Structure of a functional cap-binding domain in Rift Valley fever virus L protein

Rift Valley fever virus (RVFV) belongs to the family of Phenuiviridae within the order of Bunyavirales. The virus may cause fatal disease both in livestock and humans, and therefore, is of great economical and public health relevance. In analogy to the influenza virus polymerase complex, the bunyavirus L protein is assumed to bind to and cleave off cap structures of cellular mRNAs to prime viral transcription. However, even though the presence of an endonuclease in the N-terminal domain of the L protein has been demonstrated for several bunyaviruses, there is no evidence for a cap-binding site within the L protein. We solved the structure of a C-terminal 117 amino acid-long domain of the RVFV L protein by X-ray crystallography. The overall fold of the domain shows high similarity to influenza virus PB2 cap-binding domain and the putative non-functional cap-binding domain of reptarenaviruses. Upon co-crystallization with m7GTP, we detected the cap-analogue bound between two aromatic side chains as it has been described for other cap-binding proteins. We observed weak but specific interaction with m7GTP rather than GTP in vitro using isothermal titration calorimetry. The importance of m7GTP-binding residues for viral transcription was validated using a RVFV minigenome system. In summary, we provide structural and functional evidence for a cap-binding site located within the L protein of a virus from the Bunyavirales order

Biochemical characterization of the Lassa virus L protein

The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthomyxovirus polymerase complex. Although the L protein did not exhibit cap-snatching endonuclease activity, it synthesized RNA in vitro. RNA polymerization required manganese rather than magnesium ions, was independent of nucleotide primers, and was inhibited by viral Z protein. Maximum activity was mediated by double-stranded promoter sequences with a minimum length of 17 nucleotides, containing a nontemplated 5′-G overhang, as in the natural genome context, as well as the naturally occurring base mismatches between the complementary promoter strands. Experiments with various short primers revealed the presence of two replication initiation sites at the template strand and evidence for primer translocation as proposed by the prime-and-realign hypothesis. Overall, our findings provide the foundation for a detailed understanding of the mechanistic differences and communalities in the polymerase proteins of segmented negative-strand RNA viruses and for the search for antiviral compounds targeting the RNA polymerase of Lassa virus

Structural insights into reptarenavirus cap-snatching machinery

<div><p>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.</p></div

Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein–RNA interactions and the development of antiviral strategies against this group of emerging human pathogens

Structure and thermal stabilization of CASV endonuclease.

<p><b>A)</b> Ribbon diagram of the CASV endonuclease crystal structure. N- and C-termini are labelled and active site residues are shown as sticks. The conserved β-sheet and the long α-helix are colored in orange, a conserved helix-bundle domain in green and the remaining part with loops and α-helices in yellow. <b>B)</b> Endonuclease structures of LASV (PDB ID 5J1P), LCMV (PDB ID 3JSB) and PICV (PDB ID 4I1T) are shown as ribbon diagrams and colored according to CASV endonuclease in A). Manganese ions of LASV structure 5J1P are shown as red spheres, active site residues are shown as sticks, and N- and C-termini are marked. <b>C)</b> Electrostatic surface potential of the endonuclease structures shown in A) and B). The surface potential is shown from -5 KT/e in red to +5 KT/e in blue and was calculated using PDB2PQR and the APBS-tool of PyMOL. <b>D)</b> Thermal stability of CASV endonuclease depending on Mn²⁺ concentration. Melting temperatures are presented as mean and standard deviations of three independent measurements. Stability of the protein was tested in presence of different concentrations of Mn²⁺ and in presence of 10 mM EDTA. <b>E)</b> Close-up of the superimposed endonuclease active sites of the structures shown in A) and B). Conserved active site residues are shown as sticks, and manganese ions of LASV structure 5J1P are shown as red spheres.</p

Atomic structure of isolated CASV L-Cterm domain 2.

<p><b>A)</b> Ribbon diagram of CASV L-Cterm domain 2 structure. Chain A is shown in palegreen, chain B in grey. The N- and C-termini are marked and potential cap-binding aromatic sidechains Y1872 and W1818 are shown as sticks and colored in orange. <b>B)</b> Superimposition of SAXS derived molecular shape of L-Cterm at a concentration of 4.5 mg/ml and ribbon diagram of crystal structure. <b>C)</b> Superimposition of ribbon diagrams of chain A and B from isolated CASV L-Cterm domain 2 crystal structure (magenta and yellow, respectively) and L-Cterm crystal structure (green). Potential cap-binding aromatic sidechains are highlighted with saturate colors. <b>D)</b> Representation of chain B of L-Cterm domain 2 colored by B-factor with the highest observed B being 106 (orange) and the lowest 22 (dark blue).</p

Comparison of CASV L-Cterm structure with influenza PB2 (PDB ID 5FMM) structure.

<p><b>A)</b> Comparison of domain arrangements within PB2 and L-Cterm. Identifiers of the areas within the protein are shown in the bars. Domain 1 (D1) of L-Cterm is separated into three parts (D1-I, D1-II, and D1-III). Linkers to domain 2 or the cap-binding domain are colored in yellow. Residue numbers of the differently colored areas are given below the bars. N- and C-termini are labelled. C-terminal parts of PB2 missing in the figure are indicated by dashed lines. <b>B)</b> Structures of parts I and II of L-Cterm domain 1 (left panel) and the mid-link domains of PB2 (right panel) are shown as ribbon diagrams. Colors are coded as presented in A). Linkers to domain 2 and the cap-binding domain are shown in yellow. N-termini are labelled. <b>C)</b> Comparison of L-Cterm domain 2 (left panel) with PB2 cap-binding domain (right panel) with structures presented as ribbon diagrams. Structurally similar elements have similar colors. Linkers to other domains are colored in yellow. <b>D)</b> Structural comparison of parts II and III of L-Cterm domain 1 (left panel) and link-627 domains of PB2 (right panel). Structures are shown as ribbon diagrams. Colors are coded as in A). β-strands of part III of L-Cterm domain 1 and 627-domain of PB2 are colored in red. C-termini are labelled.</p

Atomic structure of CASV L-Cterm.

<p><b>A)</b> The structure of the protein dimer in the asymmetric unit is shown as a ribbon diagram in front and side view. Chain A is colored in blue and green, chain B is colored in dark and light grey. N- and C-termini are labelled. <b>B)</b> Chain A is shown as a ribbon diagram. N- and C-termini are labelled. Domain 1 is shown in blue, domain 2 in green. <b>C)</b> Superimposition of SAXS-derived molecular shape with the crystal structure (ribbon diagram) confirms the dimeric conformation of the protein at 1 mg/ml in solution.</p

Examination of CASV L-Cterm cap-binding in comparison to influenza virus PB2.

<p><b>A)</b> Comparison of CASV L-Cterm domain 2 (left panel) with PB2 cap-binding domain (PDB ID 2VQZ, right panel) with structures presented as ribbon diagrams. Structurally similar elements have the same color. Potential cap-binding aromatic residues in CASV L-Cterm and actual cap-binding residues in PB2 are shown as sticks and colored in orange. Bound m⁷GTP molecules are shown as sticks. <b>B)</b> The figure shows binding of m⁷GTP to the CASV L-Cterm dimer in the crystal after soaking experiments. An overview (left) and close-up (right) are shown. CASV L-Cterm is presented as a ribbon diagram with the residues relevant for binding shown as sticks. m⁷GTP is shown as sticks and the surrounding electron density (2|Fo|-|Fc| map at 2σ) as blue mesh. <b>C)</b> Interaction of W1818 with P1810 from a neighboring loop. CASV L-Cterm domain 2 is shown as green ribbon diagram, potential cap-binding residue Y1872 and W1818 are shown as orange sticks, and P1810 as blue sticks.</p