The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation.

The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell-cell, rather than virus-cell, membrane fusion. The 36-40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell-cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome-liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.

[Gene sequence analysis and prokaryotic expression of sigmaB protein of Muscovy duck reovirus YB strain].

Muscovy ducks reovirus (DRV) is an important pathogen with a high mortality rate in Muscovy ducks, the researches in the test and the immunity were useful for the prevention and control of DRV infection. In this study, the S3 genes of the three Fujian DRVs were cloned by RT-PCR and sequencing technology. It was found that DRV-YH and YJL were close to avian reovirus (ARV) in the genetic distance, with high identities ranged from 94. 6% to 98. 9%, however, the identities of DRV-YB strain and reference ARV strains in the S3 gene were only 60.6% - 61.7%. The expression vector pET-30a-S3 harboring DRV YB strain S3 gene was constructed and transformed into E. coli BL21, and then the fusion sigmaB protein expression was induced with IPTG. The SDS-PAGE of the expressed products indicated that the fusion protein of approximately 42ku in molecular weight was expressed highly in inclusion body, and made up 67. 7% of the total proteins. The most efficient concentration of IPTG and inducing time were 0. 1 mM and 5h respectively, while the best temperature for expression was 37 degrees C. After purification with the Ni2+ affinity chromatography, the fusion sigmaB protein was 93% of the total proteins, and the purified protein amounted to 0. 86g/L. The Western blot analysis showed that the fusion aB protein was recognized specifically by the antiserum against DRV, confirming that the recombinant fusion protein had good immunoreactivity.

Isolation and characterization of a turkey arthritis reovirus.

During the spring and summer of 2011, the Minnesota Veterinary Diagnostic Laboratory at the University of Minnesota received 14 submissions of 15-to-18-week-old tom turkeys that were recumbent with wing tip bruises ("wing walkers") and uni- or bilateral swelling of the hock (tibiotarsal) joints. Gastrocnemius or digital flexor tendons were occasionally ruptured. A total of five turkey arthritis reoviruses (TARV-MN1 through TARV-MN5) were isolated in specific-pathogen-free embryonated chicken eggs and QT-35 cells. The identity of the isolates was confirmed by electron microscopy, reverse transcription-polymerase chain reaction, and gene sequence analysis. BLAST analysis on the basis of a 880 bp nucleotide sequence of the S4 gene confirmed all isolates as a reovirus. Phylogenetic analysis divided the five isolates into two subgroups: subgroup I containing TARV-MN1, -2, -3, and -5, and the other subgroup containing TARV-MN4. Isolates in subgroup I had a similarity of 97%-100% with each other, while subgroup II (TARV-MN4) had a similarity of only 89.2% with subgroup I viruses. This isolate showed 90%-93% similarity with turkey enteric reoviruses in the United States, while the other four isolates in subgroup I had 89%-97.6% similarity. These results indicate divergence within TARVs as well as from enteric viruses, which needs to be confirmed by complete genome sequence analysis. Further experimental studies are planned to determine the role of these isolates in turkey arthritis and to compare them with classical chicken reovirus.

IC-tagged proteins are able to interact with each other and perform complex reactions when integrated into muNS-derived inclusions.

We have recently developed a versatile tagging system (IC-tagging) that causes relocation of the tagged proteins to ARV muNS-derived intracellular globular inclusions. In the present study we demonstrate (i) that the IC-tag can be successfully fused either to the amino or carboxyl terminus of the protein to be tagged and (ii) that IC-tagged proteins are able to interact between them and perform complex reactions that require such interactions while integrated into muNS inclusions, increasing the versatility of the IC-tagging system. Also, our studies with the DsRed protein add some light on the structure/function relationship of the evolution of DsRed chromophore.

Avian reoviruses: propagation, quantification, and storage.

Avian reoviruses (ARVs) are pathogens that cause significant morbidity among commercial poultry. ARVs are prototypic representatives of non-enveloped viruses that can cause cell-cell fusion. They belong to the Reoviridae family, which contains many highly pathogenic viruses. ARVs are ubiquitous in commercial poultry and are frequently isolated from the gastrointestinal and respiratory tracts of chickens with acute infections. The virus causes a range of disease states in chicken, including viral arthritis/tenosynovitis, gastroenteritis, hepatitis, myocarditis, "pale bird syndrome," runting-stunting syndrome, and respiratory illness. This unit describes avian reovirus propagation, quantification, and storage.

Crystallographic structure of the alpha-helical triple coiled-coil domain of avian reovirus S1133 fibre.

Avian reovirus fibre, a homo-trimer of the sigmaC protein, is a minor component of the avian reovirus outer capsid. It is anchored via a short N-terminal sequence to the inner capsid lambdaC pentamer, and its protruding globular C-terminal domain is responsible for primary host cell attachment. We have previously solved the structure of a receptor-binding fragment in which residues 160-191 form a triple beta-spiral and 196-326 a beta-barrel head domain. Here we have expressed, purified and crystallized a major sigmaC fragment comprising residues 117-326. Its structure, which was solved by molecular replacement using the previously determined receptor-binding domain structure and refined to 1.75 A (0.175 nm) resolution, reveals an alpha-helical triple coiled-coil connected to the previously solved structure by a zinc-ion-containing linker. The coiled-coil domain contains two chloride ion binding sites, as well as specific trimerization and registration sequences. The linker may act as a functionally important hinge.

Crystal structure of the avian reovirus inner capsid protein sigmaA.

Avian reovirus, an important avian pathogen, expresses eight structural and four nonstructural proteins. The structural sigmaA protein is a major component of the inner capsid, clamping together lambdaA building blocks. sigmaA has also been implicated in the resistance of avian reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed sigmaA by molecular replacement and refined it using data to 2.3-A resolution. Twelve sigmaA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of sigmaA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of sigmaA to double-stranded RNA. The minimal length of double-stranded RNA required for sigmaA binding was observed to be 14 to 18 bp.

Assignment of avian reovirus temperature-sensitive mutant recombination groups E, F, and G to genome segments.

Avian reoviruses (ARV) are less well understood than their mammalian counterparts. ARV are ubiquitous in commercial poultry and frequently isolated from acutely infected chickens. We previously described isolation of ARV temperature-sensitive (ts) mutants after nitrosoguanidine mutagenesis of wild-type ARV138, their assignment to 7 recombination groups (A-G), and genetic mapping of mutants in groups A-D to specific gene segments. For this study, wild-type serotype ARV176 was crossed with ts mutants tsE158 (Group E), tsF206 (Group F), or tsG247 (Group G) and reassortant progenies analyzed. Reassortant temperature-sensitivities were determined by efficiency of plating at permissive and non-permissive temperatures. Mapping results indicated tsE158, tsF206, and tsG247 mapped to the L1, S4, and L3 genes, respectively, which encode the lambdaA core shell, sigmaNS non-structural, and lambdaC core spike proteins, respectively. Specific amino acid substitutions in each mutant were determined and locations of structural protein alterations were placed within the 3-dimensional structure of homologous mammalian reovirus proteins. Mapping recombination groups E-G marks completion of gene assignments for all seven ts mutant groups previously generated.

Crystallization of the avian reovirus double-stranded RNA-binding and core protein sigmaA.

The avian reovirus protein sigmaA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The sigmaA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 A, alpha = 93.8, beta = 105.1, gamma = 98.2 degrees were grown and a complete data set has been collected to 2.3 A resolution. The self-rotation function suggests that sigmaA may form symmetric arrangements in the crystals.

Avian reovirus: structure and biology.

Avian reoviruses are important pathogens that cause considerable losses to the poultry industry, but they have been poorly characterized at the molecular level in the past, mostly because they have been considered to be very similar to the well-studied mammalian reoviruses. Studies performed over the last 20 years have revealed that avian reoviruses have unique properties and activities, different to those displayed by their mammalian counterparts, and of considerable interest to molecular virologists. Notably, the avian reovirus S1 gene is unique, in that it is a functional tricistronic gene that possesses three out-of-phase and partially overlapping open reading frames; the identification of the mechanisms that govern the initiation of translation of the three S1 cistrons, and the study of the properties and activities displayed by their encoded proteins, are particularly interesting areas of research. For instance, avian reoviruses are one of the few nonenveloped viruses that cause cell-cell fusion, and their fusogenic phenotype has been associated with a nonstructural 10 kDa transmembrane protein, which is expressed by the second cistron of the S1 gene; the small size of this atypical fusion protein offers an interesting model for studying the mechanisms of cell-cell fusion and for identifying fusogenic domains. Finally, avian reoviruses are highly resistant to interferon, and therefore they may be useful for investigating the mechanisms and strategies that viruses utilize to counteract the antiviral actions of interferons.

Expression of avian reovirus sigmaC protein in transgenic plants.

Avian reovirus (ARV) structural protein, sigmaC encoded by S1 genome segment, is the prime candidate to become a vaccine against ARV infection. Two plant nuclear expression vectors with expression of sigmaC-encoding gene driven by CaMV 35S promoter and rice actin promoter were constructed, respectively. Agrobacterium containing the S1 expression constructs were used to transform alfalfa, and transformants were selected using hygromysin. The integration of S1 transgene in alfalfa chromosome was confirmed by PCR and histochemical GUS staining. Western blot analysis using antiserum against sigmaC was carried out to determine the expression of sigmaC protein in transgenic alfalfa cells. The highest expression levels of sigmaC protein in the cellular extracts of selected p35S-S1 and pAct1-S1 transgenic alfalfa lines were 0.008% and 0.007% of the total soluble protein, respectively. The transgenic alfalfa cells with expression of sigmaC protein pave the way for the development of edible vaccine.

Sequences of avian reovirus M1, M2 and M3 genes and predicted structure/function of the encoded mu proteins.

We report the first sequence analysis of the entire complement of M-class genome segments of an avian reovirus (ARV). We analyzed the M1, M2 and M3 genome segment sequences, and sequences of the corresponding muA, muB and muNS proteins, of two virus strains, ARV138 and ARV176. The ARV M1 genes were 2,283 nucleotides in length and predicted to encode muA proteins of 732 residues. Alignment of the homologous mammalian reovirus (MRV) mu2 and ARV muA proteins revealed a relatively low overall amino acid identity ( approximately 30%), although several highly conserved regions were identified that may contribute to conserved structural and/or functional properties of this minor core protein (i.e. the MRV mu2 protein is an NTPase and a putative RNA-dependent RNA polymerase cofactor). The ARV M2 genes were 2158 nucleotides in length, encoding predicted muB major outer capsid proteins of 676 amino acids, more than 30 amino acids shorter than the homologous MRV mu1 proteins. In spite of the difference in size, the ARV/MRV muB/mu1 proteins were more conserved than any of the homologous proteins encoded by other M- or S-class genome segments, exhibiting percent amino acid identities of approximately 45%. The conserved regions included the residues involved in the maturation- and entry- specific proteolytic cleavages that occur in the MRV mu1 protein. Notably missing was a region recently implicated in MRV mu1 stabilization and in forming "hub and spokes" complexes in the MRV outer capsid. The ARV M3 genes were 1996 nucleotides in length and predicted to encode a muNS non-structural protein of 635 amino acids, significantly shorter than the homologous MRV muNS protein, which is attributed to several substantial deletions in the aligned ARV muNS proteins. Alignments of the ARV and MRV muNS proteins revealed a low overall amino acid identity ( approximately 25%), although several regions were relatively conserved.

Structure of the carboxy-terminal receptor-binding domain of avian reovirus fibre sigmaC.

Avian reovirus fibre, a homo-trimer of the sigmaC protein, is responsible for primary host cell attachment. The protein expressed in bacteria forms elongated fibres comprised of a carboxy-terminal globular head domain and a slender shaft, and partial proteolysis yielded a carboxy-terminal protease-stable domain that was amenable to crystallisation. Here, we show that this fragment retains receptor-binding capability and report its structure, solved using two-wavelength anomalous diffraction and refined using data collected from three different crystal forms at 2.1 angstroms, 2.35 angstroms and 3.0 angstroms resolution. The carboxy-terminal globular domain has a beta-barrel fold with the same overall topology as the mammalian reovirus fibre (sigma1). However, the monomers of the sigmaC trimer show a more splayed-out arrangement than in the sigma1 structure. Also resolved are two triple beta-spiral repeats of the shaft or stalk domain. The presence in the sequence of heptad repeats amino-terminal to these triple beta-spiral repeats suggests that the unresolved portion of the shaft domain contains a triple alpha-helical coiled-coil structure. Implications for the function and stability of the sigmaC protein are discussed.

Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus.

We have previously shown that avian reovirus (ARV) sigmaA and sigmaNS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on sigmaA (I and II) and three epitopes (A, B, and C) on sigmaNS. To further define the location of epitopes on sigmaA and sigmaNS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of sigmaA and sigmaNS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on sigmaA was located at amino acid residues 340QWVMAGLVSAA350 and epitope B on sigmaNS at amino acid residues 180MLDMVDGRP188. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of sigmaA and sigmaNS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete sigmaNS but not with truncated sigmaNS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on sigmaNS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on sigmaA was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the sigmaNS with MAb 1F9. The sigmaNS of ARV with ssRNA-binding activity could be blocked by monoclonal antibody 1F9. The epitope B on sigmaNS is required for ssRNA binding because its deletion fully abolished the ssRNA binding activity of sigmaNS.

Avian reovirus temperature-sensitive mutant tsA12 has a lesion in major core protein sigmaA and is defective in assembly.

Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperature-sensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284:113-122, 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced approximately 100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced approximately 100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein sigmaA. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the sigmaA protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu(158) substitution in ARV sigmaA lies directly under the outer face of the sigmaA protein. This may cause a perturbation in sigmaA such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly.

A molecular dynamics study of reovirus attachment protein sigma1 reveals conformational changes in sigma1 structure.

Molecular dynamics simulations were performed using the recently determined crystal structure of the reovirus attachment protein, sigma1. These studies were conducted to improve an understanding of two unique features of sigma1 structure: the protonation state of Asp(345), which is buried in the sigma1 trimer interface, and the flexibility of the protein at a defined region below the receptor-binding head domain. Three copies of aspartic acids Asp(345) and Asp(346) cluster in a solvent-inaccessible and hydrophobic region at the sigma1 trimer interface. These residues are hypothesized to mediate conformational changes in sigma1 during viral attachment or cell entry. Our results indicate that protonation of Asp(345) is essential to the integrity of the trimeric structure seen by x-ray crystallography, whereas deprotonation induces structural changes that destabilize the trimer interface. This finding was confirmed by electrostatic calculations using the finite difference Poisson-Boltzmann method. Earlier studies show that sigma1 can exist in retracted and extended conformations on the viral surface. Since protonated Asp(345) is necessary to form a stable, extended trimer, our results suggest that protonation of Asp(345) may allow for a structural transition from a partially detrimerized molecule to the fully formed trimer seen in the crystal structure. Additional studies were conducted to quantify the previously observed flexibility of sigma1 at a defined region below the receptor-binding head domain. Increased mobility was observed for three polar residues (Ser(291), Thr(292), and Ser(293)) located within an insertion between the second and third beta-spiral repeats of the crystallized portion of the sigma1 tail. These amino acids interact with water molecules of the solvent bulk and are responsible for oscillating movement of the head of approximately 50 degrees during 5 ns of simulations. This flexibility may facilitate viral attachment and also function in cell entry and disassembly. These findings provide new insights about the conformational dynamics of sigma1 that likely underlie the initiation of the reovirus infectious cycle.

Avian reovirus sigmaC protein induces apoptosis in cultured cells.

The avian reovirus (ARV) infection is associated with various disease conditions in poultry. However, the pathogenesis mechanisms are poorly characterized. In the present study, we clearly demonstrated that the sigmaC of ARV S1133 strain induced apoptosis in both BHK-21 and Vero cells. Five kinds of assays for apoptosis were used in analyzing ARV-infected BHK-21 and Vero cells: (1) assay for DNA ladders, (2) ELISA detection of cytoplasmic histone-associated DNA fragments, (3) nuclear staining with acridine orange, (4) Western blot, Northern blot, and immunofluorescent assay (IFA), and (5) flow cytometric analysis. The sigmaC protein of ARV could elicit apoptosis occurring in a dose- and time-dependent manner. The current results further our understanding of the function of sigmaC in cultured cells and suggest that sigmaC is a viral-encoded apoptin and possesses apoptosis-inducing ability. Furthermore, deletion analysis of the ARV sigmaC protein suggests that the carboxyl-terminus of sigmaC is important in mediating sigmaC-induced apoptosis because its deletion abolished the induction of apoptosis.

Subunit composition and conformational stability of the oligomeric form of the avian reovirus cell-attachment protein sigmaC.

Previous work has shown that the avian reovirus cell-attachment sigma C (sigmaC) protein is a multimer. In the first part of this study the oligomerization state of intracellularly synthesized sigmaC was analysed by different approaches, including SDS-PAGE, chemical cross-linking, sedimentation and gel filtration analysis. All these approaches indicated that protein sigmaC in its native state is a homotrimer. In the second part of the present work we investigated the effect of different factors and reagents on oligomer stability, in order to elucidate the nature of the forces that maintain the conformational stability of the homotrimer. Our results, based on the stabilizing effect conferred by reducing agents, demonstrate that the sigmaC subunits are not covalently bound via disulfide linkages. They further suggest that the formation of an intrachain disulfide bond between the two cysteine residues of the sigmaC polypeptide has a negative effect on oligomer stability. The susceptibility of the trimer to pH, temperature, ionic strength, chemical denaturants and detergents indicates that hydrophobic interactions contribute much more to oligomer stability than do ionic interactions and hydrogen bonding. Finally, our results also reveal that mammalian and avian reovirus cell attachment proteins follow different subunit dissociation pathways.

Sequence and phylogenetic analysis of the sigmaA-encoding gene of avian reovirus.

The full-length sigmaA-encoding gene nucleotide sequences (1251 bp) of ten avian reovirus (ARV) field-isolates and three vaccine strains were determined and analyzed to study the degree of genetic divergence and evolution. Strains were isolated over a 23-year period from different hosts, pathotypes, and geographic locations. A phylogenetic tree constructed from variation in the sigmaA nucleotide sequences among ARV isolates showed that Taiwanese isolates from different dates of isolation were grouped into two distant groups, indicating that they have evolved in nature. In paired identity analysis, there was over 97.3% nucleotide sequence identity in the sigmaA-encoding genes between group I Taiwanese isolates (T6, 750505, 919, and 918) and Japanese isolate OS161 as well as three US vaccine strains, suggesting that they might have descended from a common ancestor. However, the nucleotide sequences of these sigmaA-encoding genes varied extensively from those of group II Taiwanese isolates (601SI, R2/TW, 1017-1, 916, and 601G), displaying only 86% identity. These results revealed that the genetic diversity in the sigmaA-encoding gene of ARV correlated with the date of isolation and geographic locations.