Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

Abstract

Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever. Successful entry of LASV requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus (LCMV) residue (L84 ​N, K88E, L10F, and H170S) reduced the binding affinity of LASV GP1 for LAMP1. Moreover, all mutations caused decreases in glycoprotein precursor (GPC)-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPC^L84N or GPC^K88E decreased sharply in multiple cell types, while L107F and H170S had only mild effects on infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order of binding affinity being L84 ​N ​> ​L107F ​> ​K88E ​> ​H170S. The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.

Keywords: Glycoprotein; Lassa virus (LASV); Lysosome-associated membrane protein 1 (LAMP1); Membrane fusion; Receptor switch.

Fig. 1

Binding ability of the mutant GP1_LASV to LAMP1. A Multiple sequence alignment of GP1 proteins of representative Old World mammarenaviruses. The UniProt accession codes of the sequences are P08669 (LASV), P07399 (LCMV), P19240 (MOPV), Q2A069 (MOBV), and Q27YE4 (Ippy virus, IPPYV). Fully conserved residues were highlighted with a red background, and partially conserved residues were presented in red. The secondary structure observed with GP1_LASV (PDB: 4ZJF) was indicated above the sequence, and cysteine involved in the disulfide bond was numbered below the alignment in green. This graphical representation was generated using ESPript (http://espript.ibcp.fr). B Ribbon diagram of the LASV-GP1/distal-LAMP1 complex. The crystal structure of LASV-GP1 (PDB no. 4ZJF) and distal-LAMP1 (PDB no. 8ATH) was used to build the complex using the ZDOCK 3.0.2 program. Residues located in the interface of LASV-GP1 and LAMP1, and residues located spatially close to the histidine triad (H92/93/230) are colored magentas. Polar amino acids which were not conserved between LASV and LCMV are colored blue. The histidine triad are colored red. C Images of LAMP1 pull-down assays by wild-type (WT) and mutated GP1_LASV-Fc. GP1_LASV-Fc was immobilized on sepharose beads, incubated with equal amount of cell lysates at pH 5.0 and pH 8.0, pulled down, and eluted. Protein pellets were recovered and subjected to Western blot analysis. D Quantitative analysis of the pull-down efficiencies based on the intensity of the bands. Data were presented as means ± SDs from three independent experiments. ∗∗∗∗P ​< ​0.0001, ∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05.

Fig. 2

Cleavage processing of wild-type (WT) and mutant LASV GPCs. A 293T cells were transfected with WT or one of the four GPC mutants (L84 ​N, K88E, L107F, and H170S). The equal cell lysates were subjected to Western blotting and probed with an anti-LASV GP2 antibody. The image was representative of three independent experiments. B Quantitative analysis of the cleavage efficiencies based on the intensity of the bands. Data were presented as means ± SDs from three independent experiments. ∗∗∗∗P ​< ​0.0001, ∗∗∗P ​< ​0.001.

Fig. 3

Membrane fusion by wild-type (WT) and four mutant GPCs. A Quantification of membrane fusion using a dual-luciferase reporter assay at the indicated pH values. Data are presented as means ± SDs from three independent experiments. ∗∗∗∗P ​< ​0.0001, ∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05. B Qualitative analysis of membrane fusion was performed in 293T cells co-transfected with pEGFP-N1 and pCAGGS-LASV GPC plasmids. Scale bar, 200 ​μm.

Fig. 4

Infectivity of multiple cell lines by the mutant pseudoviruses. Viral copy number was normalized using quantitative RT-PCR. After normalization, A549, BHK, 293T, and Vero cells in 96-wells plate were infected with 2 ​× ​10⁶ copies/well, respectively. After 24 ​h, the cell lysates were subjected to evaluate the Renilla luciferase activities. Data were presented as means ± SDs from three independent experiments. ∗∗∗∗P ​< ​0.0001, ∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05.

Fig. 5

Infectivity of pseudoviruses on LAMP1-knockout (KO) and LAMP1 replacement A549 ​cells. A wild-type (WT) and LAMP1-KO A549 ​cells were analyzed using Western blotting with antibody against LAMP1. B LASVpv^WT, LASVpv^L84N, LASVpv^K88E, LASVpv^L107F, and LASVpv^H170S infected LAMP1-knockout and LAMP1 replacement cells, respectively, the mock group is a blank control group, and the cells were lysed and assayed for RLU at 24 ​h post-infection. Data are presented as means ± SDs from three independent experiments. ∗∗∗∗P ​< ​0.0001; ns, not significant.

Fig. 6

Analysis of GP1-LAMP1 interaction. A, B Binding of wild-type (WT) and mutant GP1_LASV to the distal domain of LAMP1 was measured using the Octet RED 96 instrument. Each column presents the maximum values of the binding curves. The response values (nm) to the indicated concentrations of GP1_LASV (1600, 800, 400, and 200 ​nmol/L) were measured. Experimental data are shown in (A); these data were fit with a 1:1 global fitting model and the fitted curves are presented in (B). C IIH6 inhibition assay was performed to investigate the effects of GP1 mutants on LASVpv binding. A549 cells were incubated with 200 ​μg/mL IIH6 or IgM control at 37 ​°C for 1 ​h. LASVpv_WT and mutant LASVpv were added and treated at 4 ​°C for 1 ​h. The unbound virus particles were washed for three times with cold PBS. After 24 ​h, cells were lysed and luciferase activity was measured using the Rluc assay system. The results are the mean of three independent determinations. Data are presented as means ± SDs from three independent experiments. ∗∗∗∗P ​< ​0.0001.

Fig. 7

Crystal structure of GP1_LASV (PDB: 5VK2), showing the positions L84, K88, L107, H170 (blue) and the histidine triad (red); these residues are magnified in the inset box.