Identification of a novel C-terminal cleavage of Crimean-Congo hemorrhagic fever virus PreGN that leads to generation of an NSM protein

The structural glycoproteins of Crimean-Congo hemorrhagic fever virus (CCHFV; genus Nairovirus, family Bunyaviridae) are derived through endoproteolytic cleavage of a 1,684-amino-acid M RNA segment-encoded polyprotein. This polyprotein is cotranslationally cleaved into the PreG(N) and PreG(C) precursors, which are then cleaved by SKI-1 and a SKI-1-like protease to generate the N termini of G(N) and G(C), respectively. However, the resulting polypeptide defined by the N termini of G(N) and G(C) is predicted to be larger (58 kDa) than mature GN (37 kDa). By analogy to the topologically similar M segment-encoded polyproteins of viruses in the Orthobunyavirus genus, the C-terminal region of PreG(N) that contains four predicted transmembrane domains may also contain a nonstructural protein, NSM. To characterize potential PreG(N) C-terminal cleavage events, a panel of epitope-tagged PreG(N) truncation and internal deletion mutants was developed. These constructs allowed for the identification of a C-terminal endoproteolytic cleavage within, or very proximal to, the second predicted transmembrane domain following the G(N) ectodomain and the subsequent generation of a C-terminal fragment. Pulse-chase experiments showed that PreG(N) C-terminal cleavage occurred shortly after synthesis of the precursor and prior to generation of the G(N) glycoprotein. The resulting fragment trafficked to the Golgi compartment, the site of virus assembly. Development of an antiserum specific to the second cytoplasmic loop of PreG(N) allowed detection of cell-associated NSM proteins derived from transient expression of the complete CCHFV M segment and also in the context of virus infection.