Fig 1.
In vitro passage of wild type SINV-WEEV did not select mutations conferring HS binding in the E2 glycoprotein.
(A) SINV-WEEV-eGFP virus stock was serially passaged 10 times on BHK-21 cells. (B) Concentrations of PFU/ml, GE/ml and GE:PFU ratios in viral stocks. Infectious viral titers and genomic RNA levels in culture medium were determined by plaque assay on BHK-21 cells and RT-qPCR, respectively. The genomic RNA:PFU ratios were calculated to indicate virion infectivity. (C) SINV-WEEV-eGFP E2 mutants replication in vitro in Vero cells. Vero cells were infected with equal genomes of WT and E2-H256R mutant corresponding to a multiplicity of infection of 1 PFU for WT SINV-WEEV-eGFP. Media were collected at the indicated times post infection, and infectious titers were determined by plaque assay on BHK-21 cells. (D) Relative binding of SINV-WEEV-eGFP WT and E2-H256R for BHK-21cells. Diluted viruses at an MOI of 5 were added to indicated cells and incubated for 45 min at 4 °C. Unbound virions were removed by washes with cold PBS, and ddPCR was used to assess the amount of bound viral particles. (E) Relative infectivity and binding of the indicated SINV-WEEV-eGFP viruses on CHO-K1, GAG-deficient pgsA-745 and HS-deficient pgsD-677 cells. For the infectivity assay, viruses were diluted and assayed for plaque formation on indicated cell monolayers. eGFP expressing foci were enumerated with a fluorescence microscope. Binding assay was performed as described above. Percent infectivity and binding were normalized to CHO-K1, which was set to 100%. (F) Effect of heparinase II treatment on plaque formation by the chimeric viruses. Confluent BHK-21 cell monolayers were treated with heparinase II at the indicated concentrations. After washing with PBS, the cells were infected with diluted viruses, and plaque assays were performed. Error bars are standard deviations. Significance (P values) was estimated by (C) two-way ANOVA with Bonferroni’s post-test, (D) Mann-Whitney test, (E,F) two-way ANOVA with Dunnett’s post-test: *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.
Table 1.
WEEV E2 positive charge substitution mutations.
Fig 2.
Structural organization of SINV-WEEV McM strain and mutational mapping of HS-binding residues on a ribbon model of the WEEV McM E2 trimer.
(A) Left panel, Icosahedral reconstruction of SINV-WEEV McM strain. The black triangle represents one asymmetric unit. The icosahedral five-, three-, and two-fold symmetry axes are shown with blue pentagon, triangle, and oval, respectively. The q3 spikes are indicated with white triangles. Right panel, central cross-section of the volume viewed down an i2-axis. Other symmetry axes are labeled. The color key represents the radial coloring of the volume in Å. (B) Three-dimensional model of the E2-E1 heterodimer of WEEV McM and (C) trimeric spike complex of E2-E1 heterodimers. Ribbon drawings show the subdomain distribution in E2 are colored according to the following scheme: domain A – cyan, domain B – teal, domain C – plum, domain D – magenta, β-ribbon – purple, N-linker - olive. The E1 glycoprotein is in grey. Locations of the mutations are highlighted in red. The figures were made using UCSF ChimeraX software.
Fig 3.
Relative infectivity and binding of SINV-WEEV E2 mutant viruses on CHO-K1, GAG-deficient pgsA-745 and HS-deficient pgsD-677 cells.
(A) For infectivity assay, wild-type and mutant viruses were serially diluted and assayed for plaque formation on the indicated cell monolayers. eGFP expressing foci were enumerated with a fluorescence microscope at 36 h post infection. (B) For binding assay, monolayers of each cell type were incubated with the indicated viruses at an MOI of 5 for 45 min at 4 °C and washed with PBS supplemented with 1% FBS to remove unbound virions. Percent infectivity/binding was normalized to CHO-K1, which was set to 100%. SINV TR339 and E2-E70K mutant are included as HS–independent and HS–dependent controls, respectively. The data are representative of two independent experiments; each performed in duplicate or triplicate. Column heights indicate mean values, and error bars denote SD. Statistical analysis: two-way ANOVA with Dunnett’s post-test: *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001, compared to CHO-K1. Only significant comparisons are represented; all others were non-significant.
Fig 4.
SINV-WEEV E2 mutants replication in vitro in Vero cells.
Vero cells were infected with equal genomes of WT SINV-WEEV and E2 mutants corresponding to a multiplicity of infection of 1 PFU for WT. Media were collected at the indicated times post infection, and infectious titers were determined by plaque assay on BHK-21 cells. Data is represented as a geometric mean and error bars represent the standard deviation (SD). Each data point is from 3 independent experiments that were performed in duplicate. (A) Mutants with replication efficiency similar to WT. (B) Mutants with slightly delayed replication rates compared to WT. (C) Mutants with significantly delayed PFU production compared to WT. Significance determined by two-way ANOVA with Dunnett’s multiple-comparison test, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. Only significant comparisons are represented, all others were non-significant.
Table 2.
Mortality rates and mean survival time (MST) of the LAV candidates in outbred CD-1 mice after primary subcutaneous infection.
Fig 5.
Mouse survival after subcutaneous LAV candidates infection.
CD-1 mice were infected subcutaneously in rear footpad with the same dose of 103 PFU of parental WEEV and the designed LAV candidate. Experiments included 4-5 animals per group. The significance between survivals of mice infected with WT WEEV and mutants was estimated using the Log-rank (Mantel Cox) test; *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001.
Table 3.
Downselected and BHK-21 cells passage-derived WEEV McM E2 positive charge mutants.
Fig 6.
Three-dimensional model of the WEEV McM trimeric spike complex of E1/E2 heterodimers.
Surface representation of WEEV McM spike trimer with E2 in light blue and E1 in grey. Locally reconstructed map obtained using newly generated 3.2Å cryo-EM reconstruction of WEEV McM strain. Location of the double mutations is highlighted in blue – D72K-K159N, green – D156K-V61D, and purple - G172R-D4N.
Fig 7.
In vitro passage of downselected E2 mutants selected second mutations that increased the yield of virions while retaining HS binding phenotypes.
SINV-WEEV E2 selected double mutants replication in vitro in Vero cells (A). Vero cells were infected with equal genomes of the indicated LAV candidates corresponding to a multiplicity of infection equal to 1 PFU per cell for WT WEEV. Media were collected at the indicated times post infection, and infectious titers were determined by plaque assay on BHK-21 cells. (B) Relative infectivity and binding of the SINV-WEEV McM E2 selected double mutants compared to parental downselected mutants on CHO-K1, GAG-deficient pgsA-745 and HS-deficient pgsD-677 cells. For the infectivity assay, viruses were diluted and assayed for plaque formation on the indicated cell monolayers. GFP expressing foci were enumerated with a fluorescence microscope. For binding assays, diluted viruses at MOI of 5 were added to indicated cells and incubated for 45 min at 4 °C. Unbound virions were removed by washes with cold PBS, and ddPCR was used to assess the amount of bound viral particles. Percent infectivity and binding were normalized to CHO-K1, which was set to 100%. Data are combined from (A) two experiments performed in triplicate and (B) three experiments performed in duplicate. Error bars show SD; significance was estimated by (A) two-way ANOVA with Tukey’s post-hoc tests on log-transformed data, (B) one-way ANOVA with Bonferroni’s post-test.: * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 8.
LAV candidates E2 protein mutations interfere with WEEV-protein receptor interactions.
K562 cells stably expressing empty vector (EV), VLDLR or PCDH10 were inoculated with the indicated chimeric eGFP reporter wild-type or mutant SINV-WEEV at an MOI of 2. Infection levels were quantified by flow cytometry and expressed as percent infectivity. Data are combined from two experiments; one performed in triplicate and the other in quadruplicate. Error bars show SD; significance was estimated by two-way ANOVA with Tukey’s post-test: * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 9.
Mouse survival after subcutaneous candidate E2 mutant LAV infection.
4–6-week-old CD-1 mice were infected subcutaneously in rear footpad with the same dose of 103 PFU of WT WEEV and WEEV E2 mutant LAV candidates. Experiments included 5 animals per group and were performed twice. Mice were monitored for survival and weight change for 14 days. (A) Survival. (B) Weight change. (C) Clinical signs. Error bars are standard error of the mean. The significance between survivals of mice infected with WT WEEV and mutants was estimated using the Log-rank (Mantel-Cox) test; *P < 0.05, **P < 0.01.
Fig 10.
Downselected single and double E2 mutant LAV candidates induce neutralizing antibodies in mice.
4–week–old CD-1 mice were infected subcutaneously with equal genomes of indicated SINV-WEEV McM chimeric LAV candidates and control SINV-VEEV TrD viruses, equivalent to 5x103 PFU (BHK-21) of WT SINV-WEEV McM and SINV-VEEV TrD viruses, respectively. Experiments included 3-5 animals per group and were performed twice for SINV-VEEV TrD controls and three times for SINV-WEEV McM LAVs. Serum samples were collected on day 21 post infection and screened for neutralization antibody levels by Vero cell plaque neutralization assay using a SINV-WEEV McM WT chimeric virus for LAV candidates and SINV-VEEV TrD WT – for controls. Sera were serially diluted 2-fold before reaction with the virus. Two independent dilutions of each sample were performed. (A, C) Nonlinear curve fits to dilution versus plaque reduction data. (B, D) Reciprocal PRNT50 values for each mouse were calculated as described in Materials and Methods. Significance of differences was determined by two-way ANOVA with Tukey’s post-test: * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 11.
E2 mutant LAV candidates protect mice from parental WT WEEV McM s.c. challenge.
CD-1 mice were challenged subcutaneously with 1x103 PFU of parental WT WEEV McM virus on day 22 post primary SINV-WEEV-eGFP LAV candidates infection. Experiments included 5 animals per group and were performed twice. Mice were monitored for survival and weight change for 14 days. (A) Survival, (B) weight change, (C) clinical signs. The significance between survivals of WT WEEV McM challenged mice infected with WT WEEV and mutants was estimated using the Log-rank (Mantel-Cox) test; * P < 0.05.
Fig 12.
Replication of WEEV McM LAV candidates in popliteal lymph nodes and brains.
CD-1 mice were infected with equal genome copies of the WEEV McM LAV candidates corresponding to 103 PFU of WT WEEV. (A) Popliteal lymph nodes were collected at 8 hpi, and viral RNA levels were quantified by qRT-PCR using primers mapping to the WEEV nsP2 region. Data are presented as log10 genome equivalents per LN. (B) Brains were collected at 4 and 5 dpi, and viral RNA levels were quantified as in panel A. Each point represents an individual mouse (PLNs: n = 12 for WT and n = 6 for each mutant; brains: n = 3 per group). Each red asterisk indicates a mouse that died on or required euthanasia due to infection on day 4 (WT) and day 5 (G172R-D4N) after infection. Dotted line indicates the limit of detection (LOD) of the assay. Statistical comparisons to WT were performed on log10 transformed values using one-way ANOVA with Bonferroni’s post-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.