Rescue of Infectivity by Sequential in Vitro Transcapsidation of Rotavirus Core Particles with Inner Capsid and Outer Capsid Proteins

Chen, Dayue; Ramig, Robert F.

doi:10.1006/viro.1993.1315

Cited by 20 publications

(16 citation statements)

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“…A systematic examination will be necessary to determine what early step of the infectious cycle, i.e., attachment, penetration, or uncoating, is enhanced by additional trypsin cleavage of TLPs. This can be tested by mutation of these sites in VP4 and transcapsidating native core particles with expressed VP4-VP6 complexes (7,21).…”

Section: Discussionmentioning

confidence: 99%

Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Crawford¹,

Mukherjee²,

Estes³

et al. 2001

J Virol

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126

103

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Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5‫ء‬ and VP8 ‫ء‬ in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8 ‫ء‬ portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5 ‫ء‬ , at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.Rotaviruses are the leading cause of severe gastroenteritis in young children worldwide (16). Structural and biochemical analyses show that rotaviruses are large, icosahedral particles having a complex architecture consisting of three concentric capsid layers surrounding a genome of 11 segments of doublestranded RNA (41, 42). The innermost capsid layer, composed of VP2, encloses the genomic double-stranded RNA. A significant portion of the genomic RNA, particularly that in close contact with the inner surface of the VP2 layer, is icosahedrally ordered (43). Anchored to the inner surface of VP2 at the icosahedral vertices are two proteins, VP1 and VP3, involved in transcription of the genome within the intact particle. The intermediate capsid layer is composed of trimers of VP6 organized on a Tϭ13 (levo) icosahedral lattice (44). The outermost layer in the infectious virus is composed of the major capsid glycoprotein VP7 and the hemagglutinin spike protein VP4 (16). VP7 is present as 780 molecules grouped as 260 trimers at the local and strict threefold axes of a Tϭ13 left-handed icosahedral lattice (44, 51). The VP7 ...

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Section: Discussionmentioning

confidence: 99%

Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Crawford¹,

Mukherjee²,

Estes³

et al. 2001

J Virol

Self Cite

126

103

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“…Chimeric viral particles composed of different viral strains or viral species, that enable us to discriminate respective proteins in the assembled particle by serological or biochemical methods are considered to prove this. Transcapsidation studies between different strains or species of rotavirus 14,15 bluetongue virus 16 and RDV, as mentioned above, 12 did not aim to see the molecular bridge that plays a critical role in constructing the unique double-layered structure.…”

Section: Introductionmentioning

confidence: 97%

Transcapsidation and the Conserved Interactions of Two Major Structural Proteins of a Pair of Phytoreoviruses Confirm the Mechanism of Assembly of the Outer Capsid Layer

Miyazaki

Hagiwara

Naitow³

et al. 2005

Journal of Molecular Biology

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“…Recoating with VP7 S-S. Recombinant VP7 coats DLPs completely and efficiently (2,56). The incorporation of VP4 in its correct conformation requires a VP4 excess, the addition of VP4 before VP7, and careful adjustment of conditions (2,35,56).…”

Section: Vp7 Neutralization Epitopes Vp7 Is a Camentioning

confidence: 99%

“…The incorporation of VP4 in its correct conformation requires a VP4 excess, the addition of VP4 before VP7, and careful adjustment of conditions (2,35,56). Selective trypsin sensitivity of VP4 is an indicator of proper recoating, as VP7 protects VP4 from nonspecific tryptic digestion and restricts cleavage to specific sites between VP8* and VP5* (2,4,18,20,35,56).…”

Section: Vp7 Neutralization Epitopes Vp7 Is a Camentioning

confidence: 99%

“…The trypsin-activated spike bears two lectinlike VP8* attachment domains at its tip, supported by three VP5*s; the spike appears to have lost the attachment domain of the third VP8* (52). The VP7 layer anchors the VP4 spikes onto the underlying double-layered particle (DLP) and allows proper proteolytic cleavage of VP4 to prime it for membrane disruption (2,56). During cell entry, the VP7-VP4 outer layer uncoats, releasing the intact DLP into the cytosol.…”

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Cross-Linking of Rotavirus Outer Capsid Protein VP7 by Antibodies or Disulfides Inhibits Viral Entry

Aoki

Trask

Coulson

et al. 2011

J Virol

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Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter-and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of ␣-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.

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Rescue of Infectivity by Sequential in Vitro Transcapsidation of Rotavirus Core Particles with Inner Capsid and Outer Capsid Proteins

Cited by 20 publications

References 0 publications

Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Trypsin Cleavage Stabilizes the Rotavirus VP4 Spike

Transcapsidation and the Conserved Interactions of Two Major Structural Proteins of a Pair of Phytoreoviruses Confirm the Mechanism of Assembly of the Outer Capsid Layer

Cross-Linking of Rotavirus Outer Capsid Protein VP7 by Antibodies or Disulfides Inhibits Viral Entry

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