Characterizing the dynamic structural changes that occur in the Lassa Virus GPC fusion machinery during viral entry and membrane fusion (Benhaim)

Significance and Specific Aims Lassa virus (LASV) is an old-world arenavirus and the causative agent of Lassa fever; a viral hemorrhagic fever endemic to West Africa [1, 2]. With an estimated mortality rate between 20-70% Lassa fever is responsible for a significant human and social impact every year [1, 2]. Despite being a significant human pathogen there are no approved vaccines against LASV and therapeutic interventions are limited [1-4]. Moreover, administration of antiviral therapeutics must be done within 6 days of infection and is often complicated by limited access to clinical care facilities and economic barriers [1, 4]. As with many human diseases endemic to the developing world, preventative immunization is the most practical and effective means of controlling and limiting LASV infection [1, 2, 4]. Past attempts at developing a vaccine against LASV have been relatively unsuccessful [1, 4].

The LASV glycoprotein complex (GPC) is the sole virally encoded antigen on the viral surface and the primary target for neutralizing antibodies and vaccine development [3-6]. Like other class 1 viral fusion proteins, GPC is a homotrimer of heterodimers with each monomer comprised of a GP1 receptor binding subunit and a GP2 fusion subunit [3, 7]. GPC is synthesized as a single polypeptide chain and is subsequently cleaved by host cellular proteases into the cleaved active GPC trimer. Recently the crystal structure for the pre-fusion state of the GPC ectodomain was solved in complex with neutralizing antibody fragments [6]. This structure revealed that the majority of neutralizing antibodies isolated from survivors of LASV infection bind to a quaternary epitope between two GPC protomers and are believed to inhibit conformational changes necessary for membrane fusion to occur [5, 6]. However, we lack an understanding of what those conformational changes are and the molecular mechanism of how LASV GPC mediates viral entry and membrane fusion [3, 8, 9]. In order to develop the most effective vaccine possible it is imperative that we understand the structure of LASV GPC and how changes in that structure enable it to function at each step of viral entry.

LASV GPC mediates entry into host cells through its receptor binding and membrane fusion activities. During infection GPC first binds the α-dystroglycan receptor on the cell surface triggering receptor mediated endocytosis and internalization of LASV [10, 11]. In the maturing endosome, as the pH of the endosomal lumen lowers to pH 6.0, GPC dissociates from the α-dystroglycan receptor and binds the LAMP1 receptor in a pH dependent manner [8, 12, 13]. GPC binding to α-dystroglycan can only occur at pH’s above 6.0, below which GPC can bind LAMP1 [3, 8, 9, 11-13]. A triad of histidine residues on GPC was identified as being critically important for GPC binding LAMP1 and are believed to function as pH sensors during GPC mediated fusion and prevent spurious and premature inactivation of GPC [8, 12, 13]. The LAMP1 bound GPC fusion protein is activated by low pH and undergoes a series of pH dependent conformational changes resulting in fusion between the viral and host membranes enabling the transfer of the viral replication machinery into the host cell [8, 12, 13]. LAMP1 binding is not required for fusion to occur, it does however raise the pH of activation for GPC and increases infectivity and membrane fusion efficiency (Figure 1)[8, 13, 14]. In the absence of LAMP1, GPC mediates fusion at pH’s >4.0, which is unusually low compared to other pH dependent viral fusion proteins and is correlated with low viral infectivity [14]. However, we do not understand how LAMP1 binding increases the pH of GPC activation. Cryo-electron tomographic reconstructions of full length GPC on vesicular stomatitis virus (VSV) virus like particles (VLPs) revealed significant morphological changes occurring throughout GPC between the neutral pH pre-fusion state and GPC at pH 5.5 with and without LAMP1 [8]. However, these models lacked sufficient resolution to fully appreciate and elucidate the structural changes that were occurring [8]. Structural characterization of membrane fusion proteins to high resolution is notoriously difficult as membrane fusion is a highly dynamic process that is not amenable to study using classical high resolution structural methods such as x-ray crystallography or cryo-electron microscopy [15]. Recently, solution state structural and biophysical methods have proven successful in the study of the molecular

Figure 1. Cell entry by Lassa virus can occur independently of LAMP1 binding. Binding to LAMP1 does however increases the pH of activation and results in increased infectivity. Hulesberg, 2018.

mechanism of influenza virus hemagglutinin membrane fusion [7, 16]. We propose to use structural mass spectrometry to characterize the dynamic and structural changes that occur throughout full length LASV GPC using VSV-VLPs displaying GPC during viral entry and during membrane fusion.

Aim 1. Characterize the dynamic structural changes that occur throughout LASV GPC during viral entry that enable membrane fusion to occur. We hypothesize that LAMP1 binding at pH 6.0 destabilizes the GPC trimer and primes it for fusion activation at elevated pH conditions. A combination of solution state structural and biophysical methods will be used to obtain a more complete structural understanding of the structural changes GPC experiences during receptor binding and their functional consequences. Hydrogen-deuterium exchange (HDX-MS) and x-ray foot printing (XRF) coupled to mass spectrometry will enable us to study the dynamic structural changes that occur throughout GPC during viral entry and receptor binding across all levels of structural organization with high structural resolution.

Aim 2. Determine the mechanism of LASV GPC fusion activation and membrane fusion. Using our established pulse labeling HDX-MS method we will monitor and characterize the conformational changes that occur throughout LASV GPC during fusion activation and membrane fusion using full length GPC displayed on VSV-VLPs with and without LAMP1 binding. Innovation

Innovative concept: The work proposed here aims to provide a complete structural characterization of the LASV GPC fusion protein during viral entry and receptor binding. By understanding how LASV GPC’s structure is changing, and the functional consequences of those changes, at each stage of viral entry we can hope to better understand how neutralizing antibodies inhibit these processes. Moreover, the LASV entry pathway is dissimilar from other viruses bearing class 1 fusion proteins and by understanding how they differ we can hope to better appreciate implications of viral diversity.

Innovative method: The combination of methods used here, namely continuous labeling HDX-MS and XRF, will result in a complete characterization of the structural changes that occur throughout GPC at all levels of structural organization. Our use of full length LASV GPC and VSV-VLPs will enable us to characterize these changes under native like conditions. Moreover, our pulse labeling HDX-MS method enables us to monitor a proteins conformational change with high structural and temporal resolution (Figure 2). To our knowledge there exists no other solution state method capable of achieving such results. Research Plan Aim 1. 1a. Identification of the optimal conditions for GPC binding to LAMP1. LASV GPC binds the LAMP1 receptor at pH’s below 6.0, however it is unclear what the optimal pH for binding is. Bio-layer interferometry (BLI) using an OCTET system will enable us to identify the optimal conditions for GPC binding to LAMP1. Soluble GPC will be immobilized and incubated in solutions containing LAMP1 at various pH conditions ranging from pH 6.0-5.0. Native PAGE gel shift assays will aid in determining the lowest pH condition where GPC bound to LAMP1 remains in the pre-fusion conformation. 1b. LAMP1 and pH induced dynamic structural changes in LASV GPC monitored by HDX-MS and XRF. VSV-VLPs displaying full length LASV GPC will be produced as previously described [8]. GPC on the surface of VSV-VLPs will be characterized and sequenced by MS2 and SDS-PAGE in order to confirm complete cleavage into GP1 and GP2. HDX-MS will be performed at the pH conditions determined by BLI for GPC-VSV-VLPs with and without LAMP1. Performing HDX-MS at low pH requires a modified exchange time course due to the pH dependence of the HDX reaction[7]. Matched XRF experiments will be performed at the Stanford Synchrotron Radiation Lightsource (SSRL) facility. Analysis of the HDX-MS and XRF data will inform on any changes in secondary structure, structural dynamics, and side chain accessibility as a result of LAMP1 binding to GPC. These methods are well established and ideally suited for a two-state comparison as proposed here [7, 15]. Aim 2. 2a. Determine optimal pH’s of fusion for GPC-VSV-VLPs with synthetic liposomes. Using well established in vitro fluorescence dequenching viral fusion assays we will determine the fusion efficiency for GPC-VSV-VLPs with and without LAMP1 across a range of pH conditions from pH 6.0-2.5 [17]. These experiments will enable us to select a comparable pH of activation for the subsequent pulse labeling HDX-MS experiments and inform on the timescale across which membrane fusion occurs.

Figure 2. Pulse labeling HDX-MS of IAV HA reveals the sequence of conformational changes that occur during membrane fusion.

2b. Determine the mechanism of GPC fusion activation. The pulse labeling HDX-MS method developed in our lab has been successfully used to determine the mechanism of influenza virus hemagglutinin membrane fusion and is well suited for the study of any pH dependent conformational change (Figure 2). GPC-VSV-VLPs (+/- LAMP1) will be rapidly acidified to the previously determined optimal conditions for fusion activation for varying amounts of time as determined in aim 2a. The pulse labeling reaction will serve to label the protein in a conformationally dependent manner. Analysis of each peptides HDX profile will reveal the local structural changes that occur and ultimately inform on the sequence of conformational changes that occur throughout all of GPC. This approach will enable us to identify any mechanistic differences between how GPC fusion activation occurs in GPC with and without LAMP1.

Expected Outcomes. We expect to determine how LAMP1 binding to GPC enables fusion activation to occur at elevated pH conditions. Specifically, our combined approach will identify any dynamic structural changes that occur in GPC as a result of LAMP1 binding and how those dynamic changes influence the mechanism of fusion activation. By understanding how the GPC fusion protein functions during viral entry and membrane fusion we can hope to better understand how our immune system interacts with and inhibits LASV GPC. References 1. Lassa fever: epidemiology, clinical features, and social consequences. Bmj, 2004. 329(7469): p. 773. 2. Hidalgo, J., et al., Viral hemorrhagic fever in the tropics: Report from the task force on tropical diseases

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14. Hulseberg, C.E., et al., Lamp1 Increases the Efficiency of Lassa Virus Infection by Promoting Fusion in Less Acidic Endosomal Compartments. MBio, 2018. 9(1).

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