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The ?1 72-96 loop controls conformational transitions during reovirus cell entry.

ABSTRACT: The reovirus outer capsid protein ?1 forms a lattice surrounding the viral core. In the native state, ?1 determines the environmental stability of the viral capsid. Additionally, during cell entry, ?1 undergoes structural rearrangements that facilitate delivery of the viral cores across the membrane. To determine how the capsid-stabilizing functions of ?1 impinge on the capacity of ?1 to undergo conformational changes required for cell entry, we characterized viruses with mutations engineered at charged residues within the ?1 loop formed by residues 72 to 96 (72-96 loop). This loop is proposed to stabilize the capsid by mediating interactions between neighboring ?1 trimers and between trimers and the core. We found that mutations at Glu89 (E89) within this loop produced viruses with compromised efficiency for completing their replication cycle. ISVPs of E89 mutants converted to ISVP*s more readily than those of wild-type viruses. The E89 mutants yielded revertants with second-site substitutions within regions that mediate interaction between ?1 trimers at a site distinct from the 72-96 loop. These viruses also contained changes in regions that control interactions within ?1 trimers. Viruses containing these second-site changes displayed restored plaque phenotypes and were capable of undergoing ISVP-to-ISVP* conversion in a regulated manner. These findings highlight regions of ?1 that stabilize the reovirus capsid and demonstrate that an enhanced propensity to form ISVP*s in an unregulated manner compromises viral fitness.

SUBMITTER: Sarkar P

PROVIDER: S-EPMC3838289 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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The μ1 72-96 loop controls conformational transitions during reovirus cell entry.

Sarkar Payel P Danthi Pranav P

Journal of virology 20131002 24

The reovirus outer capsid protein μ1 forms a lattice surrounding the viral core. In the native state, μ1 determines the environmental stability of the viral capsid. Additionally, during cell entry, μ1 undergoes structural rearrangements that facilitate delivery of the viral cores across the membrane. To determine how the capsid-stabilizing functions of μ1 impinge on the capacity of μ1 to undergo conformational changes required for cell entry, we characterized viruses with mutations engineered at ...[more]

PMID: 24089575

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Project description:Reovirus particles are covered with 200 ?1/?3 heterohexamers. Following attachment to cell surface receptors, reovirus is internalized by receptor-mediated endocytosis. Within the endosome, particles undergo a series of stepwise disassembly events. First, the ?3 protector protein is degraded by cellular proteases to generate infectious subviral particles (ISVPs). Second, the ?1 protein rearranges into a protease-sensitive conformation to generate ISVP*s and releases two virus-encoded peptides, ?1N and ?. The released peptides promote delivery of the genome-containing core by perforating the endosomal membrane. Thus, to establish a productive infection, virions must be stable in the environment but flexible to disassemble in response to the appropriate cellular cue. The reovirus outer capsid is stabilized by ?1 intratrimer, intertrimer, and trimer-core interactions. As a consequence of ISVP-to-ISVP* conversion, neighboring ?1 trimers unwind and separate. Located within the ?1 jelly roll ? barrel domain, which is a known regulator of ISVP* formation, residues 340 to 343 form a loop and have been proposed to facilitate viral entry. To test this idea, we generated recombinant reoviruses that encoded deletions within this loop (?341 and ?342). Both deletions destabilized the outer capsid. Notably, ?342 impaired the viral life cycle; however, replicative fitness was restored by an additional change (V403A) within the ?1 jelly roll ? barrel domain. In the ?341 and ?342 backgrounds, V403A also rescued defects in ISVP-to-ISVP* conversion. Together, these findings reveal a new region that regulates reovirus disassembly and how perturbing a metastable capsid can compromise replicative fitness.IMPORTANCE Capsids of nonenveloped viruses are composed of protein complexes that encapsulate, or form a shell around, nucleic acid. The protein-protein interactions that form this shell must be stable to protect the viral genome but also sufficiently flexible to disassemble during cell entry. Thus, capsids adopt conformations that undergo rapid disassembly in response to a specific cellular cue. In this work, we identify a new region within the mammalian orthoreovirus outer capsid that regulates particle stability. Amino acid deletions that destabilize this region impair the viral replication cycle. Nonetheless, replicative fitness is restored by a compensatory mutation that restores particle stability. Together, this work demonstrates the critical balance between assembling virions that are stable and maintaining conformational flexibility. Any factor that perturbs this balance has the potential to block a productive infection.

Project description:Cellular entry of nonenveloped and enveloped viruses is often accompanied by dramatic conformational changes within viral structural proteins. These rearrangements are triggered by a variety of mechanisms, such as low pH, virus-receptor interactions, and virus-host chaperone interactions. Reoviruses, a model system for entry of nonenveloped viruses, undergo a series of disassembly steps within the host endosome. One of these steps, infectious subviral particle (ISVP)-to-ISVP* conversion, is necessary for delivering the genome-containing viral core into host cells, but the physiological trigger that mediates ISVP-to-ISVP* conversion during cell entry is unknown. Structural studies of the reovirus membrane penetration protein, ?1, predict that interactions between ?1 and negatively charged lipid head groups may promote ISVP* formation; however, experimental evidence for this idea is lacking. Here, we show that the presence of polyanions (SO4(2-) and HPO4(2-)) or lipids in the form of liposomes facilitates ISVP-to-ISVP* conversion. The requirement for charged lipids appears to be selective, since phosphatidylcholine and phosphatidylethanolamine promoted ISVP* formation, whereas other lipids, such as sphingomyelin and sulfatide, either did not affect ISVP* formation or prevented ISVP* formation. Thus, our work provides evidence that interactions with membranes can function as a trigger for a nonenveloped virus to gain entry into host cells.Cell entry, a critical stage in the virus life cycle, concludes with the delivery of the viral genetic material across host membranes. Regulated structural transitions within nonenveloped and enveloped viruses are necessary for accomplishing this step; these conformational changes are predominantly triggered by low pH and/or interactions with host proteins. In this work, we describe a previously unknown trigger, interactions with lipid membranes, which can induce the structural rearrangements required for cell entry. This mechanism operates during entry of mammalian orthoreoviruses. We show that interactions between reovirus entry intermediates and lipid membranes devoid of host proteins promote conformational changes within the viral outer capsid that lead to membrane penetration. Thus, this work illustrates a novel strategy that nonenveloped viruses can use to gain access into cells and how viruses usurp disparate host factors to initiate infection.

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The ?1 72-96 loop controls conformational transitions during reovirus cell entry.

Publications

The μ1 72-96 loop controls conformational transitions during reovirus cell entry.

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