Biochemical and structural studies reveal differences and commonalities among cap-snatching endonucleases from segmented negative-strand RNA viruses

Abstract

Viruses rely on many host cell processes, including the cellular transcription machinery. Segmented negative-strand RNA viruses (sNSV) in particular cannot synthesize the 5′-cap structure for their mRNA but cleave off cellular caps and use the resulting oligonucleotides as primers for their transcription. This cap-snatching mechanism, involving a viral cap-binding site and RNA endonuclease, is both virus-specific and essential for viral proliferation and therefore represents an attractive drug target. Here, we present biochemical and structural results on the putative cap-snatching endonuclease of Crimean–Congo hemorrhagic fever virus (CCHFV), a highly pathogenic bunyavirus belonging to the Nairoviridae family, and of two additional nairoviruses, Erve virus (EREV) and Nairobi sheep disease virus (NSDV). Our findings are presented in the context of other cap-snatching endonucleases, such as the enzymatically active endonuclease from Rift Valley fever virus (RVFV), from Arenaviridae and Bunyavirales, belonging to the His− and His+ endonucleases, respectively, according to the absence or presence of a metal ion–coordinating histidine in the active site. Mutational and metal-binding experiments revealed the presence of only acidic metal-coordinating residues in the active site of the CCHFV domain and a unique active-site conformation that was intermediate between those of His+ and His− endonucleases. On the basis of small-angle X-ray scattering (SAXS) and homology modeling results, we propose a protein topology for the CCHFV domain that, despite its larger size, has a structure overall similar to those of related endonucleases. These results suggest structural and functional conservation of the cap-snatching mechanism among sNSVs.

The Cap-Snatching Mechanism of Bunyaviruses

“…The presence of an endonuclease domain has been demonstrated in the N-terminal region of several bunyaviral L proteins [18][19][20][21][22][23][24][25][26]. The active site residues and their biological relevance for bunyavirus transcription have been confirmed using in vitro biochemistry and cell-based eIF4F: Eukaryotic translation initiation factor 4F.…”

“…Whereas in His+ endonucleases this residue is involved in the coordination of the second metal ion, in His-endonucleases the distance to the active site is too large. Indeed, this Glu/Asp residue of His-endonucleases was shown to be dispensable for viral transcription [21,24]. The His+/His-classification also correlates with the in vitro activity of the isolated endonuclease domain.…”

“…minigenome systems [21,[27][28][29]. All atomic endonuclease structures of viruses from different families of the Bunyavirales share a common two-lobed, kidney-shaped architecture (Figure 2A) [18][19][20][21][22][23][24][25]. One of these lobes is mainly composed of α-helices forming a helix bundle; only Toscana virus endonuclease contains an additional small β-sheet in this region.…”

“…All bunyavirus endonucleases harbor a PD(E/D)K active site motif and coordinate two divalent metal ions crucial for catalytic activity. In addition, these endonucleases have been classified as either His-or His+, depending on whether or not they have a histidine upstream of the active site sequence motif that coordinated the first cation [21,24] ( Figure 2B,C and Table 2). His-endonucleases contain a glutamate or aspartate in a similar position as the histidine in His+ endonucleases but this acidic residue seems to be rather involved in the coordination of the second metal ion ( Figure 2B,C).…”

Structural and functional similarities in bunyaviruses: Perspectives for pan‐bunya antivirals

“…The endonuclease cleaves the 5′‐end of host mRNAs to acquire primers for viral mRNA transcription. The manganese‐dependent endonuclease domain of bunyaviruses is located at the N‐terminus of the L‐protein, with exception of the nairoviruses where it is located more downstream (Figure A) . The aa similarity of the endonuclease domain of species within the four studied bunyaviral families is 50% to 80% conserved, whereas between families, this is 30% to 45%.…”