Polypeptide Sigma Research Articles

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.

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.

Stimulation of chloramphenicol acetyltransferase mRNA translation by reovirus capsid polypeptide sigma 3 in cotransfected COS cells.

The mammalian reovirus S4 gene has been implicated in the serotype-dependent inhibition of host cell protein synthesis during viral replication in mouse L cells. To examine the effect(s) of this gene on transcription or translation or both, a DNA copy of the serotype 3 S4 gene was inserted into a eucaryotic expression vector. Cotransfection of COS cells with plasmids containing S4 and the reporter gene, chloramphenicol acetyltransferase (CAT), resulted in a marked stimulation of CAT expression, predominantly at the level of translation. The significance of these findings is discussed in relation to the double-stranded-RNA-binding activity of the S4 gene product, polypeptide sigma 3.

Biosynthesis of reovirus-specified polypeptides: effect of point mutation of the sequences flanking the 5'-proximal AUG initiator codons of the reovirus S1 and S4 genes on the efficiency of mRNA translation.

The effect on translation of site-directed nucleotide substitutions around the 5'-proximal AUG initiation codon of the reovirus s1 mRNA specifying polypeptide sigma 1 and the reovirus s4 mRNA specifying polypeptide sigma 3 was examined. The efficiency of synthesis of the S1-encoded sigma 1 polypeptide and the S4-encoded sigma 3 polypeptide was analyzed in transfected simian COS cells. Mutant s1 mRNAs possessing either GCU AUG G or GCA AUG G sequences surrounding the 5'-proximal sigma 1 AUG were translated with an efficiency comparable to that of the wild-type s1 mRNA which possesses the flanking sequence CCU AUG G. Mutant s4 mRNAs possessing either CCU AUG G or CCA AUG G sequences surrounding the 5'-proximal sigma 3 AUG were translated with an efficiency comparable to that of wild-type s4 mRNA which possesses the flanking sequence GCA AUG G. The s4 mRNAs, both wild-type and mutant, were translated in vivo about five times more efficiently than the s1 mRNAs, both wild-type and mutant. These results suggest that nucleotide positions other than the -3, -2, -1, and +4 positions relative to the 5'-proximal initiator AUG, where the A is +1, play a dominant role in determining the efficiency of translation of these two reovirus mRNAs in vivo.

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 σ 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 s3 mRNA which encodes the nonstructural RNA-binding protein sigma NS

Human reovirus serotype 1 Lang strain s3 mRNA, which encodes the nonstructural RNA-binding polypeptide sigma NS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s3 mRNA is 1198 nucleotides in length and possesses an open reading frame with a coding capacity of 366 amino acids, sufficient to account for a sigma NS polypeptide of 41,179 daltons. Comparison of the serotype 1 (Lang) s3 sequence with the serotype 3 (Dearing) s3 sequence reveals 86.8 percent homology at the nucleotide level. The predicted sigma NS polypeptides of the Lang and Dearing strains display 97 percent homology at the amino acid level.

Nucleotide sequence of reovirus genome segment S3, encoding non-structural protein sigma NS.

This report describes the complete nucleotide sequence of human reovirus (Dearing strain) genome segment S3. Previous studies indicated that this RNA encodes the major non-structural viral polypeptide sigma NS, a protein that binds ssRNAs (Huisman & Joklik, Virology 70, 411-424, 1976) and has a poly(C)-dependent poly(G) polymerase activity (Gomatos et al., J. Virol. 39, 115-124, 1981). The genome segment consists of 1,198 nucleotides and possesses an open reading frame that extends 366 codons from the first AUG triplet (residues 28-30). There is no significant sequence homology between the plus strand of genome segment S3 and that of genome segment S2 determined previously (Cashdollar et al., PNAS 79, 7644-7648, 1982). However, S3 RNA has significant dyad symmetry and regions that can potentially hybridize (delta G = -26 KCal/mole) with S2 RNA. From the predicted amino acid sequence a possible secondary structure for sigma NS protein was determined. Structural features of reovirus RNA and sigma NS are discussed in relation to their role(s) in viral genome assembly.