Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein

Olal, Daniel; Dick, Alexej; Woods, Virgil L.; Liu, Tong; Li, Sheng; Devignot, Stéphanie; Weber, Friedemann; Saphire, Erica Ollmann; Daumke, Oliver

doi:10.1093/nar/gku229

Cited by 31 publications

(40 citation statements)

References 44 publications

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“…Treatment with benzonase during protein cleanup had no effect on nucleic acid binding. Similar observations have been made with nucleoproteins from Toscana (Olal et al, 2014), Bunyamwera and Schmallenberg virus (Ariza et al, 2013). Therefore, we assumed that the acquired nucleic acid may compete with the binding of the DNA substrate.…”

Section: Pre-treatment Of Snv-n With Rnase Enhanced Its Dna-degradingsupporting

confidence: 71%

Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity

et al. 2016

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We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity.

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Section: Pre-treatment Of Snv-n With Rnase Enhanced Its Dna-degradingsupporting

confidence: 71%

Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity

et al. 2016

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“…Additionally, hantavirus NP core also exhibits homology with NPs encoded by phleboviruses, including RVFV (PDB code 4H5 L) (11-13), SFTSV (PDB code 4J4X) (14,19), and Toscana virus (PDB code 4CSG) (58). The Z score between SNV NP core and the phlebovirus NP is approximately 5.8, and the RMSD of 148 aligned residues is 4.5 Å, suggesting that they have partial structural homology (Fig.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

Guo

Wang

Sun

et al. 2016

J Virol

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Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NP core ) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NP core exclude the N-and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NP core are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCEHantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection. Hantavirus causes severe infectious diseases in human and animals (1, 2). In addition to 24 representative species, increasing numbers of antigenically and genetically distinct hantaviruses have recently been registered as new members of the genus Hantavirus (3, 4). The reservoir hosts of hantaviruses in nature are primarily rodents, shrews, moles, and bats, and the transmission of hantaviruses via aerosols of rodent excreta to humans causes two human diseases: (i) hemorrhagic fever with renal syndrome (HFRS), which is primarily caused by Hantaan virus (HTNV) in Asia, by Puumala virus (PUUV) and Dobrava virus (DOBV) in Europe, and by Seoul virus (SEOV) worldwide, and ...

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“…Prior to hydrogen-deuterium exchange experiments, the optimal concentration of guanidine hydrochloride (GuHCl) for protein fragmentation with pepsin was determined as previously described (46)(47)(48). Final GuHCl concentrations of 1.0 and 2.0 M were found to produce the best fragmentation maps of EBOV VP35 residues 81 to 340 and MARV VP35 residues 60 to 329, respectively.…”

Section: Figmentioning

confidence: 99%

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

Bruhn

Kirchdoerfer

Urata

et al. 2017

J Virol

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Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.IMPORTANCE Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics. KEYWORDS VP35, filovirus, RNA-dependent RNA polymerase, phosphoprotein, coiled coil, oligomerization, Marburg virus, Ebola virus, X-ray crystallography M arburg virus (MARV) can cause severe hemorrhagic fever in humans with high case fatality rates. Though less well known than its relative Ebola virus (EBOV), MARV was the first filovirus identified. MARV has caused sporadic outbreaks since its identification in 1968, including the 1998 to 2000 outbreak in the Democratic Republic of the Congo, which occurred with 83% lethality, and the 2004 to 2005 outbreak in Angola, which occurred with 90% lethality (1). Currently, there are no approved vaccines or therapeutics available to treat individuals infected with MARV. Despite sharing ϳ50% nucleotide sequence identity, MARV and EBOV have several striking functional differences. MARV does not produce sGP or ssGP protein and does not require VP30 for transcription. MARV and EBOV also exhibit differences in their immune evasion strategies (2).

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Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein

Cited by 31 publications

References 44 publications

Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity

Sin Nombre hantavirus nucleocapsid protein exhibits a metal-dependent DNA-specific endonucleolytic activity

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

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