Reovirus polypeptide sigma 3 and N-terminal myristoylation of polypeptide mu 1 are required for site-specific cleavage to mu 1C in transfected cells.

N-myristoylated viral polypeptide mu 1 was produced in COS cells transfected with a transient expression vector containing a DNA copy of the reovirus M2 gene. The mu 1 product was specifically cleaved to polypeptide mu 1C in cells that were cotransfected with the reovirus S4 gene and that expressed polypeptide sigma 3. Studies with site-specific mutants of the M2 gene demonstrated that conversion of mu 1 to mu 1C was dependent on myristoylation and the presence of the proteolytic cleavage sequence asparagine 42-proline 43 in mu 1, as well as on the presence of polypeptide sigma 3. The mu 1C product and polypeptide sigma 3 formed complexes that were immunoprecipitated by sigma 3-directed antibody, and a myristoylation-negative M2 double mutant, G2A-N42T, yielded mu 1 that did not undergo cleavage to mu 1C or bind sigma 3. However, the N42T single mutant did form immunoprecipitable complexes with sigma 3, indicating that binding can occur in the absence of cleavage. Polypeptide sigma 3 alternatively can bind double-stranded RNA and in COS cells stimulates translation of reporter chloramphenicol acetyltransferase mRNA translation, presumably by blocking double-stranded RNA-mediated activation of the eukaryotic initiation factor 2 alpha subunit kinase which inhibits the initiation of protein synthesis. Consistent with these observations and with the formation of mu 1C-sigma 3 complexes, coexpression of M2 with S4 DNA prevented the translational stimulatory effect of polypeptide sigma 3.     

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s2 mRNA which encodes the virion core polypeptide sigma 2

Human reovirus serotype 1 Lang strain s2 mRNA, which encodes the virion inner capsid core polypeptide sigma 2, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s2 mRNA is 1331 nucleotides in length and possesses an open reading frame with a coding capacity of 335 amino acids, sufficient to account for a sigma 2 polypeptide of 37,682 daltons. Comparison of the serotype 1 Lang s2 sequence derived from cDNA clones of s2 mRNA with the serotype 3 Dearing S2 sequence derived from cDNA clones of the S2 dsRNA genome segment reveals 86 percent homology at the nucleotide level. The predicted sigma 2 polypeptides of the Lang and Dearing strains display 98 percent homology at the amino acid level. Of 147 silent nt differences in the translated region, 136 were in the third base position of codons.     

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s4 mRNA which encodes the major capsid surface polypeptide sigma 3

Serotype 1 Lang strain s4 mRNA, which encodes the major capsid surface polypeptide sigma 3 of reovirions, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence for s4 mRNA has been determined from cDNA clones. The Lang strain s4 mRNA is 1196 nucleotides in length and possesses an open reading frame with a coding capacity of 365 amino acids, sufficient to account for a sigma 3 polypeptide of 41,212 daltons. Comparison of the serotype 1 (Lang) s4 sequence with the serotype 3 (Dearing) s4 sequence reveals 94% homology at the nucleotide level; the predicted sigma 3 polypeptides of the Lang and Dearing strains display 96% homology at the amino acid level. Two third base C codons (leu:CUC and ser:AGC) are used about one-tenth as frequently in the reovirus s4 mRNAs as compared to mammalian cellular mRNAs.     

Regulated, stable expression and nuclear presence of retrovirus double-stranded RNA-binding protein sigma3 in HeLa cells.

Reovirus genome segment S4 codes for polypeptide sigma3, a major outer capsid component of virions and a double-stranded RNA (dsRNA)-binding protein implicated in viral cytopathogenesis. We have constructed a stable HeLa cell line (S4tTA) that produces functional sigma3 under tetracycline transactivator control. In the absence of tetracycline, S4tTA cells synthesized stable dsRNA-binding sigma3 that accumulated in the nucleus as well as in the cytoplasm. However, in induced S4tTA cells also expressing reovirus outer shell polypeptide mu1/mu1C, migration of sigma3 into the nucleus was blocked, probably as a result of formation of a complex with mu1/mu1C which was exclusively in the cytoplasm. Mutant analyses indicated a correlation between dsRNA-binding activity and nuclear entry of sigma3, suggesting an additional role(s) for this capsid protein in virus-cell interactions.     

Proteolytic cleavage of the reovirus sigma 3 protein results in enhanced double-stranded RNA-binding activity: identification of a repeated basic amino acid motif within the C-terminal binding region.

The reovirus capsid protein sigma 3 was examined for double-stranded RNA (dsRNA)-binding activity by Northwestern (RNA-protein) blot analysis. Treatment of virion-derived sigma 3 protein with Staphylococcus aureus V8 protease led to an increase in the dsRNA-binding activity associated with the C-terminal fragment of the protein. Recombinant C-terminal fragments of the sigma 3 protein were expressed in Escherichia coli from the S4 cDNA of reovirus serotype 1. These truncated sigma 3 proteins displayed proteolytic processing and dsRNA-binding activity similar to those observed for native, virion-derived sigma 3 protein as measured by Northwestern blot analysis. Construction of a modified pET3c vector, pET3Exo, allowed the production of 3'-terminal deletions of the S4 cDNA by using exonuclease III and rapid screening of the induced truncated sigma 3 proteins. An 85-amino-acid domain within the C-terminal portion of the sigma 3 protein which was responsible for dsRNA-binding activity was identified. The 85-amino-acid domain possessed a repeated basic amino acid motif which was conserved in all three serotypes of reovirus. Deletion of one of the basic motifs, predicted to be an amphipathic alpha-helix, destroyed dsRNA-binding activity.     

Reovirus major capsid protein expressed in Escherichia coli

A DNA copy of the open reading frame of the S4 gene of reovirus type 3 was cloned into a temperature-regulated bacterial expression vector. Induction at 42 degrees C resulted in the synthesis of a polypeptide that comigrated with virion capsid protein sigma 3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reacted with sigma 3-specific antisera. The protein was expressed in bacteria as insoluble aggregates that accumulated in polar inclusion bodies. Aggregated product also resulted when the expression system was manipulated to induce bacterial sigma 3 (b sigma 3) synthesis at temperatures below 42 degrees C. Various methods used to solubilize b sigma 3 did not yield the monomeric protein. The results indicate that sigma 3, the major surface component of reovirions, is expressed in transfected Escherichia coli as an aggregated, disulfide cross-linked protein.     

Comparative sequence analysis of the reovirus S4 genes from 13 serotype 1 and serotype 3 field isolates.

The reovirus sigma 3 protein is a major outer capsid protein that may function to regulate translation within infected cells. To facilitate the understanding of sigma 3 structure and functions and the evolution of mammalian reoviruses, we sequenced cDNA copies of the S4 genes from 10 serotype 3 and 3 serotype 1 reovirus field isolates and compared these sequences with sequences of prototypic strains of the three reovirus serotypes. We found that the sigma 3 proteins are highly conserved: the two longest conserved regions contain motifs proposed to function in binding zinc and double-stranded RNA. We used the 16 viral isolates to investigate the hypothesis that structural interactions between sigma 3 and the cell attachment protein, sigma 1, constrain their evolution and to identify a determinant within sigma 3 that is in close proximity to the sigma 1 hemagglutination site.     

Identification of a new polypeptide coded by reovirus gene S1.

The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).     

Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.     

Genetic diversity in natural populations of mammalian reoviruses: tryptic peptide analysis of outer capsid polypeptides of murine, bovine, and human type 1 and 3 reovirus strains.

We have studied the structural relationships between the outer capsid polypeptides of eight murine, bovine, and human isolates of type 1 and 3 mammalian reoviruses. Our results show that the outer capsid polypeptides of reoviruses isolated from different mammalian species, in different years and different geographical areas, have both conserved and unique methionine-containing tryptic peptides. We found that tryptic peptides from mu 1C polypeptides of two human, one murine, and two bovine type 3 isolates and one human and two bovine type 1 reoviruses are highly conserved. Our data show that only one tryptic peptide pattern of the mu 1C polypeptide (encoded by the M2 gene) was present in reoviruses isolated from the three different mammalian species. The mu 1C polypeptide of the type 3 Dearing strain contained one tryptic peptide not found in any other reovirus isolate examined. In marked contrast to the mu 1C polypeptides, the sigma 3 polypeptides (encoded by the S4 gene) of three type 1 and three type 3 isolates were divided into two patterns based on significant differences in their tryptic peptides. In addition, at least seven tryptic peptides were conserved among the sigma 3 polypeptides of all virus strains examined. The sigma 3 polypeptide of the type 3 Dearing strain was distinguishable from the sigma 3 polypeptides of all other strains examined. The one mu 1C and two sigma 3 tryptic peptide patterns were found to occur interchangeably in isolates of type 1 or type 3. About 1/3 of the tyrosine-containing tryptic peptides of sigma 1 polypeptides of four type 3 isolates examined were conserved. Comparison of peptide differences in sigma 1 polypeptides of these isolates showed that each had one or more unique tryptic peptides, suggesting that the S1 genes coding for these polypeptides had undergone genetic drift or, alternatively, that there are at least two tryptic peptide patterns present among the sigma 1 polypeptides of these isolates. Our results suggest that genetic drift and reassortment are the most likely explanation for the extensive genetic diversity found in natural populations of mammalian reoviruses.     

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide sigma 1a and the nonstructural polypeptide sigma 1bNS

Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames. The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS. Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment.     

Identification of human galactoprotein b3, an oncogenic transformation-induced membrane glycoprotein, as VLA-3 alpha subunit: the primary structure of human integrin alpha 3.

Galactoprotein b3 is one of the cell membrane glycoproteins of fibroblasts showing enhanced expression in association with oncogenic transformation. Analysis of cDNA for this glycoprotein from hamster fibroblasts indicated that the glycoprotein is a member of the integrin superfamily [Tsuji, T., Yamamoto, F., Miura, Y., Takio, K., Titani, K., Pawar, S., Osawa, T., & hakomori, S. (1990) J. Biol. Chem. 265, 7016-7021]. In the present study, we examined the change in the amounts of mRNA for the mouse and human counterparts in fibroblasts after oncogenic transformation by Northern blot analysis using hamster galactoprotein b3 cDNA as a probe. In both human and murine fibroblasts transformed with SV-40, the homologous mRNA to galactoprotein b3 was also found to increase as compared with the progenitor cells. The human homologue of galactoprotein b3 cDNA was cloned from human bladder carcinoma cell line (T24) cDNA library. The cDNA codes for a single polypeptide of which the N-terminal sequence (21 amino acids) is identical with that of human VLA-3 alpha subunit. Based on this sequence identity and the structural similarities (i.e. the positions of most cysteine residues, the presence of a transmembrane domain near the C-terminus and the presence of metal binding sequences) with other integrins so far cloned, we conclude that human galactoprotein b3 is an integrin alpha 3 subunit. The mature integrin alpha 3 polypeptide was composed of 1,019 amino acid residues, and the overall structure was quite similar to the hamster counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

AP-4, a novel protein complex related to clathrin adaptors

Here we report the identification and characterization of AP-4, a novel protein complex related to the heterotetrameric AP-1, AP-2, and AP-3 adaptors that mediate protein sorting in the endocytic and late secretory pathways. The key to the identification of this complex was the cloning and sequencing of two widely expressed, mammalian cDNAs encoding new homologs of the adaptor beta and sigma subunits named beta4 and sigma4, respectively. An antibody to beta4 recognized in human cells an approximately 83-kDa polypeptide that exists in both soluble and membrane-associated forms. Gel filtration, sedimentation velocity, and immunoprecipitation experiments revealed that beta4 is a component of a multisubunit complex (AP-4) that also contains the sigma4 polypeptide and two additional adaptor subunit homologs named mu4 (mu-ARP2) and epsilon. Immunofluorescence analyses showed that AP-4 is associated with the trans-Golgi network or an adjacent structure and that this association is sensitive to the drug brefeldin A. We propose that, like the related AP-1, AP-2, and AP-3 complexes, AP-4 plays a role in signal-mediated trafficking of integral membrane proteins in mammalian cells.     

Sequence diversity in S1 genes and S1 translation products of 11 serotype 3 reovirus strains.

The S1 gene nucleotide sequences of 10 type 3 (T3) reovirus strains were determined and compared with the T3 prototype Dearing strain in order to study sequence diversity in strains of a single reovirus serotype and to learn more about structure-function relationships of the two S1 translation products, sigma 1 and sigma 1s. Analysis of phylogenetic trees constructed from variation in the sigma 1-encoding S1 nucleotide sequences indicated that there is no pattern of S1 gene relatedness in these strains based on host species, geographic site, or date of isolation. This suggests that reovirus strains are transmitted rapidly between host species and that T3 strains with markedly different S1 sequences circulate simultaneously. Comparison of the deduced sigma 1 amino acid sequences of the 11 T3 strains was notable for the identification of conserved and variable regions of sequence that correlate with the proposed domain organization of sigma 1 (M.L. Nibert, T.S. Dermody, and B. N. Fields, J. Virol. 64:2976-2989, 1990). Repeat patterns of apolar residues thought to be important for sigma 1 structure were conserved in all strains examined. The deduced sigma 1s amino acid sequences of the strains were more heterogeneous than the sigma 1 sequences; however, a cluster of basic residues near the amino terminus of sigma 1s was conserved. This analysis has allowed us to investigate molecular epidemiology of T3 reovirus strains and to identify conserved and variable sequence motifs in the S1 translation products, sigma 1 or sigma 1s.