Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Crawford, Sue E.; Mukherjee, Soma; Estes, Mary K.; Lawton, Jeffrey A.; Shaw, Andrea L.; Ramig, Robert F.; Prasad, B. V. Venkataram

doi:10.1128/jvi.75.13.6052-6061.2001

Cited by 126 publications

(114 citation statements)

References 52 publications

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“…There are no published structural data on the interaction of uncleaved VP4 with VP6 or VP7. The mass density for VP4, including the foot domain, is not seen in electron cryomicroscopy reconstructions of uncleaved particles (10).…”

Section: Discussionmentioning

confidence: 99%

“…Trypsin priming increases the infectivity of purified authentic TLPs approximately 20-to 66-fold (10,21). Trypsin priming increases the infectivity of recoated particles almost 1,000-fold (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…on May 12, 2018 by guest http://jvi.asm.org/ priming cleavage triggers a rearrangement that rigidifies VP4 into spikes (10). After an unknown second trigger, VP4 folds back on itself, forming a very stable umbrella-shaped trimer (18).…”

Section: Vol 80 2006 Recoated Rotavirus Particles 11301mentioning

confidence: 99%

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Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

Trask

Dormitzer

2006

J Virol

109

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Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic doublelayered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10 7 , yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.To initiate rotavirus infection, the nonenveloped, icosahedral, triple-layered particle (TLP or virion) must translocate a large transcriptionally active subviral particle, the double-layered particle (DLP), across a membrane and into the cytoplasm of a target cell. The DLP consists of concentric VP2 and VP6 icosahedral protein shells, which encapsidate 11 doublestranded RNA genome segments, the viral polymerase (VP1), and a capping enzyme (VP3). Biochemical and structural studies indicate that conformational rearrangements in VP4 and VP7, the two proteins that make up the outermost shell of the TLP, deliver the DLP into the cytoplasm. The dissociation of trimers of the plate-like protein VP7 in low-calcium environments mediates uncoating in vitro (17, 51). The spike protein VP4 is anchored in the DLP and protrudes through the VP7 layer (53,58). This protein undergoes a fold-back rearrangement that resembles the fusogenic rearrangements of enveloped virus fusion proteins (18), although the function of the VP4 conformational change has yet to be demonstrated experimentally.Because VP4 and VP7 are both targets of neutralizing antibodies, understanding the mechanism of cell entry is linked to understanding protection against rotavirus gastroenteritis, which kills approximately 500,000 children each year (43). The development of efficient techniques to incorporate recombinant VP4 and VP7 into infectious rotavirus virions would provide powerful tools to investigate how these ...

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Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

Trask

Dormitzer

2006

J Virol

109

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“…The latter, with a putative fusion peptide similar to that found in enveloped viruses such as Sindbis virus and Semiliki Forest virus, exhibits membranepermeabilizing activity (6,10). Based on the crystallographic studies of VP5*, in both the dimeric and trimeric forms, and cryoEM studies of nontrypsinized, trypisnized, and high-pHtreated rotavirus particles, it has been hypothesized that the VP4 spike undergoes a series of unique structural rearrangements from a disordered state prior to trypsinization to a dimeric state upon trypsinization and a trimeric state during the cell entry process (5,8,32,43). The underlying assumption in this hypothesis is that spikes are trimers instead of dimers as indicated by previous cryoEM studies of native mature virions and virions labeled with VP4-specific antibodies (34,40).…”

mentioning

confidence: 99%

Rotavirus Architecture at Subnanometer Resolution

Baker²,

Jiang³

et al. 2009

J Virol

Self Cite

113

101

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Rotavirus, a nonturreted member of the Reoviridae, is the causative agent of severe infantile diarrhea. The double-stranded RNA genome encodes six structural proteins that make up the triple-layer particle. X-ray crystallography has elucidated the structure of one of these capsid proteins, VP6, and two domains from VP4, the spike protein. Complementing this work, electron cryomicroscopy (cryoEM) has provided relatively low-resolution structures for the triple-layer capsid in several biochemical states. However, a complete, high-resolution structural model of rotavirus remains unresolved. Combining new structural analysis techniques with the subnanometer-resolution cryoEM structure of rotavirus, we now provide a more detailed structural model for the major capsid proteins and their interactions within the triple-layer particle. Through a series of intersubunit interactions, the spike protein (VP4) adopts a dimeric appearance above the capsid surface, while forming a trimeric base anchored inside one of the three types of aqueous channels between VP7 and VP6 capsid layers. While the trimeric base suggests the presence of three VP4 molecules in one spike, only hints of the third molecule are observed above the capsid surface. Beyond their interactions with VP4, the interactions between VP6 and VP7 subunits could also be readily identified. In the innermost T‫1؍‬ layer composed of VP2, visualization of the secondary structure elements allowed us to identify the polypeptide fold for VP2 and examine the complex network of interactions between this layer and the T‫31؍‬ VP6 layer. This integrated structural approach has resulted in a relatively high-resolution structural model for the complete, infectious structure of rotavirus, as well as revealing the subtle nuances required for maintaining interactions in such a large macromolecular assembly.Rotaviruses are double-stranded RNA viruses belonging to the family Reoviridae. They are the major causative agents of severe gastroenteritis in young children and animals (24). These relatively large (ϳ1,000 Å) nonenveloped icosahedral viruses consist of 11 segments of double-stranded RNA. Each of the 11 segments codes for one protein, with the exception of segment 11, which codes for two proteins. Of these 12 proteins encoded by the viral genome, six are structural and six are nonstructural. The proteins encoded by the rotavirus genes are well established, and their properties have been reviewed (13).Electron cryomicroscopy (cryoEM) structural analysis has shown that rotavirus has three concentric capsid layers that enclose the genomic RNA (35,36,42,45). Two outer icosahedral layers surround an inner Tϭ1 icosahedral (25, 45) layer composed of VP2, similar to the case for other members of the Reoviridae such as bluetongue virus (17), orthoreovirus (37), and rice dwarf virus (29, 46). The outermost capsid layer, composed of VP7, and the middle capsid layer, composed of VP6, both exhibit Tϭ13 icosahedral organization. Rotavirus architecture also features 132 aqueous channels, ϳ140...

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“…El primer contacto se establece a través de la VP8. 14 Los antígenos del grupo ABO están presentes en los eritrocitos y en las células epiteliales. 15 Al igual que el ácido siálico en el rotavirus VP8 animal, el rotavirus VP8 humano se une a los antígenos del grupo sanguíneo A en la misma localización.…”

Section: Discussionunclassified

¿Existe una relación entre la gastroenteritis rotavirus positivo y los grupos sanguíneos ABO? Estudio retrospectivo de cohorte

et al. 2014

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RESUMENIntroducción. El rotavirus es la causa más frecuente de diarrea grave en los niños. Son pocos los estudios clínicos sobre la relación entre la gastroenteritis por rotavirus y los grupos sanguíneos ABO. Objetivo. La meta de este estudio fue investigar la función de los grupos sanguíneos, en la incidencia de la gastroenteritis por rotavirus y la gravedad de la gastroenteritis. Métodos. Se realizó la investigación retrospectiva de las historias clínicas de los bebés que nacieron en nuestro hospital y a quienes, durante el seguimiento, se les diagnosticó gastroenteritis aguda. Resultados. En el estudio, se incluyeron 219 (36,3%) pacientes rotavirus positivos y 383 (63,6%) pacientes rotavirus negativos. El grupo sanguíneo A se detectó más frecuentemente en el grupo rotavirus positivo que en el grupo rotavirus negativo (50,6% frente a 42,2%, p= 0,047). Las tasas de hospitalización de los casos con gastroenteritis por rotavirus en los niños con el grupo sanguíneo A (30,6% frente a 8%, p < 0,001) fueron significativamente más altas. Conclusiones. Se determinó que la gastroenteritis por rotavirus fue más frecuente en los niños con el grupo sanguíneo A. Se detectó que las tasas de hospitalización de estos pacientes fueron más altas. Por lo tanto, en los niños con el grupo sanguíneo A, podría ser necesario estudiar cuidadosamente la gastroenteritis por rotavirus. INTRODUCCIÓNEl rotavirus es un virus ARN bicatenario que pertenece a la familia Reoviridae. Los rotavirus se dividen en siete grupos, dependiendo de las variaciones en la proteína VP6.1 En los seres humanos, solo se notifican las infecciones por rotavirus de los grupos A, B y C.2 La infección más frecuente por rotavirus, la del grupo A, causa entre el 21% y el 65% de las gastroenteritis graves durante la niñez. La infección por rotavirus puede manifestarse en diferentes cuadros clínicos, que varían desde una infección asintomática hasta una gastroenteritis grave acompañada por deshidratación.3 Por año, alrededor de dos millones de lactantes fallecen por gastroenteritis, principalmente en países en vías de desarrollo, y aproximadamente un tercio de estas muertes están asociadas a la gastroenteritis por rotavirus. La mayor parte de la mortalidad corresponde a los países en desarrollo. Además, en los países desarrollados, la mortalidad es más baja, mientras que la morbilidad es más alta.4 En Europa, de 29 661 de 100 000 lactantes con gastroenteritis por rotavirus que consultan al hospital; 368 de 100 000 lactantes son hospitalizados y 1 de 100 000 fallece por esta causa. 5,6 Aunque las tasas de mortalidad son más altas en los países en vías de desarrollo, el rotavirus continúa siendo importante en el mundo debido a la morbilidad, las tasas de hospitalización y la carga económica que causa en los países desarrollados.Existe evidencia de una fuerte relación entre los tipos de grupo sanguíneo y algunos agentes microbianos, como el Helicobacter pylori 7 y los norovirus.8 El propósito de este estudio fue investigar la relación de los grupos sanguíneos en la incide...

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Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Cited by 126 publications

References 52 publications

Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins

Rotavirus Architecture at Subnanometer Resolution

¿Existe una relación entre la gastroenteritis rotavirus positivo y los grupos sanguíneos ABO? Estudio retrospectivo de cohorte

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