Structure of the Lassa Virus Nucleoprotein Revealed by X-ray Crystallography, Small-angle X-ray Scattering, and Electron Microscopy

Brunotte, Linda; Kerber, Romy; Shang, Weifeng; Hauer, Florian; Haß, Meike; Gabriel, Marcin; Lelke, Michaela; Busch, Carola; Stark, Holger; Svergun, Dmitri I.; Betzel, Christian; Perbandt, Markus; Günther, Stephan

doi:10.1074/jbc.m111.278838

Cited by 51 publications

(72 citation statements)

References 49 publications

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“…Consistent with the previously reported RNA-free structures of LASV NP (16,19), NP 1-340 has a compact, mostly α-helical structure consisting of head and body regions that contain four and eight helices, respectively. The head region is formed by residues 8-24, 83-122, and 261-340, but the body is formed by residues 25-82 and 123-260 (Fig.…”

Section: Resultssupporting

confidence: 76%

“…S6). Furthermore, recent biochemical analysis of several residues previously proposed to be involved in cap-binding demonstrates that mutation of these residues results in defects in antigenome synthesis rather than defects in mRNA levels (19). Hence, the binding of ssRNA observed here combined with the positional homology of the individual, soaked-in nucleotides indicates that this site is likely not a binding site for cap, but rather is a binding site for the viral genome.…”

Section: Resultsmentioning

confidence: 79%

“…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 type I IFN (16,17).…”

mentioning

confidence: 99%

“…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 type I IFN (16,17).…”

mentioning

confidence: 99%

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Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

Hastie¹,

Liu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

121

165

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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...

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 79%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

Hastie¹,

Liu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

121

165

View full text Add to dashboard Cite

show abstract

“…Interestingly, X-ray crystallographic studies of N reveal a potential cap-binding motif in the N-terminal domain (44). Although cap-binding activity has not been directly demonstrated (8,20), it is tempting to speculate that N may replace eIF4E in recruiting eIF4G and eIG4A to viral mRNAs. Indeed, the hantavirus cap-binding nucleocapsid protein has been shown to interact directly with the small ribosomal protein RPS19 to bypass the eIF4F complex entirely (10,19).…”

Section: Discussionmentioning

confidence: 99%

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Baird

York

Nunberg

2012

J Virol

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Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic-and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.V iruses are wholly reliant on the host cell for replication. Viral proteins must access cellular systems to establish a productive environment for generating progeny virions while also evading the innate antiviral response. Viruses have therefore evolved diverse strategies and multifunctional proteins to coopt the host cell infrastructure. Elucidating these virus-cell interactions may suggest novel targets for antiviral intervention.Arenaviruses can cause acute hemorrhagic fevers with high mortality (34, 43). These viruses are endemic in rodent populations worldwide (47) and can be transmitted to humans by contact. Prototype arenavirus species include Lassa fever virus (LASV) in western Africa and Junín virus (JUNV) in the Pampas region of Argentina. These and other hemorrhagic fever arenaviruses pose ongoing threats to public health and raise concerns for biodefense planners. In the absence of licensed vaccines and specific antiviral therapies, these viruses are recognized as NIH category A priority pathogens (39). A critical understanding of the interactions of arenaviral proteins with the host cell will provide insight toward the development of effective therapies against arenavirus hemorrhagic fevers.The arenaviruses are a diverse family of enveloped negativesense RNA viruses that replicate entirely in the cell cytoplasm (9). The S and L RNA segments of the bipartite aren...

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Observation of arenavirus nucleoprotein heptamer assembly

et al. 2021

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Arenaviruses are enveloped viruses containing a segmented, negative, and ambisense single-stranded RNA genome wrapped with a nucleoprotein (NP). The NP is the most abundant viral protein in infected cells and plays a critical role in both replication/transcription and virion assembly. The NP associates with RNA to form a ribonucleoprotein (RNP) complex, and this implies self-assembly while the exact structure of this polymer is not yet known. Here, we report a measurement of the full-length Mopeia virus NP by negative stain transmission electron microscopy. We observed RNP complex particles with diameter 15 AE 1 nm as well as symmetric circular heptamers of the same diameter, consistent with previous observations.

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Structure of the Lassa Virus Nucleoprotein Revealed by X-ray Crystallography, Small-angle X-ray Scattering, and Electron Microscopy

Cited by 51 publications

References 49 publications

Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

Crystal structure of the Lassa virus nucleoprotein–RNA complex reveals a gating mechanism for RNA binding

Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Observation of arenavirus nucleoprotein heptamer assembly

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