Reoviruses have a wide geographic distribution, and virtually all mammals, including humans, serve as hosts for infection. Based on sequence analysis of reovirus strains isolated from a variety of mammalian hosts, there is no evidence that any of the gene segments confer host range restriction.⁸⁸,⁵¹⁰ Because of their near ubiquity, reoviruses are used as a marker for mammalian fecal contamination of water sources.³¹⁴ Human infections
with reovirus are common, with most children demonstrating serologic evidence of infection by the age of 5 years.⁴⁸² However, reovirus is rarely associated with disease, except in the very young. There is evidence suggesting an association between reovirus and infantile biliary atresia,³⁹³,⁴⁹⁹ but a causal link has not been demonstrated. Newborn mice are exquisitely sensitive to reovirus infection and have been used as the preferred experimental model for studies of reovirus pathogenesis.⁵¹⁰

Three mammalian reovirus serotypes have been identified by classical neutralization and hemagglutination-inhibition (HAI) tests. Four virus strains isolated from children in the 1950s—type 1 Lang (T1L), type 2 Jones (T2J), type 3 Abney (T3A), and type 3 Dearing (T3D)—are used most frequently as prototype strains.⁴²³ A distinct reovirus strain was isolated from a mouse in the Cameroon¹⁸ and may represent a fourth mammalian reovirus serotype. Nucleotide sequences indicate that nonfusogenic mammalian isolates are a distinct phylogenetic group within the genus Orthoreovirus.¹⁵³ General features of the nonfusogenic mammalian orthoreoviruses are provided in Table 44.2.

The fusogenic orthoreoviruses infect birds, mammals, and reptiles. In contrast to the nonfusogenic orthoreoviruses, fusogenic reoviruses induce the formation of large, multinucleated syncytia.¹²⁷ Fusion activity is mediated by virus-encoded, fusion-associated small transmembrane (FAST) proteins, which are not capsid components but instead expressed on the surface of infected cells.⁴⁵³ FAST proteins are small (<20 kDa), basic, acylated proteins that induce fusion in transfected cells in the absence of other viral proteins.⁶² Fusogenic members of the related genus, Aquareovirus, also encode FAST proteins.³⁸⁶ Extensive syncytium formation results in apoptosis and enhanced release of infectious virions.⁴²⁶ Expression of the p14 FAST protein by a recombinant vesicular stomatitis virus increases neuropathology.⁶⁹ The FAST proteins therefore likely contribute to the natural pathogenicity of the fusogenic orthoreoviruses. Other than this fusion activity, the replication strategies of fusogenic and nonfusogenic orthoreoviruses are similar.⁴²

The fusogenic orthoreoviruses are divided into four species: avian reovirus (ARV), Nelson Bay reovirus (NBV), reptilian reovirus (RRV), and baboon reovirus (BRV).⁸⁸ ARVs infect a variety of avian hosts and are important pathogens in the poultry industry, causing gastroenteritis, hepatitis, malabsorption, myocarditis, and pneumonia.²⁴⁹ Birds that survive acute systemic infection can develop joint and tendon inflammation that resembles the pathology of human rheumatoid arthritis. RRVs have been isolated from numerous reptile species, and infected animals often develop pneumonia and neurologic symptoms.⁵¹⁹ BRV was isolated from baboons with meningoencephalomyelitis in a colony in Texas.²⁸⁰ Therefore, it is an example of a fusogenic mammalian orthoreovirus. BRV virions, like those of the aquareoviruses, lack the classical sigma-class fiber protein used for cell adhesion by other reoviruses.⁵³⁷ NBV is the prototype orthoreovirus species isolated from bats; it is more closely related to ARV than to BRV, the other fusogenic mammalian orthoreovirus species.¹²⁷

Additional fusogenic bat reoviruses have been isolated,³⁸¹ one of which represents a new species that also lacks a sigma-class fiber attachment protein.⁴⁸⁷ Three fusogenic reoviruses isolated from humans with acute respiratory infections also may be of bat origin.⁹⁶,⁹⁷,⁹⁸ These viruses appear capable of human-to-human transmission, raising concern that fusogenic bat reoviruses may be a potential source of emerging infections in humans.

The 10 reovirus dsRNA gene segments are present in equimolar amounts in viral particles.⁴⁴⁴ The segments are grouped by size into large (L), medium (M), and small (S) classes according
to their migration in polyacrylamide gels.¹⁹⁵,³³⁰,⁴⁴⁴,⁵¹³ There are three large (L1, L2, L3), three medium (M1, M2, M3), and four small (S1, S2, S3, S4) segments. Homologous gene segments from different reovirus isolates often migrate with different mobilities in polyacrylamide gels despite being of similar or identical length³⁸⁸ (Fig. 44.3). This property allows gene segments from different isolates to be unambiguously distinguished, which is essential for the assignment of gene segments in reassortant viruses to specific parental strains (see Genome and Coding Strategies—Reassortment). The gene segments of prototype strain T3D are numbered according to their relative mobilities in Tris/acetate-buffered gels.⁴⁴¹ Each gene segment encodes at least one protein product (Table 44.4).

The [+] strand RNA of each gene segment has a dimethylated cap-1 structure on its 5′ terminus,⁹⁵,¹⁷⁹,³³² which is added by viral guanylyltransferase and methyltransferase activities present in the λ2 protein.¹⁰⁶,²⁹⁵ The viral mRNA cap is recognized by translation initiation factor eIF4E⁶¹,³⁴⁶ and protects the free 5′ ends of the viral mRNA from 5′ exonucleases.¹⁷⁷ The 5′ terminus of the [−] strand RNA of each gene segment has an unblocked diphosphate.²⁴,⁹⁵ The terminal γ-phosphate of the [−] strand is removed by a phosphohydrolase activity (possibly mediated by the μ2 or λ1 protein) within reovirus particles.²⁴,⁵¹,⁹⁵,²⁵⁶ The 3′ end of each gene segment has an unblocked hydroxyl group.³³⁰ The viral [+] strand RNA does not contain a polyadenylate tract.

Full-length sequences of the entire genomes of prototype mammalian orthoreovirus strains T1L, T2J, and T3D as well as those of several fusogenic orthoreovirus isolates have been determined (National Center for Biotechnology Information). The total length of the genome sequence is approximately 23,500 base pairs. The length of individual gene segments varies from 1,196 (T3D S4 gene segment) to 3,916 (T3D L2 gene segment) nucleotides. Most homologous gene segments of different isolates display little (±4 base pairs) or no variation in segment length. However, the S1 gene segment varies in length to a greater extent (1,463, 1,440, and 1,416 base pairs for the T1L, T2J, and T3D S1 gene segments, respectively).¹⁵⁴,³⁵⁰

Gene segment exchange (or reassortment) among reovirus strains occurs during mixed infections of cultured cells or animal hosts. The progeny of such mixed infections are commonly referred to as reassortant viruses. Many reovirus strains are distinguishable by signature electrophoretic profiles of their dsRNA gene segments in acrylamide gels (Fig. 44.3A). Co-infection of cells with such strains produces a collection of reassortant viruses in which the parental origin of each gene segment can be determined following electrophoresis of viral genomic dsRNA (Fig. 44.3B). Studies of reassortant viruses have been used to assign biological polymorphisms displayed
by different reovirus strains to specific viral gene segments. Thus, a phenotypic difference between two parental strains can be genetically mapped by screening the reassortant viruses in appropriate assays and correlating expression of the phenotype with a specific parental gene segment.

Reassortment of reovirus gene segments also occurs in vivo. Infection of mice perorally with strains T1L and T3D yields reassortant progeny in the stool and at sites of secondary replication.⁵²¹ In at least one case, a reassortant virus arising during such a mixed infection displays virulence properties that differ from both parental strains,⁴⁵¹ suggesting the selection of new mutations in vivo. Beyond experimental infection of animals, additional support for the hypothesis that reovirus gene segments reassort in nature is provided by analysis of nucleotide sequences of the L1,²⁷⁷ M1,⁵⁴¹ S1,¹³⁷ S2,⁹⁰ S3,²⁰² and S4²⁵³ gene segments derived from a panel of field-isolate strains isolated in the 1950s and 1960s by Leon Rosen.⁴¹³,⁴¹⁴,⁴¹⁷ The topologies of phylogenetic trees constructed from protein-coding sequences of these gene segments are distinct, providing convincing evidence that these segments reassort in nature.

Reassortment is not entirely random. In a study of 83 reassortant viruses derived from strains T1L and T3D, statistically significant nonrandom associations of parental alleles were observed in the L1–L2, L1–M1, L1–S1, and L3–S1 gene segment pairs.³⁵² It is possible that these gene segments or their protein products cannot accommodate exchange with gene segments from another strain without the selection of fitness-compromising mutations. Additionally, the majority of progeny viruses resulting from mixed infections retains the genome constellation of the parental strains,¹⁶⁹ suggesting that viral replication is compartmentalized in some way.

The ability to engineer viruses that contain specific sequence modifications was first accomplished for mammalian reoviruses using a strategy involving message-sense RNA transcribed in vitro and a helper virus.⁴¹⁰ This system has been used to recover infectious particles containing gene segments derived from cloned DNA copies of single reovirus gene segments.⁴⁰⁵,⁴⁰⁷ Development of a plasmid-based reverse genetics system for members of the Birnaviridae family,⁵³⁹ which contain two genomic dsRNA segments, suggested that delivery of viral positive-strand RNA alone could launch successful production of viral progeny for a dsRNA virus. This approach proved possible with the development of an entirely plasmid-based reverse genetics system for reovirus in which viable viruses are generated from a set of 10 cloned cDNAs representing the complete viral genome²⁶¹ (Fig. 44.5). Gene segment cDNAs corresponding to strains T1L²⁶⁴ and T3D²⁶¹ were introduced into plasmids at sites flanked by the promoter sequence for T7 RNA polymerase and the hepatitis delta virus (HDV) ribozyme (Fig. 44.5A). Neither helper virus nor co-expression of viral replication proteins is required for recovery of infectious virus.

Reovirus can be recovered from cells in which T7 RNA polymerase is delivered transiently by infection with a recombinant vaccinia virus²³⁶ or from cells that constitutively express the enzyme⁷³ (Fig. 44.5B). Since T7 RNA polymerase initiates transcription at a defined guanosine residue³²⁹ and all reovirus RNAs contain the nucleotide sequence, GCUA, at the 5′ terminus of the message-sense strand, T7 RNA polymerase produces transcripts with native reovirus 5′ termini. Self-cleavage of the HDV ribozyme generates RNAs with native 3′ termini.⁴⁰⁷ In cells transfected with plasmids encoding all 10 reovirus gene segments, viral gene products join with reovirus RNAs to form viral replication complexes, which mediate negative-strand synthesis and ultimately generate infectious particles. Inclusion of expression plasmids encoding reovirus proteins does not enhance recovery of recombinant reoviruses.⁵⁷ However, the efficiency of virus recovery is increased by using baby hamster kidney cells constitutively expressing T7 RNA polymerase⁷³ and reducing the plasmid number from 10 to four by combining reovirus gene segment cDNAs into multicistronic vectors.²⁶⁴

Plasmid-derived wild-type virus recapitulates properties of native virus in all cell-culture and in vivo models of reovirus infection studied to date.²⁶¹,²⁶⁴ This reverse genetics approach has been used to introduce changes into the virion capsid and replication proteins to define the roles of individual amino acids, functional domains, and structural motifs in receptor utilization,²⁵⁹,³⁹⁵ virion disassembly,¹⁴⁷,²⁶¹ membrane penetration,¹²¹,¹²³,⁴³⁰ replication biochemistry,³⁶⁹ inclusion
formation,²³⁹,²⁶³ interferon (IFN) expression,²³⁵,⁵⁵⁶ apoptosis induction,¹²¹,¹²³ viral growth and spread in vivo,⁵⁵,⁵⁶,²⁶¹ neurovirulence,¹²¹,¹²³ and myocardial injury.²³⁵ The same strategy also can be exploited to engineer recombinant reoviruses for vaccine and oncolytic applications.

A variety of complementation strategies have been developed to enable studies of viral mutations that would otherwise compromise viral viability. Stable expression in cultured cells of wild-type forms of temperature-sensitive mutant proteins allows replication of the corresponding temperature-sensitive mutant virus at nonpermissive temperature. This approach has been used to complement the replication of tsE (which segregates with the σNS-encoding S3 gene segment) and tsH (which segregates with the μ2-encoding M1 gene segment) by stable expression of the σNS³³ and μ2⁵⁵⁴ proteins, respectively. These complementing cell lines and temperature-sensitive mutant viruses could be used to define sequences in σNS and μ2 required for viral replication.

Recombinant reovirus outer-capsid proteins μ1, σ1, and σ3 can bind and “recoat” purified subvirion particles (ISVPs or cores) in vitro to generate infectious virion-like particles.⁸³,⁸⁴,²⁴⁴ These recoated particles have been used to identify sequences in μ1, σ1, and σ3 proteins that function during cell entry and disassembly.⁸¹,⁸⁴,²⁴³,⁵³¹

Reovirus replication is blocked in cell lines stably expressing small interfering RNAs (siRNAs) specific for reovirus transcripts.²⁶² RNAi-mediated inhibition of viral gene expression can be complemented by vectored expression of viral genes in which the siRNA target sequence has been altered to prevent siRNA-mediated degradation. This approach has been used to identify sequences in μ2, μNS, and σNS proteins that function in viral inclusion formation and RNA synthesis.¹⁶,⁷⁷,²⁶²,²⁶³ Limitations of this strategy include the efficiency of viral RNA knockdown and transfection efficiency of the complementation plasmids.

The S1-encoded σ1 protein (49–51 kDa, 455–470 aa) mediates viral binding to cellular receptors²⁷,⁸⁹,²⁷⁹,⁵¹⁴ and influences target-cell selection in the infected host.²⁸,⁵¹⁵,⁵¹⁷ It is a homotrimer present in 36 copies per virion.¹¹⁴,⁴⁷⁸ Structural information is available for the σ1 protein of prototype type 3 strain, T3D. The 455 amino acids of T3D σ1 fold into a filamentous trimer approximately 480 Å long and 90 Å wide at its broadest point, with a globular C-terminal head, a central body, and a slender N-terminal tail⁹²,¹⁷⁴,³⁹⁵ (Fig. 44.6A). Residues 310 to 455 comprise the head, which is constructed from two Greek-key motifs that assemble into an eight-stranded β-barrel.⁹²,⁴³¹ With the exception of the loop connecting β-strands D and E (D-E loop), which contains a 3₁₀ helix, loops connecting individual strands of the β-barrel are very short. Residues 170 to 309 form the body domain, which is constructed primarily from repeating units of two antiparallel β-strands connected by short loops. Three such units assemble into a triple β-spiral, which is a motif observed to date only in viral fibers, including the adenovirus fiber,⁵⁰⁵ bacteriophage PRD1 P5,³²¹ avian reovirus attachment protein σC,²⁰³ and mammalian reovirus T3D σ1.⁹²,³⁹⁵ The body domain features four β-spiral repeats at the N-terminus (β1–β4, residues 170–235), a short α-helical coiled-coil (residues 236–251), and three additional β-spiral repeats (β5–β7, residues 252–309) at the C-terminus (Fig. 44.6A). The short α-helical coiled-coil corresponds to a narrowing in the body domain in a composite image of negative-stained electron micrographs of purified σ1.¹⁷⁴ Residues 1 to 160 form the tail domain, the structure of which is unknown. However, a repeating heptad sequence motif suggests that this region forms an amphipathic α-helix, which likely assembles into an α-helical coiled-coil in the trimer.⁹²,³⁵⁰ The extreme N-terminus of σ1 does not contain any obvious sequence motifs. It is hydrophobic and anchors the protein into the pentameric turret formed by λ2 in the reovirus virion.¹⁵¹,¹⁷⁶,²⁸³ This symmetry mismatch (a trimer of σ1 engaged to a pentamer of λ2) suggests an interaction of limited strength, which may aid in σ1 release during viral disassembly.⁴⁷²

The σ1 molecule possesses discrete regions of flexibility⁹²,¹⁷⁴,³⁹⁵ (Fig. 44.6A, arrows). One site of substantial flexibility in T3D σ1 is contributed by a four-residue insertion between the two most C-terminal β-spiral repeats (β6 and β7).⁷⁸,⁹² Sequence alignments suggest that σ1 of reovirus prototype strains T1L and T2J each contain a six-residue insertion at the same position.⁹² This insertion appears to correspond to a region of flexibility observed just below the σ1 head in electron micrographic images.¹⁷⁴ A second region of flexibility is observed at the midpoint of σ1 and corresponds to the transition between the predicted α-helical coiled-coil region of the tail and the β-spiral–containing body. A final region of flexibility close to the N-terminus likely represents the virion insertion domain.⁹²,¹⁷⁴,³⁵⁰

Reoviruses display the capacity to agglutinate erythrocytes of several mammalian species.²⁸⁴ For type 3 reoviruses, hemagglutination is mediated by interactions of the σ1 protein with terminal α-linked sialic acid residues on several glycosylated erythrocyte proteins such as glycophorin A.¹⁸³,³⁷⁶ Strain T3D exhibits a reduced capacity to agglutinate erythrocytes following treatment with neuraminidase, which removes terminal sialic acid moieties.¹⁸³ Preincubation of L cells with neuraminidase or virus with sialosides also significantly diminishes T3D binding.¹⁸⁴,³⁷⁵ Sialic acid residues linked in α2,3 or α2,6 configurations effectively block type 3 reovirus binding to L cells.³⁷⁵ Reovirus T3D binds to sialoglycophorin, but not to asialoglycophorin, with an avidity of approximately 5 × 10⁻⁹ M,²⁶ which is a property mediated by the σ1 protein.⁸⁹

Binding to sialic acid is essential for reovirus infection of some types of cultured cells, such as MEL cells.⁹¹,⁴²² Sequence polymorphisms within the σ1 body domain at residues 198, 202, and 204 determine the capacity of field-isolate reovirus strains to bind sialic acid and infect MEL cells.¹³⁸,⁴²¹ Furthermore, nonsialic acid–binding type 3 variants can be adapted to growth in MEL cells during serial passage. These variants have gained the capacity to bind sialic acid and contain sequence changes within the polymorphic region of the σ1 body. In addition, sialic acid binding also serves an important role in reovirus tropism and pathogenesis in vivo. A sialic acid–binding strain of reovirus, but not a nonsialic acid–binding strain, causes bile
duct injury in newborn mice and exhibits 1,000-fold greater binding capacity for human cholangiocarcinoma cells²⁸ (see Pathogenesis and Immunity—Hepatobiliary System).

The structure of a T3D σ1 construct encompassing the body and head domains in complex with α-2,3-sialyllactose has been determined.³⁹⁵ The oligosaccharide binds in a shallow groove next to the loop connecting the second and third β-spiral repeats (β2 and β3). The σ1 protein contains three identical binding sites, one on each chain, and all three are occupied by α-2,3-sialyllactose molecules in the crystal structure, with the sialic acid making identical and extensive contacts in each chain (Fig. 44.6B). Remarkably, σ1 also can form complexes with α-2,6-sialyllactose and α-2,8-di-siallylactose.³⁹⁵ In each case, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. Studies using point-mutant viruses indicate that Asn198, Arg202, Leu203, Pro204, and Gly205 are required for hemagglutination and infection of MEL cells,³⁹⁵ suggesting that these residues serve a functional role in T3D σ1–sialic acid interactions.

Type 1 reoviruses also appear to bind sialic acid in some contexts. T1L, but not T3D, binds the apical surface of microfold (M) cells, but not enterocytes, in tissue sections of rabbit Peyer patches.²¹⁷ Binding is inhibited by preincubation of the tissue sections with neuraminidase or with lectins that specifically recognize α-2-3–linked sialic acid. The capacity of T1L to bind the apical surface of M cells segregates with the S1 gene segment using reassortant genetics and with reovirus particles recoated with recombinant σ1 protein. The interaction between T1L σ1 and sialic acid is especially intriguing as type 1 reoviruses are
incapable of infecting MEL cells, a property dependent on sialic acid binding that segregates with the S1 gene segment,⁴²² and are insensitive to the growth-inhibitory effects of neuraminidase treatment of L cells.³⁴⁹ These findings suggest that the functional glycans engaged by types 1 and 3 reoviruses differ, even though such glycans appear to terminate in α-2,3–linked sialic acid. Concordantly, studies using expressed protein suggest that T1L engages carbohydrates using a domain that differs from that used by T3D to bind carbohydrates.⁸⁹

Type 2 reoviruses also are capable of producing hemagglutination,²⁸⁴ but little is known about the basis for this property.

Reovirus strains of all three serotypes use junctional adhesion molecule-A (JAM-A, also known as F11R/JAM/JAM1), a member of the immunoglobulin superfamily,²⁷,³⁰⁹,⁵²⁹ as a receptor.²⁷,⁷⁶,³⁸² JAM-A was identified as a reovirus receptor using a genetic screen and subsequently shown to bind directly to the σ1 head domain with nanomolar affinity.²⁷,⁴³¹ Human and murine homologs of JAM-A, but not JAM family members JAM-B or JAM-C, serve as receptors for all reovirus serotypes and strains tested to date.²⁷,⁷⁶,³⁸² The role of JAM-A as a reovirus receptor in vivo has been examined using JAM-A–null mice¹³ (see Pathogenesis and Immunity – Spread of Virus). Following peroral inoculation, JAM-A is dispensable for reovirus growth in the intestine. However, it is required for infection of vascular endothelial cells and promotes efficient hematogenous dissemination of reovirus to sites of secondary infection. Thus, JAM-A serves as a high-affinity reovirus receptor in cultured cells and in vivo. Receptors for reovirus at other sites within the host, including the central nervous system (CNS), have not been conclusively identified.

Structural and biochemical studies highlight the regions and specific interactions that mediate reovirus engagement of JAM-A⁷⁶,⁹²,¹⁷³,²⁰⁴,²⁵⁹,³⁸² (Fig. 44.6C). The largest area of conserved residues in σ1 forms the D-E and F-G loops in the head domain.⁷⁶,⁹² The crystal structure of the σ1 head domain in complex with the JAM-A D1 domain reveals that residues in this region, centered at the D-E loop and its 3₁₀ helix, form the largest area of JAM-A contact.²⁵⁹ Interactions in this area are polar and involve residues Thr380, Gly381, and Asp382. A second area of JAM-A contact includes residues within the σ1 body, just N-terminal to the head domain. Interactions in this region are largely hydrophobic and involve Arg316 in β-strand A of the head, β-spiral residues Arg297 and Tyr298, the α-helical turn that connects the β-spiral with the β-barrel, and Pro377.

The D1 domain of JAM-A is required for high-affinity binding to σ1.¹⁷³,²⁰⁴,³⁸² Mutation of individual JAM-A D1 domain residues Arg59, Glu61, Lys63, Leu72, Tyr75, and Asn76 that lie in or adjacent to the dimer interface diminishes or abolishes σ1 binding and reovirus infectivity.²⁰⁴ Concordantly, the structure of the σ1–JAM-A complex shows that each σ1 trimer binds three independent JAM-A monomers. Contacts primarily involve the JAM-A dimer interface and a conserved region at the base of the σ1 head⁹²,²⁵⁹ (Fig. 44.6C). In addition, the structure of the σ1-JAM-A complex also identifies residues bound by σ1 that are found just outside the dimer interface of JAM-A.²⁵⁹ These residues may serve as initial contact points for σ1 and facilitate disruption of the JAM-A homodimer to allow interaction of σ1 with the JAM-A dimer interface. It is also possible that a cavity in the JAM-A dimer interface renders the homodimer intrinsically unstable, thereby promoting its disruption by σ1. Regardless of the mechanism, the σ1-JAM-A interaction is thermodynamically favored, as the K_D is ∼ 1,000-fold lower than the K_D of the JAM-A homodimer interaction.²⁰⁴,²⁵⁹,⁵⁰⁶

The presence of discrete receptor-binding domains in σ1 suggests that reoviruses employ a multiple-step binding process similar to that used by some herpesviruses¹⁰⁹,⁴⁶⁵ and retroviruses.⁴⁶,⁵⁰³ Binding studies using isogenic point-mutant viruses T3SA+ and T3SA-, which vary only in the capacity to engage sialic acid,²⁶ support this hypothesis. Kinetic analyses using inhibitors of sialic acid and JAM-A binding demonstrate that sialic acid is engaged first in the adsorption process, as the inhibitory effect of sialic acid analogs on infection by T3SA+ occurs at early but not late time points.²⁶ However, a σ1-specific monoclonal antibody that blocks virus binding to JAM-A inhibits viral infectivity at both early and late times during adsorption.²⁶ Thus, reovirus binding to sialic acid enhances virus attachment through rapid adhesion of virus to the cell surface where access to JAM-A is thermodynamically favored. It is not known whether binding to sialic acid induces structural changes in σ1 that affect its capacity to interact with JAM-A. However, it is clear that sialic acid binding is not a necessary prerequisite for JAM-A binding, as reoviruses incapable of binding sialic acid are capable of binding JAM-A.²⁷ It is possible that engagement of JAM-A by the σ1 head leads to conformational changes in other regions of σ1 or in other capsid components.¹⁶⁸

Treatment of virions of some reovirus strains with intestinal proteases in vitro to generate ISVPs leads to cleavage of the σ1 protein.⁸⁷,³⁴⁹ Strain-specific differences in cleavage susceptibility segregate with a single amino acid polymorphism in the short α-helical coiled-coil in the body domain of σ1 (Fig. 44.6A, asterisk). Strains like T3D with a threonine at position 249 in σ1 are susceptible to cleavage by trypsin after Arg245, whereas those with an isoleucine at position 249 are resistant to cleavage.⁸⁷ It is possible that Thr249 disrupts the α-helical coiled-coil and allows access to sites that otherwise would be shielded from protease.⁸⁷,³⁴⁹ Conversion of T3D virions to ISVPs by intestinal proteases leads to loss of the σ1 head domain and a resultant 90% reduction in infectivity.³⁴⁹ Since cleavage of σ1 occurs C-terminal to the sialic acid–binding pocket, residual infectivity of T3D ISVPs is dependent on this carbohydrate.³⁴⁹ Susceptibility of the T3D σ1 protein to cleavage likely accounts for the attenuated virulence of this strain after oral inoculation.⁵³,²⁵⁴,⁴¹⁹

In addition to its function in receptor engagement, the σ1 protein is the target of serotype-specific neutralizing antibodies.⁷⁵,⁴⁶⁹,⁵¹⁶ For example, serotype-specific monoclonal antibodies 5C6⁵⁰⁸ and 9BG5⁷⁵ neutralize infectivity of type 1 and type 3 reovirus strains, respectively, and bind the σ1 head domain in a serotype-specific fashion.⁸⁹ Variants of T1L selected for resistance to neutralizing monoclonal antibodies have mutations at
Ala415, Gln417, Asn445, and Gly447,²¹⁶ which are located in the σ1 head. Similarly, variants of T3D selected for resistance to neutralizing monoclonal antibodies have mutations at Asp340 and Glu419,³⁰ which are also located in the σ1 head. Interestingly, for both T1L and T3D, the sites in σ1 altered in the neutralization-resistant variants are in close proximity on adjacent monomers,⁹²,²¹⁶ suggesting an epitope that spans two monomers in the σ1 trimer. Neutralization-resistant variants of T3D display diminished neurovirulence and altered CNS tropism in mice,⁴⁶⁷,⁴⁶⁸ perhaps as a consequence of altered σ1 receptor recognition in the murine CNS.

Following attachment to cell-surface glycans and JAM-A, reovirus is internalized by receptor-mediated endocytosis (Fig. 44.7). Thin-section electron micrographs show virions in structures that appear to be clathrin-coated pits on the cell surface and in clathrin-coated vesicles in the cytoplasm,⁵⁹,⁶⁰,²⁹⁸,⁴²¹,⁴⁸⁰ suggesting clathrin-dependent uptake. This finding was confirmed using video fluorescence microscopy in which reovirus virions and clathrin are observed to colocalize during internalization.¹⁵⁸ Treatment of cells with chlorpromazine, which impairs clathrin-mediated endocytosis, inhibits reovirus internalization and infection,²⁹⁸ suggesting a functional role for clathrin in reovirus entry. However, there is mounting evidence that both clathrin- and caveolin-dependent mechanisms can be employed by some viruses to enter host cells.²⁷⁵,³⁸⁵ Although there are no published reports of clathrin-independent uptake strategies for reovirus, a role for caveolae or other entry mechanisms in reovirus cell entry has not been conclusively excluded.
Regardless of the internalization mechanism, reovirus particles are sorted into vacuoles resembling endosomes and lysosomes during the entry process.⁵⁹,⁶⁰,¹⁵⁸,²⁹⁸,⁴²¹,⁴⁸⁰

Expression of a JAM-A truncation mutant lacking a cytoplasmic tail allows reovirus to infect nonpermissive cells,²⁹⁷ suggesting that molecules other than JAM-A mobilize the internalization apparatus that promotes reovirus cell entry. The L2-encoded λ2 protein (144 kDa, 1288–1289 aa) contains conserved integrin-binding motifs, RGD and KGE,⁶⁴,⁴³⁸ suggesting that reovirus employs integrin-dependent internalization mechanisms to enter cells. The RGD and KGE sequences are displayed on surface-exposed loops of λ2,³⁹⁴ where they could interact with integrins to promote internalization. However, direct interactions between λ2 and integrins have not been reported. Interestingly, the L2 gene segment is genetically linked to viral shedding in infected mice and spread to littermates,²⁵⁴ suggesting a role for λ2 in reovirus-induced disease.

Treatment of cells with antibodies specific for β1 integrin reduces reovirus infection, while antibodies specific for other integrin subunits expressed on permissive cells, including those specific for α integrin subunits, have no effect.²⁹⁷ However, antibodies specific for β1 integrin do not alter infection by in vitro–generated ISVPs,²⁹⁷ which directly penetrate the plasma membrane and do not require endocytosis.²²⁶,²⁹⁴ Concordantly, β1 integrin–specific antibodies do not alter the binding of reovirus to JAM-A.²⁹⁷ These findings suggest that β1 integrin blockade inhibits endocytic uptake of virions following JAM-A engagement. In comparison to β1 integrin-expressing cells, β1-null cells are substantially less susceptible to infection by reovirus virions, while infection by ISVPs is equivalent in both cell types.²⁹⁷ Diminished reovirus replication in β1-null cells correlates with diminished viral uptake, indicating that β1 integrin is required for efficient reovirus cell entry.

NPXY motifs in the β1 integrin cytoplasmic tail play a key role in sorting reovirus within the endocytic compartment. NPXY motifs are found in the cytoplasmic domains of many receptors⁹⁴,¹²⁵,³⁶⁷ and recruit adaptor protein 2 or disabled protein 2³⁴¹,³⁶⁷ to initiate clathrin assembly at the plasma membrane. Substitution of a tyrosine with a phenylalanine residue in either or both β1 integrin NPXY motifs (NPXF) results in inefficient internalization of reovirus virions and diminished infectivity.²⁹⁸ Infection of cells expressing NPXF β1 integrin results in distribution of virions to lysosomes where they are degraded, suggesting that the β1 integrin NPXY motifs target reovirus to the precise endocytic organelle that permits functional disassembly.

The Src family of kinases contains eight members, Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, and Yes, three of which—Fyn, Src, and Yes—are expressed in most cell types.⁴⁹¹ Src-family kinases regulate numerous cellular processes including proliferation, differentiation, migration, adhesion, and cytoskeletal rearrangements⁴⁹¹ and transduce signals from a variety of receptors.¹⁴⁵,¹⁸²,³⁰⁸,⁴²⁷ In a small-molecule inhibitor screen, genistein, a broad-spectrum tyrosine-kinase inhibitor, and PP2, a specific Src-family-kinase inhibitor, were found to diminish reovirus infectivity by inhibiting an early step in the viral life cycle.²⁹⁹ Although neither inhibitor impedes internalization of reovirus virions, both inhibitors target virions to lysosomes. At early times following infection, Src co-localizes with reovirus virions and is phosphorylated at the activation residue, Tyr416, suggesting that reovirus induces activation of Src during entry and traffics with Src during sorting in the endocytic compartment. Reduction of Src expression by RNAi decreases reovirus infectivity.²⁹⁹ These findings suggest that Src is part of a signaling network that targets reovirus to endocytic organelles for viral disassembly and thus promotes functional entry into host cells. The precise mechanism by which Src mediates these effects is unknown.

In cellular endosomes, reovirus virions undergo stepwise disassembly to form discrete intermediates, the first of which is the ISVP²¹,⁶⁰,⁸⁵,⁴⁵⁶,⁴⁸⁰ (Fig. 44.7). ISVPs are characterized by the loss of σ3, a conformational change in σ1, and cleavage of μ1 to form δ and φ. The rate-limiting step in reovirus disassembly is the proteolytic removal of the S4-encoded σ3 protein (41 kDa, 365 aa).²¹,⁴⁸⁰ In some cell types, proteolysis of σ3 is dependent on acidic pH¹³⁹,⁴⁸⁰ and endocytic cysteine proteases.²¹ Murine L929 fibroblast cells treated with inhibitors of endosomal acidification, such as ammonium chloride⁴⁸⁰ or bafilomycin A1,³¹⁰ or inhibitors of cysteine proteases, such as E64,²¹ do not support reovirus replication when infection is initiated by virions but do so when infection is initiated by ISVPs. Thus, the block to reovirus replication imposed by these inhibitors in murine L929 cells occurs following internalization but prior to disassembly, which is coincident with the proteolysis of σ3.

Cathepsins B and L, which reside in late endosomes and lysosomes,⁸⁶ catalyze reovirus disassembly in fibroblasts.¹⁵⁶ Both enzymes are optimally active at acidic pH and serve functions in extracellular matrix formation, antigen presentation, and apoptosis.⁸⁶ These enzymes also mediate cell entry of several other viruses, including Ebola virus⁸² and SARS coronavirus.²²⁹ Cathepsin S, an acid-independent cysteine protease required for processing internalized antigens,⁴⁰¹ and the acid-independent serine protease neutrophil elastase can mediate uncoating of some reovirus strains in monocyte and macrophage cell lines.¹⁹¹,¹⁹³ It is possible that the broad tissue tropism displayed by reovirus is determined in part by the multiple host proteases capable of mediating its disassembly.²⁴⁷

Proteolytic enzymes also are required for reovirus infection following peroral inoculation of mice²⁹,⁵⁴ (see Pathogenesis and Immunity—Entry into the Host). Reovirus virions are converted to ISVPs in the intestinal lumen by the resident serine proteases chymotrypsin and trypsin. ISVPs produced in this fashion enter intestinal M cells to allow systemic dissemination of reovirus in the host.¹² ISVPs generated by chymotrypsin or trypsin in vitro or in the gut lumen²⁹,⁵⁴ display a protein profile (loss of σ3 and cleavage of μ1) indistinguishable from that of ISVPs generated in the endocytic compartment of cells.²²,¹⁵⁶

Sequences in σ3 that influence its susceptibility to proteolysis have been identified through studies of viruses selected
during persistent infection (PI viruses) or mutant viruses selected for resistance to either cysteine protease inhibitor E64 (D-EA viruses)¹⁵⁷ or ammonium chloride (ACA-D viruses).⁹⁹ These viruses exhibit accelerated kinetics of disassembly and harbor a tyrosine-to-histidine mutation at amino acid 354 (Y354H) near the C-terminus of the protein⁹⁹,¹⁵⁷,⁵²⁴ (Fig. 44.8). Cryo-EM image analysis of a PI virus with an isolated Y354H mutation reveals a structural alteration in σ3 at a hinge region located between its two major domains.⁵³¹ These findings suggest that the C-terminus of σ3 influences susceptibility of the protein to cleavage. Interestingly, the σ3 amino acid sequence of strain T3A differs from that of T3D at eight positions, including Y354H. However, a glycine-to-glutamate substitution at position 198 in T3A σ3 suppresses the disassembly-enhancing effects of His354,¹⁴⁷ suggesting the existence of an allosteric network regulating σ3 cleavage.

The σ3 C-terminus also dictates strain-specific differences in the susceptibility of σ3 to proteolytic attack.²⁴³,²⁴⁴ The σ3 protein of strain T1L is cleaved more rapidly than that of T3D. Analysis of ISVPs recoated with chimeric σ3 proteins generated from T1L and T3D revealed that the C-terminus is primarily responsible for the rate of σ3 proteolysis. Moreover, sequence polymorphisms at residues 344, 347, and 353 in σ3 contribute to this effect.²⁴³

Treatment of reovirus virions in vitro with either cathepsin B or cathepsin L leads to an initial cleavage of σ3 at a terminus.¹⁵⁶ Since sequence polymorphisms in the σ3 C-terminus determine susceptibility to proteolysis, the initial cleavage of σ3 probably occurs in this region. During proteolysis by cathepsin L, subsequent cleavages occur between residues 243 to 244 and 250 to 251¹⁵⁶ (Fig. 44.8A). These cleavage sites are physically located near the C-terminus in the σ3 crystal structure³⁶⁸ (Fig. 44.8B and C). Because of this proximity, the small end fragment released following initial cathepsin L cleavage likely exposes the cleavage sites between residues 243 to 244 and 250 to 251, rendering them sensitive to proteolysis. The C-terminus therefore appears to control access to internal, proteolytically sensitive sites in σ3. Because reovirus disassembly in some cell types is acid-dependent,¹³⁹,⁴⁸⁰ the C-terminus might be primed for movement at acidic pH. Mutations near the C-terminus, like Y354H, may alter the conformation of the protein to allow improved access to these cleavage sites and thus accelerate outer capsid disassembly.⁵³¹

The disassembly of reovirus virions to ISVPs is accompanied by a dramatic conformational change in σ1. Electron micrographs of negatively stained reovirus virions and ISVPs reveal filamentous projections extending up to 400 Å from the surface of ISVPs but not virions.¹⁷⁶ These images suggest that σ1 adopts a compact form in the virion and a more extended one in the ISVP. Cryo-EM image reconstructions of virions and cores lack a discernable density corresponding to σ1 at the icosahedral vertices¹⁵¹,³²³ (Fig. 44.2). However, cryo-EM image analysis of
ISVPs demonstrates discontinuous density for σ1 extending ∼ 100 Å from each vertex (Fig. 44.2). Presumably, the full length of σ1 is not visible in reovirus particles because the molecule is flexible and icosahedral averaging was employed for the cryo-EM image reconstructions. As further evidence of the conformational alterations in σ1 during viral disassembly, proteolytic cleavage of T3D σ1 during virion-to-ISVP conversion increases viral hemagglutination capacity,³⁴⁹ suggesting enhanced access to the sialic acid–binding region as a consequence of extension of the σ1 tail and body domains. Interdomain regions of flexibility in σ1¹⁷⁴ (Fig. 44.6) may be required to allow such conformational rearrangements.

Electron micrographs of purified σ1 show molecules with either single- or multilobed head domains,¹⁷⁴ suggesting another type of structural alteration in σ1 in which the head can exist in both “open” and “closed” conformations. Although neither the mechanism nor the functional significance of this σ1 conformational change is understood, structural studies of σ1 provide clues about how this change might occur. A cluster of six conserved aspartic acid residues on a rigid β-hairpin at the base of the σ1 head, sandwiched between hydrophobic residues that block access to solvent, forms the main contact area between monomers in the trimer.⁹²,⁴³¹ Of the two aspartic acid residues contributed by each monomer, one (Asp346) is neutralized by a salt-bridge interaction with a nearby residue, while the other (Asp345) is not.⁹²,⁴³¹ The three Asp345 side chains closely appose each other at the center of the trimer in an otherwise hydrophobic environment. Since accumulation of negative charge in this region is predicted to destabilize the trimer,⁷⁸ and an aspartate-to-asparagine mutation results in σ1 trimers with a structure indistinguishable from wild-type,⁴³¹ it is likely that Asp345 is protonated in the σ1 crystal structure,⁹² thus representing the “closed” conformation of σ1. This conformation might form during crystallization at near-neutral pH and physiologically in conditions of low pH, similar to those encountered in the endocytic compartment during reovirus entry.⁴³¹ Thus, the aspartic acid sandwich motif may contribute to σ1 conformational rearrangements by acting as a molecular switch that mediates the oligomeric state of the σ1 head, depending on environmental pH.⁴³¹

Studies to assess the capacity of reovirus entry intermediates to penetrate artificial lipid bilayers, model membranes of erythrocytes, or membranes of cells that support reovirus infection indicate that ISVPs but not virions or cores mediate membrane penetration.⁵⁹,⁸⁰,⁸³,⁸⁴,²²⁶,²⁹⁴,⁴⁹² Such studies led to the idea that ISVPs or related subviral particles are the membrane-active intermediates in the reovirus entry pathway. Since ISVPs differ from cores by the presence of outer-capsid proteins σ1 and μ1,¹¹⁴,¹⁵¹ and because cores recoated with μ1 and σ3 in vitro and then treated with chymotrypsin to remove σ3 are capable of membrane penetration,⁸³ these findings point to a role for the M2-encoded μ1 protein (76 kDa, 708 aa) in membrane penetration. This biochemical evidence also is supported by several genetic studies. Differences in membrane-penetration efficiency displayed by reovirus strains T1L and T3D segregate with the M2 gene segment.⁷⁹,²⁹⁴,⁴³⁰ Additionally, viruses selected for resistance to denaturants such as ethanol contain mutations within the M2 gene segment and display alterations in membrane penetration capacity.⁷⁹,¹²³,²²⁶,⁵²² Together, these data demonstrate a function for the μ1 protein in membrane penetration.