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. IMPORTANCECHIKV 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.

Chikungunya virus: epidemiology, replication, disease mechanisms, and prospective intervention strategies

“…However, residue 82 is also a major determinant of CHIKV clearance, as it influences neutralization by CHIKV-specific antibodies (150). Indeed, reversion at residue 82 in 181/25 was observed in mice (75,180) and humans (183), indicating instability of attenuation and raising concerns about safety. Therefore, other strategies for development of live-attenuated vaccines for CHIKV have been investigated.…”

“…Strain 181/25 showed no adverse effects in a phase I clinical trial (181), and although it was highly immunogenic in phase II clinical trials, approximately 8% of subjects developed mild, transient arthralgia (182). Attenuation of 181/25 is attributable to only two point mutations in E2 (T12I and G82R) (180); the mutation at residue 82, which enhances glycosaminoglycan binding (74,75), is the dominant attenuating factor (180). However, residue 82 is also a major determinant of CHIKV clearance, as it influences neutralization by CHIKV-specific antibodies (150).…”

“…However, CHIKV displays broad tropism, replicating in many vertebrate and invertebrate cells (70)(71)(72)(73); bona fide proteinaceous receptors have not been identified for CHIKV. Glycosaminoglycans, which are expressed on many susceptible cell types, appear to serve as attachment factors to enhance infectivity (64,(74)(75)(76)(77). After E2-mediated attachment to cells, receptor-bound particles are internalized mainly by clathrin-mediated endocytosis ( Figure 2 and refs.…”

Mxra8 is a receptor for multiple arthritogenic alphaviruses

“…As CHIKV-181/25 is a cell culture-adapted vaccine strain 12 that has acquired heparan sulfate (HS) binding activity 13 , we evaluated Mxra8 with other CHIKV strains. Infection of CHIKV-AF15561, the parental Asian strain of CHIKV-181/25, which binds poorly to HS 14 , and CHIKV-37997, a West African strain, was abolished in Δ Mxra8 3T3 cells, reduced in Δ Mxra8 MEFs (Fig 1a), and restored in trans-complemented Δ Mxra8 3T3 cells (Fig 1b–c). However, the dependence on Mxra8 was less with CHIKV-LR 2006, an East Central South African strain (Fig 1a and d).…”

Chikungunya virus outbreak expansion and microevolutionary events affecting epidemiology and epidemic potential

“…In particular, a change of amino acid at E2 position 82 affected swelling, inflammation, and necrosis induced by infection. 99 The authors found that an arginine in this position (as in the vaccine strain) reduced virulence, while replacing the arginine with a glycine as in the virulent AF15561 strain resulted in muscle destruction and more severe tendonitis. Interestingly, the presence of the arginine residue also affected the ability of the virus to replicate in lymphoid tissues.…”