Cellular Localization and Antigenic Characterization of Crimean-Congo Hemorrhagic Fever Virus Glycoproteins

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins GN and GC. To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both GN and GC. Using these MAbs, we found that GN predominantly colocalized with a Golgi marker when expressed alone or with GC, while GC was transported to the Golgi apparatus only in the presence of GN. Both proteins remained endo-β-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the GN ectodomain, because a soluble version of GN lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of GN and GC also resulted in localization of soluble GC to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the GN precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of GN when it was expressed alone but were dispensable when GC was coexpressed. In neutralization assays on SW-13 cells, MAbs to GC, but not to GN, prevented CCHFV infection. However, only a subset of GC MAbs protected mice in passive-immunization experiments, while some nonneutralizing GN MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to GC

Crimean-Congo hemorrhagic fever: History, epidemiology, pathogenesis, clinical syndrome and genetic diversity

“…5 and 6) (Altamura et al, 2007;Bertolotti-Ciarlet et al, 2005). The polyprotein is first cleaved by a host signal peptidase into 140 kDa PreG N and 85 kDa PreG C segments.…”

“…CCHFV glycoproteins contain an exceptionally large number of cysteine residues, suggesting the presence of many disulfide bonds and a complex secondary structure. The N-terminus of G N has mucin-like features, with the potential for extensive O-glycosylation (Sanchez et al, 2002;Papa et al, 2002c;Bertolotti-Ciarlet et al, 2005). Interestingly, the C-terminal cytoplasmic tail of G N contains two zinc fingers, which can bind viral RNA (Estrada and De Guzman, 2011).…”

Crimean-Congo Hemorrhagic Fever-Treatment and Preventive Strategies

“…Scientists are now attempting to develop anti-CCHFV specific monoclonal antibodies (mAbs) for the treatment of patients. Monoclonal antibodies specific to both the CCHF Gn and Gc surface glycoproteins were generated to evaluate their neutralization and protective properties (26). It was concluded that neutralization of CCHFV depends not only on the properties of the used antibody, but also on host factors and non-neutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity.…”

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus

“…In addition, not all of the Gc MAbs that neutralized CCHFV infection in vitro conferred protection in vivo. Thus, there was not a strict correlation between in vitro neutralization and in vivo protection [26].…”

“…In a recent study, monoclonal antibodies (MAbs) to CCHFV Gc, but not to Gn, neutralized virus in plaque reduction neutralization tests (PRNT) [26]. However, MAbs directed against Gn were generally more effective at protecting mice from a lethal CCHFV challenge than MAbs to Gc when administered either 24 h before or after infection even though these Gn MAbs did not neutralize in the cell-culture assays.…”