Complete genome sequence analysis of candidate human rotavirus vaccine strains RV3 and 116E

Rippinger, Christine M.; Patton, John T.; McDonald, Sarah

doi:10.1016/j.virol.2010.06.005

Cited by 39 publications

(33 citation statements)

References 55 publications

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“…Reaction mixtures contained 50 mM TrisHCl (pH 7.1); 1.5% polyethylene glycol; 2 mM dithiothreitol; 1.5 units of RNasin (Promega); 20 mM magnesium acetate; 4 mM MnCl 2 ; 1.25 mM each ATP, CTP, UTP, and GTP; 10 Ci of [␣- Sequence and structural analyses. The VP1 and VP2 gene sequences of murine rotavirus strain EB were determined according to a method described previously by Rippinger et al (22). Briefly, viral RNA was harvested from intestinal homogenates of EB-infected neonatal BALB/c mice (gift from H. Greenberg, Stanford University).…”

Section: Methodsmentioning

confidence: 99%

Rotavirus VP2 Core Shell Regions Critical for Viral Polymerase Activation

McDonald

Patton

2011

J Virol

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The innermost VP2 core shell of the triple-layered, icosahedral rotavirus particle surrounds the viral genome and RNA processing enzymes, including the RNA-dependent RNA polymerase (VP1). In addition to anchoring VP1 within the core, VP2 is also an essential cofactor that triggers the polymerase to initiate double-stranded RNA (dsRNA) synthesis using packaged plus-strand RNA templates. The VP2 requirement effectively couples packaging with genome replication and ensures that VP1 makes dsRNA only within an assembling previrion particle. However, the mechanism by which the rotavirus core shell protein activates the viral polymerase remains very poorly understood. In the current study, we sought to elucidate VP2 regions critical for VP1-mediated in vitro dsRNA synthesis. By comparing the functions of proteins from several different rotaviruses, we found that polymerase activation by the core shell protein is specific. Through truncation and chimera mutagenesis, we demonstrate that the VP2 amino terminus, which forms a decameric, internal hub underneath each 5-fold axis, plays an important but nonspecific role in VP1 activation. Our results indicate that the VP2 residues correlating with polymerase activation specificity are located on the inner face of the core shell, distinct from the amino terminus. Based on these findings, we predict that several regions of VP2 engage the polymerase during the concerted processes of rotavirus core assembly and genome replication.

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Section: Methodsmentioning

confidence: 99%

Rotavirus VP2 Core Shell Regions Critical for Viral Polymerase Activation

McDonald

Patton

2011

J Virol

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“…Our sequence and FJ998273 both differ from the original published sequence (GenBank accession no. U16299) in showing serine rather than proline at amino acid position 71 (48,60). The proteins were soluble, and the same batches proved suitable for structural studies (7,31,61).…”

Section: Methodsmentioning

confidence: 98%

Relative Roles of GM1 Ganglioside, N -Acylneuraminic Acids, and α2β1 Integrin in Mediating Rotavirus Infection

Fleming

Böhm

Dang

et al. 2014

J Virol

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N-acetyl-and N-glycolylneuraminic acids (Sia) and ␣2␤1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl ␣-D-N-acetylneuraminide treatment, but did not alter ␣2␤1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidasetreated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of ␣2␤1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCERotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

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“…49 The vaccine is based on a G9P [11] strain called 116E, originally isolated from a child in Delhi, with a VP4 similar to various bovine RVs. [50][51][52][53] There is also a second candidate based on live attenuated rotavirus strain called RV3, a G3P [6], which contains human RV-like VP7 and VP4 proteins and was isolated from neonates. 50 However, there is a newer approach reported from Wen et al in 2012 based on truncated recombinant VP8* (ΔVP8*) proteins of human rotavirus strain Wa P [8], DS-1 P [4] or 1076 P [6], which was expressed in E. coli in high yield.…”

Section: Rotavirus Immunizationmentioning

confidence: 99%