The X-ray crystal structure of the N protein from the serious human pathogen CCHFV reveals a compact globular domain from which an extended arm domain protrudes. The globular domain contains positively charged surfaces that, because of their exposed and contiguous position on the exterior of the molecule and by the strict requirement of several residues on these surfaces for CCHFV gene expression, we propose are involved in RNA binding. In support of our identification of these putative RNA binding regions, the CCHFV N globular domain shows a high degree of structural similarity with the N-terminal RNA binding domain of the LASV N protein. The consequences of this close structural relationship are discussed further below.
In LASV, there is a proposed gating mechanism of RNA binding where helices α5 and α6 are repositioned to reveal the RNA binding surface (17
). The concept of a flexible arm being involved in gating RNA binding has also been proposed for RVFV N, which has an N-terminal arm interacting with an oligomerization groove on adjacent monomers and consequently exposing an RNA binding cleft (14
). CCHFV N may operate via a similar mechanism, although the structural elements involved in the gating process are likely different. The position of helix α5 in LASV corresponds to the beginning of the flexible loop leading to the CCHFV arm domain; thus, one possibility is that the arm of CCHFV N may be involved in switching between RNA-bound and unbound conformations.
Structural alignment () revealed that the CCHFV RNA binding pocket corresponds to the domain within LASV N that was initially thought to bind the cap (34
) but was later suggested to represent a binding pocket involved in the interaction with a single nucleotide of a bound RNA strand (17
). This raises the possibility either that CCHFV N binds RNA via two surfaces (platform and pocket) or that conformational rearrangements create a continuous RNA binding surface. It is quite possible that a combination of arm movement and structural rearrangements is required in order to reveal the appropriate RNA binding surfaces, which may not be evident in the apo crystal structures.
Because we do not know the length of RNA bound by each N monomer, or indeed the oligomeric form of N in the RNP, we cannot currently discriminate between any plausible RNA binding mechanisms or deduce what any required conformational changes might be. However, it seems reasonable, based on previous studies of RNP formation, to anticipate more details of the RNA binding mechanism to be revealed by a structure of the N protein in complex with RNA, possibly via a gating mechanism analogous to that seen for LASV.
The possibility that the arm domain can adopt different positions is supported by comparison of our N structure for strain Baghdad-12 with that for strain YL04057, reported recently (15
). While the globular domains align very closely, the arm domains adopt radically different positions, being rotated by nearly 180 degrees and with the arm apex being translated by 40 Å (). This rearrangement may have important consequences, as the two arm positions may represent interchangeable forms that possess different activities in critical N protein functions such as oligomerization or RNA binding. However, one possibility is that the arm positions are dictated by the different primary sequences of the respective strains.
Strain differences could account for a change in the preferred lowest energy state. However, the arm is more likely to be more mobile and free to adopt a number of conformations, taking into account the likeliness that an exposed α helix is not a stable structure in isolation. The single α helix linking the globular and arm domains is unlikely to provide a rigid link. Alternatively, these differences may have been forced by crystal packing. However, should the strain differences we observed also be reflected in solution, one consequence of the shifted arm position and sequence differences between the two N structures is an altered distribution of electrostatic surface potential, which may affect the RNA binding ability of the respective proteins. It is possible that the arm represents part of a gating mechanism allowing a switch between RNA-bound and unbound states. However, it is also plausible that the switch in arm position is responsible for conversion of monomeric N into higher-order multimers, a property that is required for RNP formation.
In addition to the radically different arm positions posing possible functional significance, the differing arm conformations of these two strains may also have a bearing on strategies for structure-based design of small-molecule inhibitors for use as antivirals.
A striking feature of the CCHFV N structure is that the arm domain displays a DEVD caspase cleavage motif at its apex, in the most accessible position on the entire molecule (). This exposed position, along with its strict conservation in all CCHFV strains, suggests that the virus has evolved to present the cleavage motif to the cellular environment, which is somehow beneficial to the virus life cycle. If possession of the exposed cleavage site were not beneficial to the virus or if caspase cleavage were a host defense mechanism, a fast-mutating RNA virus such as CCHFV would be predicted to quickly eliminate the motif. The functional significance of this caspase cleavage site is therefore an intriguing feature of the CCHFV N protein. To investigate the fate of the N protein fragments following cleavage, we performed caspase cleavage of the N protein in vitro and showed that the cleavage products remained associated as a single unit. This raises the interesting possibility that N protein functions may remain unaltered following cleavage. The cleaved N protein could of course have an altered tertiary or quaternary structure, which may influence function in a variety of ways, including interaction with different protein partners, the adoption of different oligomeric states, or alteration of RNA binding affinity. In order to best understand the functional relevance of this DEVD motif in the CCHFV life cycle, we need to manipulate the CCHFV genome with a view to studying the consequence of such a change in the context of a live virus infection of intact organisms. Unfortunately, such a system currently does not exist.
Intriguingly, the nucleocapsid protein (NP) of human-infecting strains of influenza A virus also possesses caspase cleavage sites which have been shown to possess important roles in the virus life cycle (51
). Infectious influenza viruses bearing mutations that abrogate caspase cleavage at an N-terminal recognition site could not be rescued, indicating that such alterations are lethal to virus viability, whereas mutations within a C-terminal caspase cleavage site rapidly reverted to the wild type to restore cleavability. These findings establish an important precedent that links caspase cleavability with virus fitness, and thus pathogenesis, suggesting a second functional surface (in addition to the RNA binding site) that could be targeted by antivirals.
Structural comparisons indicate that the CCHFV N protein globular domain exhibits a high degree of homology with the N-terminal domain of the N protein of LASV, a member of the Arenaviridae
family, whose members uniformly possess two RNA segments. In contrast, CCHFV N shows negligible structural similarity with the only other bunyavirus (RVFV) N protein for which high-resolution structural data are available. The finding that these bunyavirus N proteins appear essentially structurally unrelated yet the CCHFV N protein shows extremely high homology with the N protein from a virus of a different taxonomic family with a different number of genome segments is intriguing. Previous phylogenetic analyses based on L protein sequences have also described a close relationship between nairoviruses and arenaviruses (48
), and this conclusion is further supported by the phylogenetic analysis of N and RdRp protein sequences presented here ( and ). Further supporting this close relationship, arenaviruses and nairoviruses also possess unique aspects of cellular biology that are absent from other bunyaviruses, such as the dependence of cellular SKI-1 protease processing of their respective glycoprotein precursors (23
). However, the high degree of structural similarity we observed between the CCHFV and LASV N proteins is the most compelling evidence yet that arenaviruses have an ancestor in common with a current or past member of the Nairovirus
genus. This suggests that the current classification of all three-segment negative-strand RNA viruses as bunyaviruses may be oversimplistic and that the evident diversity within the Bunyaviridae
family may warrant reevaluation of its current taxonomic status.