Structural insights into reptarenavirus cap-snatching machinery

Abstract

Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed.

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.…”

“…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.…”

“…The His+/His-classification also correlates with the in vitro activity of the isolated endonuclease domain. Whereas His+ endonucleases show high activity in vitro [18,21,22,25], His-endonucleases have been found to be either inactive or display very low in vitro activity [19][20][21]23,24], although from cell-based assays it is clear that they are functionally active [21].…”

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

“…LACV-L CBD shares a conserved minimal fold with equivalent structures from RVFV ( Phenuiviridae ) 8 , CASV ( Arenaviridae ) 9 and influenza virus ( Orthomyxoviridae ) 14 , consisting in an antiparallel β-sheet stacked against an α-helix ( Supplementary Figure 5 ). In addition, LACV-L CBD contains an insertion consisting of the three-stranded β-sheet (β38, β39, β40), the α-helix 78 and charged loops (1932-1936 and 1956-1963).…”

“…The mid-domain fold is conserved between Orthomyxoviridae and Arenaviridae polymerases 9, 11 ( Supplementary Figure 6b ), whereas the distal C-terminal region of the polymerase differs between the three families. Orthomyxoviridae and Arenaviridae respectively have a PB2 627-domain and D1-III domain that are structurally related 9, 11 , whereas the ZBD present in LACV has a different fold. This suggests that Orthomyxoviridae and Arenaviridae polymerases are more closely related ( Supplementary Figure 6c ).…”