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We determined that the highly pathogenic avian reovirus strain 176 (ARV-176) possesses an enhanced ability to establish productive infections in HD-11 avian macrophages compared to avian fibroblasts. Conversely, the weakly pathogenic strain ARV-138 shows no such macrophagotropic tendency. The macrophage infection capability of the two viruses did not reflect differences in the ability to either induce or inhibit nitric oxide production. Moderate increases in the ARV-138 multiplicity of infection resulted in a concomitant increase in macrophage infection, and under such conditions the kinetics and extent of the ARV-138 replication cycle were equivalent to those of the highly infectious ARV-176 strain. These results indicated that both viruses are apparently equally capable of replicating in an infected macrophage, but they differ in the ability to establish productive infections in these cells. Using a genetic reassortant approach, we determined that the macrophagotropic property of ARV-176 reflects a post-receptor-binding step in the virus replication cycle and that the ARV-176 M2 genome segment is required for efficient infection of HD-11 cells. The M2 genome segment encodes the major mu-class outer capsid protein (muB) of the virus, which is involved in virus entry and transcriptase activation, suggesting that a host-specific influence on ARV entry and/or uncoating may affect the likelihood of the virus establishing a productive infection in a macrophage cell.

Previous studies which used intertypic reassortants of the wild-type reovirus serotype 1 Lang and the temperature-sensitive (ts) serotype 3 mutant clone tsA279 identified two ts lesions; one lesion, in the M2 gene segment, was associated with defective transmembrane transport of restrictively assembled virions (P. R. Hazelton and K. M. Coombs, Virology 207:46-58, 1995). In the present study we show that the second lesion, in the L2 gene segment, which encodes the lambda2 protein, is associated with the accumulation of a core-like particle defective for the lambda2 pentameric spike. Physicochemical, biochemical, and immunological studies showed that these structures were deficient for genomic double-stranded RNA, the core spike protein lambda2, and the minor core protein micro2. Core particles with the lambda2 spike structure accumulated after temperature shift-down from a restrictive to a permissive temperature in the presence of cycloheximide. These data suggest the spike-deficient, core-like particle is an assembly intermediate in reovirus morphogenesis. The existence of this naturally occurring primary core structure suggests that the core proteins lambda1, lambda3, and sigma2 interact to initiate the process of virion capsid assembly through a dodecahedral mechanism. The next step in the proposed capsid assembly model would be the association of the minor core protein mu2, either preceding or collateral to the condensation of the lambda2 pentameric spike at the apices of the primary core structure. The assembly pathway of the reovirus double capsid is further elaborated when these observations are combined with structures identified in other studies.

A temperature-sensitive reovirus mutant, tsG453, whose defect was mapped to major outer capsid protein sigma 3, makes core particles but fails to assemble the outer capsid around the core at non-permissive temperature. Previous studies that made use of electron cryo-microscopy and image reconstructions showed that mu 1, the other major outer capsid protein, but not sigma 3, interact extensively with the core capsid. Although wild-type sigma 3 and mu 1 interact with each other, immunocoprecipitation studies showed that mutant sigma 3 protein was incapable of interacting with mu 1 at the non-permissive temperature. In addition, restrictively-grown mutant sigma 3 protein could not be precipitated by some sigma 3-specific monoclonal antibodies. These observations suggest that in a wild-type infection, specific sigma 3 and mu 1 interactions result in changes in mu 1 conformation which are required to allow mu 1/sigma 3 complexes to condense onto the core capsid shell during outer capsid assembly, and that sigma 3 in non-permissive tsG453 infections is misfolded such that it cannot interact with mu 1.

Temperature-sensitive mutants provide an ideal means for dissecting viral assembly pathways. The morphological variants produced by and biological characteristics of tsA279, a previously uncharacterized mutant from the Fields' panel of temperature-sensitive mutants of reovirus, were determined under restrictive growth conditions. The mutant showed a distinctive pattern of increased temperature sensitivity as the temperature was raised from 39 degrees to 40 degrees. Wild-type reovirus type 1 Lang and the mutant were crossed to generate reassortants. Efficiency of plating analyses of the reassortants showed that tsA279 has temperature-sensitive lesions in two genes, a mildly temperature-sensitive one in L2, which encodes core spike protein lambda 2, and a stronger, dominant lesion in M2, which encodes major outer capsid protein mu 1. Electron microscopic examination of thin-sectioned tsA279-infected cells showed three ways in which the mutant phenotypes were expressed. The mutant appeared to be blocked in transmembrane transport of virions, a phenotype that mapped to the M2 gene; the mutant produced significantly reduced amounts of identifiable particles; and those particles that were produced appeared to be morphological variants. Immunofluorescent microscopy and immunoprecipitations of tsA279- and various T1L x tsA279 reassortant-infected cells suggested that the reduction in observed progeny was caused by a decreased production of viral proteins at the nonpermissive temperature. This phenotype also mapped to the mutant M2 gene.

Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations less than or equal to 1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of greater than 1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.