Thermostability of Reovirus Disassembly Intermediates (ISVPs) Correlates with Genetic, Biochemical, and Thermodynamic Properties of Major Surface Protein μ1

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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...

“…Particle concentrations were estimated by measuring the A 260 (22). Infection of L929 cells and plaque assays to determine viral titers were carried out as described previously (30,49).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Chandran¹,

Parker²,

Ehrlich

et al. 2003

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138

“…Factors that promote ISVP3ISVP* conversion in vitro include increased temperature, larger monovalent cations such as K ϩ and Cs ϩ , and higher particle concentration (2,6,7,8,10,36,37). The promoting factor in the cell is unknown, but low pH is clearly not required (23,24,51).…”

mentioning

confidence: 99%

“…The core remains intact, and mRNA synthesis and 5Ј capping are primarily accomplished by core proteins 3 and 2, respectively (18,34,44,52). Translocation of the reovirus core from an extracellular milieu to the cytoplasm probably occurs in an endosome or related structure, and 1 appears to be a principal agent of this process (1,3,10,11,19,27,29,31,33,36,37,38,42,56).…”

mentioning

confidence: 99%

Requirements for the Formation of Membrane Pores by the Reovirus Myristoylated μ1N Peptide

Zhang

Agosto

Ivanovic

et al. 2009

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The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the 1 protein, each complexed with three copies of the protector protein 3. Conformational changes in 1 following the proteolytic removal of 3 lead to release of the myristoylated N-terminal cleavage fragment 1N and ultimately to membrane penetration. The 1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant 1 trimers and synthetic 1N peptides with both RBCs and liposomes. The 1 trimer mediates hemolysis and liposome disruption under conditions that promote the 1 conformational change, and mutations that inhibit 1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free 1 trimer. Autolytic cleavage to form 1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that 1N cleavage is not required when steric barriers are removed. Synthetic myristoylated 1N peptide forms size-selective pores in liposomes, as measured by fluorescence dequenching of labeled dextrans of different sizes. Addition of a C-terminal solubility tag to the peptide does not affect activity, but sequence substitution V13N or L36D reduces liposome disruption. These substitutions are in regions of alternating hydrophobic residues. Their locations, the presence of an N-terminal myristoyl group, and the full activity of a C-terminally extended peptide, along with circular dichroism data that indicate prevalence of ␤-strand secondary structure, suggest a model in which 1N ␤-hairpins assemble in the membrane to form a ␤-barrel pore.

“…The central region of 1 that we identified as controlling the cell attachment efficiency of T1L 1 also affects 1 intratrimer interactions. Changes in this region alter the efficiency with which 1 undergoes conformational transitions (17,18,(43)(44)(45)(46). We previously demonstrated that a single polymorphic difference between T1L and T3D 1 (at amino acid 305) within this region controls strainspecific differences in the cell penetration function of T1L and T3D 1 (18).…”

Section: Discussionmentioning

confidence: 99%

Reovirus μ1 Protein Affects Infectivity by Altering Virus-Receptor Interactions

Thete

Snyder

Mainou

et al. 2016

Proteins that form the reovirus outer capsid play an active role in the entry of reovirus into host cells. Among these, the 1 protein mediates attachment of reovirus particles to host cells via interaction with cell surface glycans or the proteinaceous receptor junctional adhesion molecule A (JAM-A). The 1 protein functions to penetrate the host cell membrane to allow delivery of the genome-containing viral core particle into the cytoplasm to initiate viral replication. We demonstrate that a reassortant virus that expresses the M2 gene-encoded 1 protein derived from prototype strain T3D in an otherwise prototype T1L background (T1L/T3DM2) infects cells more efficiently than parental T1L. Unexpectedly, the enhancement in infectivity of T1L/T3DM2 is due to its capacity to attach to cells more efficiently. We present genetic data implicating the central region of 1 in altering the cell attachment property of reovirus. Our data indicate that the T3D 1-mediated enhancement in infectivity of T1L is dependent on the function of 1 and requires the expression of JAM-A. We also demonstrate that T1L/T3DM2 utilizes JAM-A more efficiently than T1L. These studies revealed a previously unknown relationship between two nonadjacent reovirus outer capsid proteins, 1 and 1. IMPORTANCEHow reovirus attaches to host cells has been extensively characterized. Attachment of reovirus to host cells is mediated by the 1 protein, and properties of 1 influence the capacity of reovirus to target specific host tissues and produce disease. Here, we present new evidence indicating that the cell attachment properties of 1 are influenced by the nature of 1, a capsid protein that does not physically interact with 1. These studies could explain the previously described role for 1 in influencing reovirus pathogenesis. These studies are also of broader significance because they highlight an example of how genetic reassortment between virus strains could produce phenotypes that are distinct from those of either parent.A ttachment of virus is the first step in the infection of host cells. Cell attachment occurs via interactions of viral attachment factors with host cell receptors. For enveloped viruses, viral glycoproteins embedded in the lipid membrane serve as attachment factors (1). For nonenveloped viruses, specific structural features on the capsid or sequences within the exposed portion of the viral structural proteins bind host receptors (1). Mutations within the receptor-binding site can alter the efficiency with which virus attaches to host cells and consequently modulate the capacity of the virus to establish infection. In viral systems where capsids are formed from multiple structural proteins, these proteins fit together in a precise geometric arrangement. Thus, changes to the properties of one capsid protein can influence the function of other capsid proteins. In this report, we highlight one such example by demonstrating a previously unknown functional relationship between two nonadjacent viral capsid proteins of mammalian orthoreo...