Localization of membrane permeabilization and receptor binding sites on the VP4 hemagglutinin of rotavirus: implications for cell entry

During rotavirus entry, a virion penetrates a host cell membrane, sheds its outer capsid proteins, and releases a transcriptionally active subviral particle into the cytoplasm. VP5*, the rotavirus protein believed to interact with the membrane bilayer, is a tryptic cleavage product of the outer capsid spike protein, VP4. When a rotavirus particle uncoats, VP5* folds back, in a rearrangement that resembles the fusogenic conformational changes in enveloped-virus fusion proteins. We present direct experimental evidence that this rearrangement leads to membrane binding. VP5* does not associate with liposomes when mounted as part of the trypsinprimed spikes on intact virions, nor does it do so after it has folded back into a stably trimeric, low-energy state. But it does bind liposomes when they are added to virions before uncoating, and VP5* rearrangement is then triggered by addition of EDTA. The presence of liposomes during the rearrangement enhances the otherwise inefficient VP5* conformational change. A VP5* fragment, VP5CT, produced from monomeric recombinant VP4 by successive treatments with chymotrypsin and trypsin, also binds liposomes only when the proteolysis proceeds in their presence. A monoclonal antibody that neutralizes infectivity by blocking a postattachment entry event also blocks VP5* liposome association. We propose that VP5* binds lipid bilayers in an intermediate conformational state, analogous to the extended intermediate conformation of enveloped-virus fusion proteins.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A Rotavirus Spike Protein Conformational Intermediate Binds Lipid Bilayers

Trask

Kim

Harrison

et al. 2010

“…Passage of large nonenveloped particles across the cellular membrane barrier during entry is commonly thought to require localized disruption of the plasma membrane or lysis of an endocytic compartment (5,8,41). A "reverse budding" model in which particle translocation is mechanistically coupled to disassembly and membrane insertion of viral capsid proteins also has been proposed (66). Definitive evidence for any mechanism is nonetheless lacking.…”

Section: Vol 77 2003 Entry-related Structural Changes In Reovirus Imentioning

confidence: 99%

The δ Region of Outer-Capsid Proteinμ1 Undergoes Conformational Change and Release from ReovirusParticles during CellEntry

Chandran¹,

Parker²,

Ehrlich

et al. 2003

138

Cell entry by reoviruses requires a large, transcriptionally active subvirion particle to gain access to the cytoplasm. The features of this particle have been the subject of debate, but three primary candidates-the infectious subvirion particle (ISVP), ISVP*, and core particle forms-that differ in whether putative membrane penetration protein 1 and adhesin 1 remain particle bound have been identified. Experiments with antibody reagents in this study yielded new information about the steps in particle disassembly during cell entry. Monoclonal antibodies specific for the ␦ region of 1 provided evidence for a conformational change in 1 and for release of the ␦ proteolytic fragment from entering particles. Antiserum raised against cores provided evidence for entry-related changes in particle structure and identified entering particles that largely lack the ␦ fragment inside cells. Antibodies specific for 1 showed that it is also largely shed from entering particles. Limited coimmunostaining with markers for late endosomes and lysosomes indicated the particles lacking ␦ and 1 did not localize to those subcellular compartments, and other observations suggested that both the particles and free ␦ were released into the cytoplasm. Essentially equivalent findings were obtained with native ISVPs and highly infectious recoated particles containing wild-type proteins. Poorly infectious recoated particles containing a hyperstable mutant form of 1, however, showed no evidence for the in vitro and intracellular changes in particle structure normally detected by antibodies, and these particles instead accumulated in late endosomes or lysosomes. Recoated particles with hyperstable 1 were also ineffective at mediating erythrocyte lysis in vitro and promoting ␣-sarcin coentry and intoxication of cells in cultures. Based on these and other findings, we propose that ISVP* is a transient intermediate in cell entry which mediates membrane penetration and is then further uncoated in the cytoplasm to yield particles, resembling cores, that largely lack the ␦ fragment of 1.The entry of animal viruses into host cells is accompanied by proteolytic cleavage, protein conformational changes, and/or protein shedding events that result in partial or complete disassembly of entering particles. One key aspect of disassembly is the conversion of virions, which often have greater stability in the aqueous environments between cells, to particle forms with more hydrophobic surfaces that can interact with a cellular lipid bilayer and mediate the passage of viral components into the cytoplasm. Other key elements include the activation of viral particles or nucleocapsids for genome transcription, translation, and/or targeting to particular subcellular sites. We have been studying the disassembly cascade and cell entry mechanisms used by a group of large nonenveloped viruses, the mammalian orthoreoviruses (reoviruses).Reoviruses belong to the family Reoviridae, an evolutionarily divergent group of double-stranded RNA viruses whose human-infecting members...

“…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). Upon trypsinization, two subunits associate to form dimers as seen in the reconstructions of the trypsinized particles, and the other subunit remains disordered and associates to form a trimer only during the cell entry processes.…”

mentioning

confidence: 98%

Rotavirus Architecture at Subnanometer Resolution

Baker²,

Jiang³

et al. 2009

113

101

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...