Three-Dimensional Organization of Rift Valley Fever Virus Revealed by Cryoelectron Tomography

Freiberg, Alexander N.; Sherman, Michael B; Morais, Marc C.; Holbrook, Michael R.; Watowich, Stanley J.

doi:10.1128/jvi.01191-08

Cited by 121 publications

(140 citation statements)

References 49 publications

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“…Respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV), and rabies virus have highly symmetric, helical RNPs, the structures of which determine the morphology of the rod-or bullet-shaped virus particles (22)(23)(24)(25). In contrast, spherical phlebovirus particles have T = 12 icosahedral symmetry that does not include the RNP (26). N-RNA multimers from (Fig.…”

Section: Discussionmentioning

confidence: 99%

Phleboviruses encapsidate their genomes by sequestering RNA bases

Raymond¹,

Piper

Gerrard

et al. 2012

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

102

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Rift Valley fever and Toscana viruses are human pathogens for which no effective therapeutics exist. These and other phleboviruses have segmented negative-sense RNA genomes that are sequestered by a nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes of irregular, asymmetric structure, previously uncharacterized at high resolution. N binds nonspecifically to single-stranded RNA with nanomolar affinity. Crystal structures of Rift Valley fever virus N-RNA complexes reconstituted with defined RNAs of different length capture tetrameric, pentameric and hexameric N-RNA multimers. All N-N subunit contacts are mediated by a highly flexible α-helical arm. Arm movement gives rise to the three multimers in the crystal structures and also explains the asymmetric architecture of the RNP. Despite the flexible association of subunits, the crystal structures reveal an invariant, monomeric RNP building block, consisting of the core of one N subunit, the arm of a neighboring N, and four RNA nucleotides with the flanking phosphates. Up to three additional RNA nucleotides bind between subunits. The monomeric building block is matched in size to the repeating unit in viral RNP, as visualized by electron microscopy. N sequesters four RNA bases in a narrow hydrophobic binding slot and has polar contacts only with the sugar-phosphate backbone, which faces the solvent. All RNA bases, whether in the binding slot or in the subunit interface, face the protein in a manner that is incompatible with base pairing or with "reading" by the viral polymerase. transmitted by arthropod vectors and cause a variety of severe diseases worldwide. The Rift Valley fever virus is a highly infectious, mosquito-borne pathogen endemic to sub-Saharan Africa. RVFV infects livestock and humans and generally causes a flu-like illness; however, 1% of cases result in hemorrhagic fever disease, which has a 50% case-fatality rate (1). The closely related Toscana virus (TOSV) is endemic to the Mediterranean basin, is transmitted by infected phlebotomine sandflies and causes neurological dysfunction in humans (2). The membrane envelope of bunyaviruses encloses a three-segment, negative-sense RNA genome that is encapsidated by a nucleocapsid protein (N), forming the ribonucleoprotein (RNP) (3). However, the size and sequence of N vary extensively among the five genera of the Bunyaviridae family. Phlebovirus N are highly similar, but they appear unrelated to N of the other four bunyavirus genera.We showed previously that phleboviruses have a unique genome-packaging strategy and an RNP that lacks the helical symmetry observed in some other negative-sense RNA viruses (NSVs) (4, 5). However, the detailed interactions between phlebovirus N and the viral RNA genome are unknown. The crystal structure of a RVFV N monomer revealed a compact helical fold with two lobes (5). The structure of an RVFV N hexamer demonstrated conformational flexibility in N and showed a putative RNA binding site on the inner surface of the hexameric ring (6). An α-helical arm, which is...

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Section: Discussionmentioning

confidence: 99%

Phleboviruses encapsidate their genomes by sequestering RNA bases

Raymond¹,

Piper

Gerrard

et al. 2012

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

102

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“…The Gn-Gc heterodimer is the building block of 110 hexamers and 12 pentamers containing six and five of the Gn-Gc heterodimeric units, respectively. Together, these 122 capsomers make up the icosahedral shell of the virion, following a T ϭ 12 triangulation (13,24). Exposure of virions to the activating pH will lead to rearrangement of the metastable Gn-Gc heterodimer and concomitantly enforce the interactions between Gc subunits, resulting in the formation of a Gc homooligomer.…”

Section: Discussionmentioning

confidence: 99%

“…In the case of the RVFV virion, a 78-kDa protein consisting of pre-Gn and Gn regions has been identified as a third but minor structural component (27,60). Cryoelectron microscopy studies of RVFV virions showed that the viral envelope comprises 720 heterodimers of Gn (54 kDa) and Gc (56 kDa), forming 110 cylinder-shaped hexamers and 12 pentamers according to a Tϭ12 icosahedral lattice (13,24).…”

mentioning

confidence: 99%

Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein

Boer

Kortekaas

Spel

et al. 2012

J Virol

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bThe entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin-and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (

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“…However, the limited resolution (6.1 nm) impeded determination of the compositions of the capsomers. It remained unclear whether a single structural unit composed of both G N and G C forms both the five-and six-coordinated glycoprotein capsomers or whether G N alone forms some capsomers and G C alone forms the other capsomers (10). In addition, the organization of RNPs inside the virions could not be addressed.…”

mentioning

confidence: 99%

“…In fact, there exist conflicting reports about isolation of both heterodimers (32) and homodimers (36) from Triton X-100-solubilized UUKV virions. The structure of RVFV virions studied recently using electron cryo-tomography revealed five-and six-coordinated glycoprotein capsomers organized on a Tϭ12 lattice (10). However, the limited resolution (6.1 nm) impeded determination of the compositions of the capsomers.…”

mentioning

confidence: 99%

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for G _N -G _C Glycoprotein Heterodimers

Huiskonen

Överby

Weber

et al. 2009

J Virol

121

117

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Rift Valley fever virus (RVFV) is a member of the genusPhlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins G N and G C , organized on a T‫21؍‬ icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either G N and G C homodimers or G N -G C heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five-and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a G N -G C heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens.

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Three-Dimensional Organization of Rift Valley Fever Virus Revealed by Cryoelectron Tomography

Cited by 121 publications

References 49 publications

Phleboviruses encapsidate their genomes by sequestering RNA bases

Phleboviruses encapsidate their genomes by sequestering RNA bases

Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for G _N -G _C Glycoprotein Heterodimers

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Three-Dimensional Organization of Rift Valley Fever Virus Revealed by Cryoelectron Tomography

Cited by 121 publications

References 49 publications

Phleboviruses encapsidate their genomes by sequestering RNA bases

Phleboviruses encapsidate their genomes by sequestering RNA bases

Acid-Activated Structural Reorganization of the Rift Valley Fever Virus Gc Fusion Protein

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for G N -G C Glycoprotein Heterodimers

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Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for G _N -G _C Glycoprotein Heterodimers