Mammalian arenaviruses are divided into two subgroups based on geographic distribution: Old World and New World [1
]. Both subgroups contain human pathogens capable of causing severe hemorrhagic fever with high morbidity and mortality. Lassa virus (LASV), the pathogen that causes Lassa fever, is an Old World arenavirus endemic to West Africa. Each year, LASV infects several hundred-thousand people resulting in nearly 5000 deaths [2
]. The 2018 outbreak in Nigeria was more extensive and had a higher case fatality rate (CFR) than normally recorded (CFR of confirmed cases was approximately 25% as of July 2018) [4
], which exemplifies the need to develop LASV antivirals and vaccines. Human infections predominantly occur through zoonotic spread from the rodent host Mastomys natalensis
, and potentially Hylomyscus pamfi
and Mastomys erythroleucus
]. Transmission can occur through direct contact with infected rodent hosts or exposure to rodent excreta/blood. In addition, person-to-person spread can occur through contact with infectious bodily fluids, putting healthcare workers at higher risk [7
]. Due to the lack of vaccines and effective therapeutics, LASV is categorized as a class A pathogen [9
The arenavirus particle consists of a host cell-derived lipid envelope encasing a bi-segmented RNA genome in an ambisense orientation. The envelope contains mature trimeric viral glycoprotein (GP) spikes that are responsible for attachment and entry into the host cell. The glycoprotein precursor (GPC) is produced as a type I membrane protein and is processed twice by host cell peptidases. First, a cellular peptidase in the endoplasmic reticulum (ER) cleaves the stable signal peptide (SSP) subunit from the precursor. Second, subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) in the cis-Golgi cleaves GP1 from GP2 [10
]. The arenavirus signal peptide is not degraded; instead, it becomes part of the trimeric glycoprotein complex serving as a chaperone assisting with protein processing, trafficking, and pH sensing [13
]. The GP1 and GP2 subunits mediate receptor interactions and membrane fusion, respectively [17
To enter the host cell, enveloped viruses must mediate fusion between the viral envelope and cellular membrane. The arenavirus glycoprotein contains two heptad repeat (HR) domains and an amino-terminal fusion peptide (N-FP) [22
], characteristic of class I fusion proteins [19
], and similar to those of retroviruses, filoviruses, paramyxoviruses, and influenza [17
]. Unlike typical class I fusion proteins, the arenavirus GP2 also contains an internal fusion loop (I-FP), which helps mediate fusion [23
]. Under low-pH, the arenavirus glycoprotein undergoes major conformational changes prior to initiating viral fusion [25
]. Interaction between GP1 and lysosomal associated membrane protein 1 (LAMP1) dissociates GP1 from the trimer, activating the GP2 fusion protein [26
]. During GP2 rearrangement, the fusion peptide/loop inserts into the host membrane. Once multiple GP2 subunits are triggered, the glycoproteins collapse into an energetically favorable conformation known as a six-helix bundle (6HB) [19
]. Full collapse of the glycoprotein complex forms a fusion pore, which enables genome release into the cytoplasm [28
The pre-fusion LASV GP and post-fusion GP2 of lymphocytic choriomeningitis virus (LCMV), a closely related Old World arenavirus, have been crystalized [19
]. These two structures illustrate the major GP2 conformational changes that occur during fusion. Previous studies on arenavirus GP2 subunits have characterized both the C-terminal domain, required for interactions with SSP, and hydrophobic amino acids within the fusion peptide and fusion loop [23
]. However, there has not been an extensive characterization of the GP2 subunit as a whole. Therefore, we produced a panel of GP mutants using either insertional or alanine-scanning mutagenesis and functionally characterized them to identify conserved residues that are critical for the fusion process. We identified several residues, that when changed to alanine, did not affect protein processing but inhibited GP2-mediated-fusion, suggesting these residues may be important for GP2 structural rearrangement or lipid interactions.
In this study, we produced and characterized a library of 59 LASV GP2 mutants to identify residues involved in GP2 function. We identified 14 residues (E308, F309, I334, I337, L344/I345, L355, K368, L372, L382, K417, H467/R468, and L469) that are critical for GP folding, trafficking, or SKI/S1P recognition, evidenced by the lack of GP2 present in cell surface material. Twenty-one charged or hydrophobic residues could be changed to alanine without significantly altering protein production, localization or function, suggesting that GP2 can tolerate mutations at those locations. In total, nine constructs were efficiently cleaved (at least 80% of parental GP), yet failed to produce syncytia at levels similar to parental GP. Many of these residues, including D268, L280, R282, and L285/I286, are part of the fusion peptide domain. Both L280 and L285-I286 within I-FP impaired both cell-to-cell and virus-to-cell fusion, suggesting these hydrophobic residues may be critical for I-FP insertion, as expected (Figure 5
). Surprisingly, GP-D268A and GP-R282A mutant proteins failed to induce cell-to-cell fusion, but were efficient in virus-to-cell fusion when incorporated onto VSV particles. These data suggest the charged residues are required for mediating fusion at the plasma membrane that is predominantly saturated lipids and sterols, but are less important when fusing in the lysosomal membrane, which contains low levels of cholesterol [36
]. Similar lipid-dependent fusion occurs with other viruses; efficient insertion of the Dengue fusion peptide requires specific lipids within the late endosomes and fails to mediate low-pH fusion at the plasma membrane [39
While many of the residues that impacted fusion localized to the fusion peptide, five residues were part of other GP2 domains (Figure 5
). I323 is part of the HR1 domain, I403 and L415 are in the HR2 domain, and L394 is the amino acid preceding HR2 (Figure 5
). These residues may be critical for 6HB formation [19
]. Residue R422 falls right next to the membrane but was not resolved in either crystal structure. Removal of the charged residue may impact the final 6HB structure and inhibit fusion.
Of the seven constructs that produced cleaved GP2 but failed to transduce cells (GP-L280A, GP-L285A/I286A, GP-I323A, GP-L394A, GP-I403A, GP-L415A, and GP-R422A), six were hydrophobic residues. While all of the constructs retained high cleavage efficiencies, several (GP-L280A, GP-L285A/I286A, GP-L394A, GP-I403A, and GP-L415A) produced decreased levels of GP on the surface, which may contribute to the decreased transduction.
Three of our hydrophobic constructs, GP-F262A, GP-L266A, and GP-L280A were previously characterized [23
]. Klewitz et al. found GP-F262A and GP-L266A produced very little protein on the surface and had no fusion activity [23
]. However, our cleavage data suggests that both GP-F262A and GP-L266A are surface-expressed near or above parental GP levels and induced both cell-to-cell and virus-to-cell fusion (Figure 3
A). These phenotypic differences may be due to a difference in our transfection protocols, harvesting cells at 36 h versus 24 h, which allows more time for GP to traffic to the cell surface. We both found GP-L280A produced cleaved GP2, albeit at a decreased level, but was unable to induce fusion. Therefore, while the hydrophobic nature of the N-FP and I-FP is important, some individual residues can be made less hydrophobic while preserving functionality. Both data sets agree that the internal fusion peptide region is important for protein function.
Taken together, these data demonstrate that both conserved hydrophobic and charged residues throughout GP2 are required for optimal protein function. Specifically, the data highlighted specific residues near or within the I-FP, HR1, and HR2 domains that play critical roles in fusion.
4. Materials and Methods
Cell Lines and Transfections
Vero cells stably expressing human SLAM were maintained in Dulbecco−s modified Eagle−s medium (DMEM) with 5% (v
) fetal bovine serum (FBS) and incubated at 37 °C and 5% CO2
]. HAP1 and HAP1-ΔDAG1 cells (Horizon Discovery, Cambridge, UK) were maintained in Iscove’s media supplemented with 10% (v
) FBS and incubated at 37 °C and 5% CO2
. All transfections were performed with GeneJuice (Millipore, Burlington, MA) according to the manufacturer’s instructions.
Expression Vectors and Mutagenesis
The LASV GPC protein coding sequence was codon optimized for mammalian expression and cloned into a pcDNA3.1 intron vector. A CMV promoter initiated gene expression, and we included a β-globin intron in the 5’ untranslated region (UTR) to increase protein production [31
]. We added a carboxy-terminal 3xFLAG tag to the GP2 cytoplasmic tail to biochemically detect HA and charged constructs. HA insertions and point mutations were created with QuikChange mutagenesis and PfuTurbo-HS polymerase (Agilent, Santa Clara, CA). We verified the presence of each mutation with DNA sequence analysis, and a complete sequence information is available upon request.
Vero cells were transfected (as described above) with plasmid DNA encoding the indicated LASV GPC construct. Thirty-six hours following transfection, cells were washed with cold PBS and incubated with 0.5 mg/mL sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (ThermoFisher, Waltham, MA) for 30 min on ice to tag cell surface proteins with biotin [41
]. The reaction was quenched with Tris-HCl, and cells were lysed in M2 lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) at 4°C, then centrifuged (20,000× g, 15 min, 4°C). The clarified lysate was rotated with streptavidin sepharose beads (GE Healthcare, Chicago, IL) for 60 min. Following incubation, the streptavidin sepharose beads were washed in buffer 1 (100 mM Tris, 500 mM lithium chloride, 0.1% Triton X-100) and then in buffer 2 (20 mM HEPES (pH 7.2), 2 mM EGTA, 10 mM magnesium chloride, 0.1% Triton X-100). The samples were then incubated in urea buffer (200 mM Tris, pH 6.8, 8 M urea, 5% sodium dodecyl sulfate (SDS), 0.1 mM EDTA, 0.03% bromophenol blue, 1.5% dithiothreitol) for 30 min at 55°C and analyzed using an immunoblot.
Antibodies and Immunoblots
After surface biotinylation, samples were separated by gel electrophoresis on 4-20% Nu-PAGE gels (ThermoFisher, Waltham, MA) and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare). HA and charged GP constructs were detected with an antibody against the Flag epitope tag (M2; Sigma, Burlington, MA) and a mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch, West, Grove, PA). Hydrophobic constructs were detected with an antibody against LASV GP2 (22.5D), kindly provided by Dr. James Robinson (Tulane University), and a human IgG HRP-conjugated secondary antibody (Jackson). Immunoblots were visualized with SuperSignal West Dura Extended Duration Substrate (ThermoFisher, Waltham, MA) and a ChemiDoc digital imaging system (Bio-Rad, Hercules, CA). Immunoblots were quantified using ImageLab software (Bio-Rad, Hercules, CA).
Cell-to-Cell Fusion Assay
Vero cells were co-transfected with LASV GP mutants and pmaxGFP (4:1 ratio). Forty hours following transfection, media was removed and replaced with PBS (pH 4) and incubated (37 °C and 5% CO2
) for 30 min to allow glycoprotein triggering. The PBS was replaced with warm DMEM and cells were incubated for an additional 3 h to enable membrane rearrangement and syncytia formation. Four representative pictures of the fusion were taken using Zoe microscope (Bio-Rad) (20× magnification) and unfused cells were counted. Fusion efficiency was quantified using the following equation:
Each mutant was assessed for fusion in at least three independent experiments.
VSV Pseudoparticle Production and Transductions
GP constructs lacking the C-terminal 3xFlag tag were used to make vesicular stomatitis virus (VSV) pseudotyped particles. Vero cells were transfected with LASV GP DNA. Thirty-six hours following transfection the cells were transduced with VSVΔG-GFP particles pseudotyped with VSV-G (MOI 1) for one hour (courtesy of Dr. Michael Whitt; KeraFAST, Boston, MA) [42
]. The particle-containing media was then replaced with fresh DMEM. VSVΔG-GFP particles displaying the LASV GP were collected 8 h following the transduction. These particles were applied onto HAP1 and HAP1-ΔDAG1 cells. A larger volume of particles (4 times as much) was used to transduce HAP1-ΔDAG1 cells to overcome the decreased transduction efficiency when cells are missing the primary α-DG receptor [18
]. The number of GFP positive cells was enumerated in a flow cytometer. Results are displayed as the percent of GFP positive cells present in a population of 10,000 live cell events compared to parental GP transduction.