Residue 82 of the Chikungunya virus E2 attachment protein modulates viral dissemination and arthritis in mice

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has reemerged to cause profound epidemics of fever, rash, and arthralgia throughout sub-Saharan Africa, Southeast Asia, and the Caribbean. Like other arthritogenic alphaviruses, mechanisms of CHIKV pathogenesis are not well defined. Using the attenuated CHIKV strain 181/25 and virulent strain AF15561, we identified a residue in the E2 viral attachment protein that is a critical determinant of viral replication in cultured cells and pathogenesis in vivo. Viruses containing an arginine at E2 residue 82 displayed enhanced infectivity in mammalian cells but reduced infectivity in mosquito cells and diminished virulence in a mouse model of CHIKV disease. Mice inoculated with virus containing an arginine at this position exhibited reduced swelling at the site of inoculation with a concomitant decrease in the severity of necrosis in joint-associated tissues. Viruses containing a glycine at E2 residue 82 produced higher titers in the spleen and serum at early times postinfection. Using wild-type and glycosaminoglycan (GAG)-deficient Chinese hamster ovary (CHO) cell lines and soluble GAGs, we found that an arginine at residue 82 conferred greater dependence on GAGs for infection of mammalian cells. These data suggest that CHIKV E2 interactions with GAGs diminish dissemination to lymphoid tissue, establishment of viremia, and activation of inflammatory responses early in infection. Collectively, these results suggest a function for GAG utilization in regulating CHIKV tropism and host responses that contribute to arthritis.

Importance: CHIKV is a reemerging alphavirus of global significance with high potential to spread into new, immunologically naive populations. The severity of CHIKV disease, particularly its propensity for chronic musculoskeletal manifestations, emphasizes the need for identification of genetic determinants that dictate CHIKV virulence in the host. To better understand mechanisms of CHIKV pathogenesis, we probed the function of an amino acid polymorphism in the E2 viral attachment protein using a mouse model of CHIKV musculoskeletal disease. In addition to influencing glycosaminoglycan utilization, we identified roles for this polymorphism in differential infection of mammalian and mosquito cells and targeting of CHIKV to specific tissues within infected mice. These studies demonstrate a correlation between CHIKV tissue tropism and virus-induced pathology modulated by a single polymorphism in E2, which in turn illuminates potential targets for vaccine and antiviral drug development.

FIG 1

Schematic depiction of polymorphic residues in CHIKV strains AF15561 and 181/25. Distribution of amino acid polymorphisms between strains AF15561 and 181/25 across the ∼12-kb genome. Numbers correspond to the amino acid positions within each protein.

FIG 2

Residue 82 of the E2 attachment protein is a determinant of CHIKV infectivity in mammalian cells. (A) Vero cells were adsorbed with CHIKV strains AF15561 (AF), 181/25 (181), or the variant viruses shown at an MOI of 1 PFU/cell and incubated for 24 h in medium containing 20 mM NH₄Cl. The cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percent infected cells for triplicate experiments. Error bars indicate standard errors of the means. (B) Vero cells were adsorbed with virus strain AF15561, 181/25, AF15561 E2 G82R, or 181/25 E2 R82G at an MOI of 0.01 PFU/cell. At the times shown, viral titers in culture supernatants were determined by plaque assay using Vero cells. Results are presented as the mean viral titers for triplicate samples. Error bars indicate standard deviations. Titers of virus strains AF15561 and AF15561 E2 G82R were significantly different at 6, 12, and 18 h postinfection, and titers of virus strains 181/25 and 181/25 E2 R82G were significantly different at 12, 18, 24, 30, and 36 h postinfection. (C) Vero cells were adsorbed with ∼3 × 10¹⁰ genomes of wild-type (WT) virus strain AF15561, 181/25, AF15561 E2 G82R, or 181/25 E2 R82G for 30 min. After 30-min incubation, cells were stained with CHIKV E2-specific MAb, and virus-bound cells were quantified by flow cytometry. Results are presented as percent bound cells for triplicate experiments normalized to cell autofluorescence in the absence of CHIKV MAb. No CHIKV-bound cells were detected for virus strain 181/25 E2 R82G. Error bars indicate standard deviations. Values that are significantly different, as determined by ANOVA, followed by Bonferroni's posthoc test (A and C) and Student's t test (B), are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Residue 82 of the E2 attachment protein is a determinant of CHIKV infectivity in mosquito cells. (A) C6/36 mosquito cells were adsorbed with CHIKV strains AF15561 and 181/25 or the variant viruses shown at an MOI of 1 PFU/cell and incubated for 24 h in medium containing 20 mM NH₄Cl. The cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percent infected cells for quadruplicate experiments. Error bars indicate standard errors of the means. (B) C6/36 cells were adsorbed with virus strain AF15561, 181/25, AF15561 G82R, or 181/25 E2 R82G at an MOI of 0.01 PFU/cell. At the times shown, viral titers in culture supernatants were determined by plaque assay using Vero cells. Results are presented as the mean viral titers for triplicate samples. Error bars indicate standard deviations. The titers of virus strains 181/25 and 181/25 E2 R82G were significantly different at 12, 18, and 24 h postinfection. Values that are significantly different, as determined by ANOVA, followed by Bonferroni's posthoc test (A) and Student's t test (B), are indicated by asterisks as follows: **, P < 0.01; ***, P < 0.001.

FIG 4

An arginine at E2 residue 82 confers greater dependence on glycosaminoglycans. (A) CHO-K1 and CHO-pgsA745 cells were adsorbed with virus strain AF15561, 181/25, AF1561 E2 G82R, or 181/25 E2 R82G at an MOI of 10 PFU/cell and incubated for 24 h. Wild-type (WT) CHIKV or variant strains containing substitutions at E2 residue 82 were tested. The cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percent infected cells for triplicate experiments normalized to the values for parental CHO-K1 cells. Error bars indicate standard errors of the means. (B) Strains AF15561, 181/25, AF1561 E2 G82R, and 181/25 E2 R82G were treated with BSA at 1,000 μg/ml or heparin at the concentrations shown for 30 min and adsorbed to Vero cells at an MOI of 2.5 PFU/cell. After incubation for 24 h, cells were stained with CHIKV-specific antiserum and DAPI to detect nuclei and imaged by fluorescence microscopy. Results are presented as percent infected cells for triplicate experiments normalized to the values for mock-treated virus. Error bars indicate standard errors of the means. (C) The virus strains shown at 5 × 10⁹ genome copies each were incubated with heparin-conjugated or unconjugated agarose beads for 30 min, resolved by SDS-PAGE, and detected by immunoblotting with CHIKV E2-specific MAb (left). Twenty-five percent of input virus is shown as a control. The percentage of virus bound to beads was quantified by optical densitometry for triplicate experiments (right). Error bars indicate standard deviations. Values that are significantly different, as determined by Student's t test (A and C) and Kruskal-Wallis analysis followed by Dunn's posthoc test (B), are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

CHIKV E2 residue 82 modulates virus-induced pathology. C57BL/6J mice (20 to 22 days old) were inoculated with 10³ PFU of CHIKV strain AF15561, 181/25, AF1561 E2 G82R, or 181/25 E2 R82G in the left rear footpad. (A) Mice were weighed at 24-h intervals postinoculation. Results are presented as the mean percent starting weight (weight on day 0). Error bars indicate standard deviations. The number of mice at different time points follow: day 0 to day 1 (D0-D1), n = 17; D2-D3, n = 15; D4-D5, n = 8; D6-D7, n = 3. (B) Swelling of the left and right hind feet was quantified using calipers at the times shown (in days). Error bars indicate standard deviations. The number of mice at different time points follow: D1, n = 2; D3, n = 7; D5, n = 5; D7, n = 3. Values that are significantly different as determined by ANOVA followed by Bonferroni's posthoc test are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

An arginine at E2 residue 82 diminishes CHIKV-induced arthritis. C57BL/6J mice (20 to 22 days old) were inoculated with PBS or 10³ PFU of virus strain AF15561, 181/25, AF1561 E2 G82R, or 181/25 E2 R82G in the left rear footpad. At day 7 postinoculation, mice were euthanized and perfused with 4% PFA. Consecutive 5-μm sections of the left hind limb were stained with H&E. (A) Representative sections of three mice per group are shown for mock-infected mice or mice inoculated with the parental strains and the reciprocal E2 82 variant strains. Bars, 50 μm. (B) H&E-stained sections were scored for histological evidence of inflammation and necrosis in the metatarsal muscle and tendonitis. Results are expressed as pathology score of tissues from individual animals. Horizontal black lines indicate mean pathology score. Scores were assigned based on the following scale: 0, no lesions; 1, minimal, 0 to 24% of tissue affected; 2, mild, 25 to 49% of tissue affected; 3, moderate, 50 to 75% of tissue affected; 4, marked, >75% of tissue affected.

FIG 7

Viral loads following inoculation of parental strains and the reciprocal E2 82 variant strains. C57BL/6J mice (20 to 22 days old) were inoculated with PBS or 10³ PFU of virus strain AF15561, 181/25, AF1561 E2 G82R, or 181/25 E2 R82G in the left rear footpad. At days 1, 3, and 5 postinoculation, mice were euthanized, their ankles, quadriceps (Quad), and spleens were excised, and serum was collected. Viral titers in tissue and serum homogenates were determined by plaque assay using BHK-21 cells. Results are expressed as the mean PFU/gram (tissue) or PFU/ml (serum). Error bars indicate standard errors of the means. Dashed lines indicate the limit of detection. The number of mice at different time points follow: D1, n = 5; D3, n = 7; D5, n = 5. Values that are significantly different as determined by ANOVA followed by Bonferroni's posthoc test are indicated by asterisks and bars as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.