Arenaviruses cause disease in industrialized and developing nations alike. Among them, the hemorrhagic fever virus Lassa is responsible for ∼300,000-500,000 infections/y in Western Africa. The arenavirus nucleoprotein (NP) forms the protein scaffold of the genomic ribonucleoprotein complexes and is critical for transcription and replication of the viral genome. Here, we present crystal structures of the RNA-binding domain of Lassa virus NP in complex with ssRNA. This structure shows, in contrast to the predicted model, that RNA binds in a deep, basic crevice located entirely within the N-terminal domain. Furthermore, the NP-ssRNA structures presented here, combined with hydrogen-deuterium exchange/MS and functional studies, suggest a gating mechanism by which NP opens to accept RNA. Directed mutagenesis and functional studies provide a unique look into how the arenavirus NPs bind to and protect the viral genome and also suggest the likely assembly by which viral ribonucleoprotein complexes are organized.structural biology | virology T he arenavirus family has a worldwide distribution and contains significant human pathogens such as Lassa (LASV), Machupo, Junin, Lujo (1, 2), and lymphyocytic choriomeningitis virus. Of these arenaviruses, LASV carries the largest disease burden, causing 300,000 to 500,000 infections per year in Western Africa. It is also the hemorrhagic fever most frequently transported out of Africa to the United States and Europe (2-4).Arenaviruses have a bisegmented, negative-sense, singlestranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) (2). Of these proteins, NP is the most abundant in an infected cell. NP associates with L to form the ribonucleoprotein (RNP) core for RNA replication and transcription (5) and the matrix protein Z for viral assembly (6-8). The arenavirus NP also plays an important role in the suppression of the innate immune system (9-11).Genome and antigenome RNAs of negative-strand RNA viruses (NSV) do not exist as naked RNA, but rather as a RNP complex in which the RNA is encapsidated by the viral nucleoprotein. During replication of many negative-strand RNA viruses, the nascent nucleoprotein (usually termed N) is bound by a polymerase cofactor (often a phosphoprotein, termed P), which prevents polymerization of N and nonspecific encapsidation of host cell RNAs (12-15). The resulting complex is termed N 0 -P, in which N 0 denotes RNA-free N. The arenavirus, orthomyxovirus (flu), and bunyavirus (Hanta, Rift Valley Fever) families (i.e., segmented NSV) do not encode an analogous P protein, and the mechanism by which the nucleoprotein controls RNA binding during virus infection is not yet understood.The arenavirus nucleoprotein (termed NP instead of N) has distinct N-and C-terminal domains connected by a flexible linker (16)(17)(18)(19). The C-terminal domain functions as an exonuclease (16, 17) specific for dsRNA (17) and linked to antagonism of t...

Lassa virus causes hemorrhagic fever characterized by immunosuppression. The nucleoprotein of Lassa virus, termed NP, binds the viral genome. It also has an additional enzymatic activity as an exonuclease that specifically digests double-stranded RNA (dsRNA). dsRNA is a strong signal to the innate immune system of viral infection. Digestion of dsRNA by the NP exonuclease activity appears to cause suppression of innate immune signaling in the infected cell. Although the fold of the NP enzyme is conserved and the active site completely conserved with other exonucleases in its DEDDh family, NP is atypical among exonucleases in its preference for dsRNA and its strict specificity for one substrate. Here, we present the crystal structure of Lassa virus NP in complex with dsRNA. We find that unlike the exonuclease in Klenow fragment, the double-stranded nucleic acid in complex with Lassa NP remains base-paired instead of splitting, and that binding of the paired complementary strand is achieved by “relocation” of a basic loop motif from its typical exonuclease position. Further, we find that just one single glycine that contacts the substrate strand and one single tyrosine that stacks with a base of the complementary, non-substrate strand are responsible for the unique substrate specificity. This work thus provides templates for development of antiviral drugs that would be specific for viral, rather than host exonucleases of similar fold and active site, and illustrates how a very few amino acid changes confer alternate specificity and biological phenotype to an enzyme.

Summary Since its first identification 50 years ago, Marburgviruses have emerged several times, with 83–90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in nonhuman primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific “wing” antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure.

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucinlike domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem ␤-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.