Melina A. Agosto

Mammalian reovirus, a nonfusogenic nonenveloped virus, forms size-selective pores in a model membrane

During cell entry, reovirus particles with a diameter of 70–80 nm must penetrate the cellular membrane to access the cytoplasm. The mechanism of penetration, without benefit of membrane fusion, is not well characterized for any such nonenveloped animal virus. Lysis of RBCs is an in vitro assay for the membrane perforation activity of reovirus; however, the mechanism of lysis has been unknown. In this report, osmotic-protection experiments using PEGs of different sizes revealed that reovirus-induced lysis of RBCs occurs osmotically, after formation of small size-selective lesions or “pores.” Consistent results were obtained by monitoring leakage of fluorophore-tagged dextrans from the interior of resealed RBC ghosts. Gradient fractionations showed that whole virus particles, as well as the myristoylated fragment μ1N that is released from particles, are recruited to RBC membranes in association with pore formation. We propose that formation of small pores is a discrete, intermediate step in the reovirus membrane-penetration pathway, which may be shared by other nonenveloped animal viruses.

Peptides released from reovirus outer capsid form membrane pores that recruit virus particles

Nonenveloped animal viruses must disrupt or perforate a cell membrane during entry. Recent work with reovirus has shown formation of size‐selective pores in RBC membranes in concert with structural changes in capsid protein μ1. Here, we demonstrate that μ1 fragments released from reovirus particles are sufficient for pore formation. Both myristoylated N‐terminal fragment μ1N and C‐terminal fragment ϕ are released from particles. Both also associate with RBC membranes and contribute to pore formation in the absence of particles, but μ1N has the primary and sufficient role. Particles with a mutant form of μ1, unable to release μ1N or form pores, lack the ability to associate with membranes. They are, however, recruited by pores preformed with peptides released from wild‐type particles or with synthetic μ1N. The results provide evidence that docking to membrane pores by virus particles may be a next step in membrane penetration after pore formation by released peptides.

A Role for Molecular Chaperone Hsc70 in Reovirus Outer Capsid Disassembly

After crossing the cellular membrane barrier during cell entry, most animal viruses must undergo further disassembly before initiating viral gene expression. In many cases, these disassembly mechanisms remain poorly defined. For this report, we examined a final step in disassembly of the mammalian reovirus outer capsid: cytoplasmic release of the central, δ fragment of membrane penetration protein μ1 to yield the transcriptionally active viral core particle. An in vitro assay with reticulocyte lysate recapitulated the release of intact δ molecules. Requirements for activity in this system were shown to include a protein factor, ATP, and Mg2+ and K+ ions, consistent with involvement of a molecular chaperone such as Hsc70. Immunodepletion of Hsc70 and Hsp70 impaired δ release, which was then rescued by addition of purified Hsc70. Hsc70 was associated with released δ molecules not only in the lysate but also during cell entry. We conclude that Hsc70 plays a defined role in reovirus outer capsid disassembly, during or soon after membrane penetration, to prepare the entering particle for gene expression and replication.

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

ABSTRACT The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the μ1 protein, each complexed with three copies of the protector protein σ3. Conformational changes in μ1 following the proteolytic removal of σ3 lead to release of the myristoylated N-terminal cleavage fragment μ1N and ultimately to membrane penetration. The μ1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant μ1 trimers and synthetic μ1N peptides with both RBCs and liposomes. The μ1 trimer mediates hemolysis and liposome disruption under conditions that promote the μ1 conformational change, and mutations that inhibit μ1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free μ1 trimer. Autolytic cleavage to form μ1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that μ1N cleavage is not required when steric barriers are removed. Synthetic myristoylated μ1N 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 β-strand secondary structure, suggest a model in which μ1N β-hairpins assemble in the membrane to form a β-barrel pore.

The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration.

Rhodopsin recycling via the retromer, rather than degradation through lysosomes, can alleviate light-induced photoreceptor degeneration in Drosophila.

A positive-feedback mechanism promotes reovirus particle conversion to the intermediate associated with membrane penetration

Membrane penetration by reovirus is associated with conversion of a metastable intermediate, the ISVP, to a further-disassembled particle, the ISVP*. Factors that promote this conversion in cells are poorly understood. Here, we report the in vitro characterization of a positive-feedback mechanism for promoting ISVP* conversion. At high particle concentration, conversion approximated second-order kinetics, and products of the reaction operated in trans to promote the conversion of target ISVPs. Pore-forming peptide μ1N, which is released from particles during conversion, was sufficient for promoting activity. A mutant that does not undergo μ1N release failed to exhibit second-order conversion kinetics and also failed to promote conversion of wild-type target ISVPs. Susceptibility of target ISVPs to promotion in trans was temperature dependent and correlated with target stability, suggesting that capsid dynamics are required to expose the interacting epitope. A positive-feedback mechanism of promoting escape from the metastable intermediate has not been reported for other viruses but represents a generalizable device for sensing a confined volume, such as that encountered during cell entry.

Thermolabilizing Pseudoreversions in Reovirus Outer-Capsid Protein μ1 Rescue the Entry Defect Conferred by a Thermostabilizing Mutation

ABSTRACT Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein μ1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to μ1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of μ1-based thermostabilization can occur by a general mechanism of μ1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between μ1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in μ1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.

Oligomeric State of Purified Transient Receptor Potential Melastatin-1 (TRPM1), a Protein Essential for Dim Light Vision

Background: TRPM1 is essential for the light response of retinal depolarizing bipolar cells. Results: Recombinant purified TRPM1 is mostly dimeric, and a low resolution cryo-EM structure is presented. Conclusion: Because most TRP channels function as tetramers, active TRPM1 channels likely require additional partner subunits. Significance: The results suggest a novel paradigm for structure and regulation within the TRP channel family. Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.

Domain organization and conformational plasticity of the G protein effector, PDE6.

Background: PDE6, the rod photoreceptor phosphodiesterase, is the key effector enzyme in phototransduction. Results: EM reconstructions of PDE6 complexed with various probes are presented. Conclusion: Fitting of x-ray structures yielded an atomic model of the catalytic subunits, and the locations of other structural features are indicated. Significance: These data offer the most complete view to date of the PDE6 holoenzyme. The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6α and -β, each contain one catalytic domain and two non-catalytic GAF domains, whereas two small inhibitory PDE6γ subunits allow tight regulation by the G protein transducin. The structure of holo-PDE6 in complex with the ROS-1 antibody Fab fragment was determined by cryo-electron microscopy. The ∼11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. A structure of PDE6 in complex with prenyl-binding protein (PrBP/δ) indicated the location of the PDE6 C-terminal prenylations. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6γ revealed that PDE6γ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. Removal of PDE6γ caused dramatic structural rearrangements, which were reversed upon its restoration.

Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods.

Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.