Glycoprotein N-linked glycans play a critical role in arenavirus pathogenicity

Abstract

Several arenaviruses cause hemorrhagic fevers in humans with high case fatality rates. A vaccine named Candid#1 is available only against Junin virus (JUNV) in Argentina. Specific N-linked glycans on the arenavirus surface glycoprotein (GP) mask important epitopes and help the virus evade antibody responses. However the role of GPC glycans in arenavirus pathogenicity is largely unclear. In a lethal animal model of hemorrhagic fever-causing Machupo virus (MACV) infection, we found that a chimeric MACV with the ectodomain of GPC from Candid#1 vaccine was partially attenuated. Interestingly, mutations resulting in acquisition of N-linked glycans at GPC N83 and N166 frequently occurred in late stages of the infection. These glycosylation sites are conserved in the GPC of wild-type MACV, indicating that this is a phenotypic reversion for the chimeric MACV to gain those glycans crucial for infection in vivo. Further studies indicated that the GPC mutant viruses with additional glycans became more resistant to neutralizing antibodies and more virulent in animals. On the other hand, disruption of these glycosylation sites on wild-type MACV GPC rendered the virus substantially attenuated in vivo and also more susceptible to antibody neutralization, while loss of these glycans did not affect virus growth in cultured cells. We also found that MACV lacking specific GPC glycans elicited higher levels of neutralizing antibodies against wild-type MACV. Our findings revealed the critical role of specific glycans on GPC in arenavirus pathogenicity and have important implications for rational design of vaccines against this group of hemorrhagic fever-causing viruses.

Machupo virus with mutations in the transmembrane domain and glycosylation sites is attenuated and immunogenic in animal models of Bolivian Hemorrhagic Fever

“…6A). This is consistent with previous data demonstrating that MACV mutants lacking specific glycans on GPC are more susceptible to antibody neutralization 29 . We further performed ELISAs to measure the level of MACV GP1 and NP specific antibodies with purified MACV (Carvallo) GP1 and NP proteins.…”

“…In this study, we characterized MACV mutant containing the TMD F438I substitution and mutations at glycosylation sites on GP1. Our previous research revealed that neither the F438I substitution nor the glycosylation site mutations alone are sufficient for full attenuation in IFN-αβ/γ R -/mice 29,30 . This work demonstrates that the combination of the F438I substitution in TMD with the glycan deficiency at N83 and N166 of GPC led to full attenuation in our IFN-αβ/γ R -/mouse model.…”

“…Our previous work demonstrates that disruption of glycosylation sites at GPC N83 and N166 partially attenuate MACV in IFN-αβ/γ R -/mice 29 . Though all mice survive challenge with this mutant, all mice still develop neurological signs of disease.…”

“…HEK-293T cells (Life Technologies) were maintained in Dulbecco's modified Eagles medium (DMEM) (GE Healthcare Life Sciences) with 10% FBS (Life Technologies) and 1% P/S (Life Technologies) at 37°C with 5% CO2 . The recombinant MACV Carvallo strain 31 , MACV GPCF438I 30 , MACV GPCΔN83/ΔN166 29 , MACV GPCΔN83/F438I, MACV GPCΔN166/F438I, and MACV GPCΔN83/ΔN166/F438I were rescued by using a reverse genetics system previously described 31,54 . The wild-type Chicava strain was a generous gift from Dr. Thomas Ksiazek.…”

“…Our previous work has demonstrated that a MACV strain expressing the whole GPC of Cd#1 is fully attenuated 27 . Likewise, a MACV strain containing the Cd#1 GPC ectodomain is also partially attenuated, though reversions at two N-linked glycans were frequently observed 28,29 . Removal of the two N-linked glycans at N83 and N166 (MACV GPCΔN83/ΔN166) results in a strain that is partially attenuated in IFN-αβ/γ R -/mice 29 .…”