Heparan sulfate binding by natural eastern equine encephalitis viruses promotes neurovirulence

Abstract

The Alphavirus genus of the family Togaviridae contains mosquito-vectored viruses that primarily cause either arthritogenic disease or acute encephalitis. North American eastern equine encephalitis virus (NA-EEEV) is uniquely neurovirulent among encephalitic alphaviruses, causing mortality in a majority of symptomatic cases and neurological sequelae in many survivors. Unlike many alphaviruses, NA-EEEV infection of mice yields limited signs of febrile illness typically associated with lymphoid tissue replication. Rather, signs of brain infection, including seizures, are prominent. Use of heparan sulfate (HS) as an attachment receptor increases the neurovirulence of cell culture-adapted strains of Sindbis virus, an arthritogenic alphavirus. However, this receptor is not known to be used by naturally circulating alphaviruses. We demonstrate that wild-type NA-EEEV strain FL91-4679 uses HS as an attachment receptor and that the amino acid sequence of its E2 attachment protein is identical to those of natural isolates sequenced by RT-PCR amplification of field samples. This finding unequivocally confirms the use of HS receptors by naturally circulating NA-EEEV strains. Inactivation of the major HS binding domain in NA-EEEV E2 demonstrated that the HS binding increased brain replication and neurologic disease but reduced lymphoid tissue replication, febrile illness signs, and cytokine/chemokine induction in mice. We propose that HS binding by natural NA-EEEV strains alters tropism in vivo to antagonize/evade immune responses, and the extreme neurovirulence of wild-type NA-EEEV may be a consequence. Therefore, reinvestigation of HS binding by this and other arboviruses is warranted.

Fig. 1.

HS dependence and E2 gene sequences of selected alphaviruses. (A) Relative infectivity of various normal (VEEV, CHIKV) or chimeric (SV, EEEV, WEEV, RRV) replicons for WT CHOK1 cells (filled boxes) or HS-deficient pgsA745 cells (open boxes). Infectivity of each replicon for CHOK1 cells was set to 100%. Error bars are SDs. *P < 0.01; **P < 0.001. (B) Comparison of the amino acid sequences of the indicated SV, RRV, CHIKV, VEEV, WEEV, WT EEEV, and mutant 71-77 EEEV strains between amino acids 67 and 82 of the mature E2 glycoprotein. Positively charged residues are shown in red. Substitution mutations to positively charged residues (24, 28, *) that confer HS binding to cell culture-adapted strains of SV, VEEV, or CHIKV are shown above the sequences.

Fig. 2.

HS dependence of WT and 71-77 NA-EEEV infectivity in comparison with WT SV TR339 and the HS binding SV 39K70 mutant. (A) Relative infectivity of replicons for WT CHOK1 (filled bars) and HS-deficient pgsA745 cells (open bars). (B) Relative infectivity of viruses for BHK cells in the presence of 200 μg/mL BSA (filled bars) or 200 μg/mL soluble heparin (open bars). (C) Relative infectivity of replicons for BHK-21 cells in RPMI infection medium supplemented to 220 mM NaCl (open bars). Infectivity for CHOK1 cells, infection in BSA, and normal RPMI (solid bars) was set to 100%. (D) Plaque titer data from a single-step (multiplicity of infection = 1) growth curve of WT and 71-77 mutant viruses on BHK-21 cells. WT, filled squares; 71-77, open squares. Error bars are SDs, and some are too small to be seen. *P < 0.01; **P < 0.001.

Fig. 3.

Morbidity and mortality data for individual CD-1 mice inoculated with equal particles of WT NA-EEEV and the 71-77 mutant sc (A–C) or ic (D–F). WT, filled squares; 71-77, open squares (A and D). Seizures were defined as uncontrolled spasmodic limb and trunk movements, usually observed when mice had assumed a laterally recumbent posture. Seizures were measured for half-hour intervals during twice-daily collection of morbidity and mortality data. The presence of seizures with WT infection and absence with 71-77 mutant infection was reproduced in multiple independent experiments.

Fig. 4.

Tissue plaque titer data from CD-1 mice infected sc (A–E) or ic (F) with equal particles of WT NA-EEEV or the 71-77 mutant. WT, filled squares; 71-77, open squares. LD, detection limit. Error bars are SDs, and some are too small to be seen.

Fig. 5.

Results of IVIS analysis of mice inoculated sc (A and B) or ic (D and E) with equal particles of fLuc-expressing chimeric replicons packaged in WT NA-EEEV (A and D) or 71-77 mutant (B and E) structural proteins. Quantitation of fLuc signal in homogenized brain (C) or DLN (F) is shown. Sensitivity of each image (footpad/DLN or brain) was set equally such that pseudocolor images of light emitted could be directly compared. Error bars are SDs, and some are too small to be seen. *P = 0.01; **P = 0.01.

Fig. 6.

Serum levels of IFN-α/β measured by bioassay (A; WT, filled square; 71-77, open square) or IFN-α ELISA (B; WT, filled bar; 71-77, open bar) at various times after sc inoculation of CD-1 mice with equal particles of WT EEEV or the 71-77 mutant or levels measured by bioassay 12 h after chimeric replicon infection of CD-1 mice (C). Error bars are SDs, and some are too small to be seen. LD, detection limit for the bioassay. *P < 0.05; **P < 0.01; ***P < 0.001.