Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

Abstract

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins-1, 1, and 3-to enter host cells. 1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of 1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked 1, 1, and 3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to 1. The recoated particles bound to and infected cultured cells in a 1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant 1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the 1 protein. Additional experiments showed that recoated particles containing 1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound 1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of 1 with respect to its structure, assembly into particles, and roles in entry.

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

“…(Virions and ISVPs of some reovirus strains can bind to RBCs via 1 attachment to cell-surface sialic acid; however, particles of the strain used in this study do not bind in this way to bovine RBCs.) Similar results with recoated virus-like particles lacking 1 (32) confirmed that RG association is not 1 dependent (data not shown). In addition, viral cores lacking all outer-capsid proteins sedimented to the bottom, confirming that an outer-capsid protein is required for RG association (data not shown).…”

“…Digestion was stopped by addition of 300 g͞ml soybean trypsin inhibitor (Sigma) and incubation on ice for at least 15 min. T1L cores and cores recoated with WT T1L 1 and 3 were made as described (32), except that virions were digested for 3-4 h, cores were purified by banding on CsCl step gradients (1.30-1.53 g͞cm 3 ), and cores were recoated by incubating with insect cell lysates for Ϸ4 h, followed by banding on CsCl step gradients (1.30-1.45 g͞cm 3 ).…”

“…The membrane-perforation activity of ISVP*s can be assayed in vitro by lysis of RBCs (12,19,32,33). In this study we demonstrated that hemolysis occurs osmotically, after formation of small size-selective lesions, or ''pores,'' in the RBC membrane.…”

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

“…Because particle-free 1(N42A) can disrupt liposomes (Fig. 2B), we investigated the disruption activity of cores recoated with 1(N42A) (12,13). Previous work has shown that recoating with 1(N42A) yields correctly assembled virion-like particles, which undergo normal disassembly steps, including the structural rearrangement of 1 to a protease-sensitive form (42).…”

Atomic model of a cypovirus built from cryo-EM structure provides insight into the mechanism of mRNA capping

“…3D) In contrast to the flap-like structure on the orthoreovirus λ2 or the aquareovirus VP1, the spike-like complex of CPV sits on the turret substituting for the flap-like structure of orthoreoviruses or aquareoviruses. The turret of orthoreoviruses and aquareoviruses can adopt both closed and open states (8,20,(33)(34)(35). The biological mechanism behind the open/closed state of the turret is still elusive.…”