because some components are unknown or difficult to reconstitute, such as the fusion of exosomes to the plasma or the endosomal membrane. Dissecting Zika and influenza fusion reactions benefitted from the ability to decouple binding from fusion using the pH drop. Other membrane fusion processes, including those of other viruses, may lack a clear dependence on experimentally accessible factors, which would limit the extent to which modeled parameters could be assigned to distinct mechanistic steps. Nevertheless, this latest study further underscores viral fusion as a rich area for exploration with this approach. Introducing variables such as membrane lipid composition and the presence and concentration of host receptors will uncover the contribution of these additional factors to fusion for this and other enveloped viruses. Also, exploration of various viruses even in the receptor-free setting will provide well-controlled comparisons and test the assumption that results can be extrapolated between individual related viruses, such as among the flaviviruses to which Zika belongs. Identification of the off-pathway state is an unexpected result uniquely enabled by access to fusion rates and efficiencies. A caveat of the model is that the existence of an off-pathway state suggests that fusion efficiency will depend on the length of time between the binding and acidification steps, but this was not observed. Nevertheless, this initial observation has intriguing implications. The model provides a modified framework for interpreting, for example, structures of different E protein conformations and their relevance to the fusion reaction. Further down the road, the relationship of the off-pathway state to viral inactivation, which may be irreversible, could present an avenue of exploration for antiviral therapies. Acid trips tend to have negative connotations, but discovering that Zika virus goes off-pathway could prove advantageous for human health.