Mammalian reoviruses contain a myristoylated structural protein.

The structural protein mu 1 of mammalian reoviruses was noted to have a potential N-myristoylation sequence at the amino terminus of its deduced amino acid sequence. Virions labeled with [3H]myristic acid were used to demonstrate that mu 1 is modified by an amide-linked myristoyl group. A myristoylated peptide having a relative molecular weight (Mr) of approximately 4,000 was also shown to be a structural component of virions and was concluded to represent the 4.2-kDa amino-terminal fragment of mu 1 which is generated by the same proteolytic cleavage that yields the carboxy-terminal fragment and major outer capsid protein mu 1C. The myristoylated 4,000-Mr peptide was found to be present in reovirus intermediate subviral particles but to be absent from cores, indicating that it is a component of the outer capsid. A distinct large myristoylated fragment of the intact mu 1 protein was also identified in intermediate subviral particles, but no myristoylated mu-region proteins were identified in cores, consistent with the location of mu 1 in the outer capsid. Similarities between amino-terminal regions of the reovirus mu 1 protein and the poliovirus capsid polyprotein were noted. By analogy with other viruses that contain N-myristoylated structural proteins (particularly picornaviruses), we suggest that the myristoyl group attached to mu 1 and its amino-terminal fragments has an essential role in the assembly and structure of the reovirus outer capsid and in the process of reovirus entry into cells.     

Incorporation of epitope-tagged viral sigma3 proteins to reovirus virions

Tagging of viral capsid proteins is a powerful tool to study viral assembly; it also raises the possibility of using viral particles to present exogenous epitopes in vaccination or gene therapy strategies. The ability of reoviruses to induce strong mucosal immune response and their large host range and low pathogenicity in humans are some of the advantages of using reoviruses in such applications. In the present study, the feasibility of introducing foreign epitopes, "tags", to the sigma3 protein, a major component of the reovirus outer capsid, was investigated. Among eight different positions, the amino-terminal end of the protein appeared as the best location to insert exogenous sequences. Additional amino acids at this position do not preclude interaction with the micro1 protein, the other major constituent of the viral outer capsid, but strongly interfere with micro1 to micro1C cleavage. Nevertheless, the tagged sigma3 protein was still incorporated to virions upon recoating of infectious subviral particles to which authentic sigma3 protein was removed by proteolysis, indicating that micro1 cleavage is not a prerequisite for outer capsid assembly. The recently published structure of the sigma3- micro1 complex suggests that the amino-terminally inserted epitope could be exposed at the outer surface of viral particles.     

Cell entry-associated conformational changes in reovirus particles are controlled by host protease activity

Membrane penetration by reovirus requires successive formation of two cell entry intermediates, infectious subvirion particles (ISVPs) and ISVP*s. In vitro incubation of reovirus virions with high concentration of chymotrypsin (CHT) results in partial digestion of the viral outer capsid to form ISVPs. When virions are instead digested with low concentrations of chymotrypsin, the outer capsid is completely proteolyzed to form cores. We investigated the basis for the inverse relationship between CHT activity and protease susceptibility of the reovirus outer capsid. We report that core formation following low-concentration CHT digestion proceeds via formation of particles that contain a protease-sensitive form of the μ1C protein, a characteristic of ISVP*s. In addition, we found that both biochemical features and viral genetic requirements for ISVP* formation and core formation following low-concentration CHT digestion are identical, suggesting that core formation proceeds via a particle resembling ISVP*s. Furthermore, we determined that intermediates generated following low-concentration CHT digestion are distinct from ISVPs and convert to ISVP*-like particles much more readily than ISVPs. These results suggest that the activity of host proteases used to generate ISVPs can influence the efficiency with which the next step in reovirus cell entry, namely, ISVP-to-ISVP* conversion, occurs.     

Beta1 integrin mediates internalization of mammalian reovirus

Reovirus infection is initiated by interactions between the attachment protein sigma1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein lambda2, which serves as the structural base for sigma1, suggests that integrins mediate reovirus endocytosis. A beta1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a beta1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in beta1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing beta1. However, reovirus bound equivalently to cells that differed in levels of beta1 expression, suggesting that beta1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into beta1-deficient cells was substantially diminished in comparison to viral uptake into beta1-expressing cells. These data provide evidence that beta1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.     

Cathepsin S supports acid-independent infection by some reoviruses

In murine fibroblasts, efficient proteolysis of reovirus outer capsid protein sigma3 during cell entry by virions requires the acid-dependent lysosomal cysteine protease cathepsin L. The importance of cathepsin L for infection of other cell types is unknown. Here we report that the acid-independent lysosomal cysteine protease cathepsin S mediates outer capsid processing in macrophage-like P388D cells. P388D cells supported infection by virions of strain Lang, but not strain c43. Genetic studies revealed that this difference is determined by S4, the viral gene segment that encodes sigma3. c43-derived subvirion particles that lack sigma3 replicated normally in P388D cells, suggesting that the difference in infectivity of Lang and c43 virions is at the level of sigma3 processing. Infection of P388D cells with Lang virions was inhibited by the broad spectrum cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane but not by NH(4)Cl, which raises the endocytic pH and thereby inhibits acid-dependent proteases such as cathepsins L and B. Outer capsid processing and infection of P388D cells with Lang virions were also inhibited by a cathepsin S-specific inhibitor. Furthermore, in the presence of NH(4)Cl, cell lines engineered to express cathepsin S supported infection by Lang, but not c43, virions. Our results thus indicate that differences in susceptibility to cathepsin S-mediated sigma3 processing are responsible for strain differences in reovirus infection of macrophage-like P388D cells and other cathepsin S-expressing cells. Additionally, our data suggest that the acid dependence of reovirus infections of most other cell types may reflect the low pH requirement for the activities of most other lysosomal proteases rather, than some other acid-dependent aspect of cell entry.     

In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3

Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.     

Interferon-inducible Transmembrane Protein 3 (IFITM3) Restricts Reovirus Cell Entry

Reoviruses are double-stranded RNA viruses that infect the mammalian respiratory and gastrointestinal tract. Reovirus infection elicits production of type I interferons (IFNs), which trigger antiviral pathways through the induction of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, the functions of many of these genes are unknown. The interferon-inducible transmembrane (IFITM) proteins are one class of ISGs that restrict the cell entry of some enveloped viruses, including influenza A virus. One family member, IFITM3, localizes to late endosomes, where reoviruses undergo proteolytic disassembly; therefore, we sought to determine whether IFITM3 also restricts reovirus entry. IFITM3-expressing cell lines were less susceptible to infection by reovirus, as they exhibited significantly lower percentages of infected cells in comparison to control cells. Reovirus replication was also significantly reduced in IFITM3-expressing cells. Additionally, cells expressing an shRNA targeting IFITM3 exhibited a smaller decrease in infection after IFN treatment than the control cells, indicating that endogenous IFITM3 restricts reovirus infection. However, IFITM3 did not restrict entry of reovirus infectious subvirion particles (ISVPs), which do not require endosomal proteolysis, indicating that restriction occurs in the endocytic pathway. Proteolysis of outer capsid protein μ1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions. These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry.     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids

The capsid proteins of adeno-associated viruses (AAV) have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s) of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.     

Visualization of Individual Reovirus Particles by Low-Temperature, High-Resolution Scanning Electron Microscopy

We used low-temperature, high-resolution scanning electron microscopy (cryo-HRSEM) to visualize surface structures on individual reovirus particles. Both intact virions and two forms of subvirion particles—infectious subvirion particles and cores—were examined, and despite some distortion of particles during specimen preparation and viewing in the microscope, the images obtained by cryo-HRSEM exhibited a level of interpretable detail not routinely achieved by other methods without image averaging. Cryo-HRSEM images of discrete reovirus particles were used to characterize and confirm features of the outer protein capsid of this virus by comparison with image reconstructions previously derived from cryotransmission electron microscopy. Distinct surface features attributable to each of the four outer-capsid proteins were identified. In addition, cryo-HRSEM images confirmed that significant changes occur on the surfaces of individual reovirus particles during disassembly and entry of cells and that the reovirus outer capsid is organized as a left-handed T=13 icosahedron. Several unique capabilities and potential uses suggest that cryo-HRSEM has a place alongside other, more established methods for molecular characterizations of virus particles.     

Adaptation of reovirus to growth in the presence of protease inhibitor E64 segregates with a mutation in the carboxy terminus of viral outer-capsid protein sigma3

Reovirus virions are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis leading to degradation of sigma3 protein and proteolytic cleavage of micro1/micro1C protein. E64 is a specific inhibitor of cysteine-containing proteases that blocks disassembly of reovirus virions. To identify domains in reovirus proteins that influence susceptibility to E64-mediated inhibition of disassembly, we selected variant viruses by serial passage of strain type 3 Dearing (T3D) in murine L929 cells treated with E64. E64-adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yields than T3D did after infection of cells treated with 100 microM E64. Viral genes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses of E64-sensitive strain type 1 Lang and two prototype D-EA viruses. Growth of reassortant viruses in the presence of E64 segregated with the S4 gene, which encodes outer-capsid protein sigma3. Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a common tyrosine-to-histidine mutation at amino acid 354 in the deduced amino acid sequence of sigma3. Proteolysis of D-EA virions by endocytic protease cathepsin L occurred with faster kinetics than proteolysis of wild-type T3D virions. Treatment of D-EA virions, but not T3D virions, with cathepsin D resulted in proteolysis of sigma3, a property that also was found to segregate with the D-EA S4 gene. These results indicate that a region in sigma3 protein containing amino acid 354 influences susceptibility of sigma3 to proteolysis during reovirus disassembly.     

Proteolytic processing of reovirus is required for adherence to intestinal M cells.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes.     

Features of reovirus outer capsid protein mu1 revealed by electron cryomicroscopy and image reconstruction of the virion at 7.0 Angstrom resolution

Reovirus is a useful model for addressing the molecular basis of membrane penetration by one of the larger nonenveloped animal viruses. We now report the structure of the reovirus virion at approximately 7.0 A resolution as obtained by electron cryomicroscopy and three-dimensional image reconstruction. Several features of the myristoylated outer capsid protein mu1, not seen in a previous X-ray crystal structure of the mu1-sigma3 heterohexamer, are evident in the virion. These features appear to be important for stabilizing the outer capsid, regulating the conformational changes in mu1 that accompany perforation of target membranes, and contributing directly to membrane penetration during cell entry.     

Functional structure of the somatomedin B domain of vitronectin

The N-terminal somatomedin B domain (SMB) of vitronectin binds PAI-1 and the urokinase receptor with high affinity and regulates tumor cell adhesion and migration. We have shown previously in the crystal structure of the PAI-1/SMB complex that SMB, a peptide of 51 residues, is folded as a compact cysteine knot of four pairs of crossed disulfide bonds. However, the physiological significance of this structure was questioned by other groups, who disputed the disulfide bonding shown in the crystal structure (Cys5-Cys21, Cys9-Cys39, Cys19-Cys32, Cys25-Cys31), notably claiming that the first disulfide is Cys5-Cys9 rather than the Cys5-Cys21 bonding shown in the structure. To test if the claimed Cys5-Cys9 bond does exist in the SMB domain of plasma vitronectin, we purified mouse and rat plasma vitronectin that have a Met (hence cleavable by cyanogen bromide) at residue 14, and also prepared recombinant human SMB variants from insect cells with residues Asn14 or Leu24 mutated to Met. HPLC and mass spectrometry analysis showed that, after cyanogen bromide digestion, all the fragments of the SMB derived from mouse or rat vitronectin or the recombinant SMB mutants are still linked together by disulfides, and the N-terminal peptide (residue 1-14 or 1-24) can only be released when the disulfide bonds are broken. This clearly demonstrates that Cys5 and Cys9 of SMB do not form a disulfide bond in vivo, and together with other structural evidence confirms that the only functional structure of the SMB domain of plasma vitronectin is that seen in its crystallographic complex with PAI-1.     

Association of reovirus outer capsid proteins sigma 3 and mu 1 causes a conformational change that renders sigma 3 protease sensitive.

Association of the reovirus proteins sigma 3 and mu 1 influences viral entry, initiation of outer capsid assembly, and modulation of the effect of sigma 3 on cellular translation. In this study, we have addressed whether structural changes occur in sigma 3 as a result of its interaction with mu 1. Using differences in protease sensitivity to detect conformationally distinct forms of sigma 3, we showed that association of sigma 3 with mu 1 caused a conformational change in sigma 3 that converted it from a protease-resistant to a protease-sensitive structure and occurred posttranslationally. The effect of mu 1 on the structure of sigma 3 was stoichiometric. Our results are consistent with a model in which sigma 3's association with mu 1 shifts its function from translational control to assembly of an outer capsid in which sigma 3 is folded into the protease-sensitive conformation that is required for its cleavage during the next round of infection.