Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.     

Genome analysis of MG virus, a human papovavirus.

The single late 26S mRNA of Semliki Forest virus (SFV) directs the synthesis of the four viral structural proteins, C, E3, E2, and E1, and the recently described nonstructural protein, 6K. We report here partial NH2-terminal amino acid sequences of the SFV polypeptides E3 and 6K and of p62, the precursor to E3 and E2. In addition, were have determined a partial NH2-terminal sequence of the Sindbis virus homolog of 6K, the 4.2K protein. p62 and E3 of SFV have identical NH2-terminal amino acid sequences. Comparison of the partial NH2-terminal sequences of 6K of SFV and 4.2K of Sindbis virus with the deduced amino acid sequence encoded by the 26S mRNA of each virus reveals that the genes for these peptides are located in each case between those for E2 and E1. The order of the genes on the 26S mRNA of the alphaviruses is therefore 5'-C-E3-E2-6K-E1-3'. We discuss two mechanisms by which the nascent viral glycoproteins may be inserted into the membrane of the endoplasmic reticulum.     

The oligomerization reaction of the Semliki Forest virus membrane protein subunits

The Semliki Forest virus (SFV) spike is composed of three copies of a membrane protein heterodimer. The two subunits of this heterodimer (p62 and E1) are synthesized sequentially from a common mRNA together with the capsid (C) in the order C-p62-E1. In this work heterodimerization of the spike proteins has been studied in BHK 21 cells. The results indicate that: (a) the polyprotein is cotranslationally cleaved into individual chains; (b) the two membrane protein subunits are initially not associated with each other in the endoplasmic reticulum (ER); (c) heterodimerization occurs predominantly between subunits that originate from the same translation product (heterodimerization in cis); (d) the kinetics of subunit association are very fast (t1/2 = 4 min); and (e) this heterodimerization is highly efficient. To explain the cis- directed heterodimerization reaction we suggest that the p62 protein, which is made before E1 during 26S mRNA translation, is retained at its translocation site until also the E1 chain has been synthesized and translocated at this same site. The mechanism for p62 retention could either be that the p62 anchor sequence cannot diffuse out from an "active" translocation site or that the p62 protein is complexed with a protein folding facilitating machinery that is physically linked to the translocation apparatus.     

Membrane biogenesis. In vitro cleavage, core glycosylation, and integration into microsomal membranes of sindbis virus glycoproteins

Sindbis virus 26S RNA has been translated in a cell-free protein-synthesizing system from rabbit reticulocytes. When the system was supplemented with EDTA-stripped dog pancreas microsomal membranes, the following results were obtained: (a) Complete translation of 26S RNA, resulting in the production, by endoproteolytic cleavage, of three polypeptides that are apparently identical to those forms of C, PE2, and E1 that are synthesized in vivo by infected host cells during a 3-min pulse with [35S]methionine. (b) Correct topological deposition of the three viral polypeptides--in vitro-synthesized PE2 and E1 forms are inserted into dog pancreas microsomal membranes in a orientation which, by the criterion of their limited (or total) inaccessibility to proteolytic probes, is indistinguishable from that of their counterparts in the rough endoplasmic recticulum of infected host cells; in vitro-synthesized C is not inserted into membranes and therefore is accessible to proteolytic enzymes, like its in vivo-synthesized counterpart. (c) Core glycosylation of in vitro-synthesized PE2 and E1 forms, as indicated by binding to concanavalin A Sepharose and subsequent elution by alpha-methylmannoside.     

Envelope proteins of Semliki Forest virus synthesized in Xenopus oocytes are transported to the cell surface.

The mRNA coding for the structural proteins of Semliki Forest virus, the 26S RNA, was injected into Xenopus oocytes. Synthesis of the capsid protein and the three envelope glycoproteins E1, E2 and E2 was observed. The proteins, which are normally incorporated into the plasma membrane of infected cells, are transported to the surface of the oocytes. The transport of the membrane proteins takes place in the presence of tunicamycin. The results show that the proteins foreign to the oocyte reach their destination in the plasma membrane. Consequently, the mRNA contains the information for the transport and proteolytic cleavage of the polypeptides.     

Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.     

Reinitiation of translocation in the Semliki Forest virus structural polyprotein: identification of the signal for the E1 glycoprotein.

The biosynthesis of the Semliki Forest virus (SFV) structural proteins provides an interesting model system to study the reinitiation of translocation of membrane proteins into the endoplasmic reticulum membrane. The two transmembrane spike proteins, p62 and E1, are derived from a single polypeptide precursor. Once the first protein, p62, has been anchored and its cytoplasmic tail has been synthesized, translocation must be reinitiated to account for the insertion of the E1 protein. We have used deletion mutagenesis of the SFV cDNA to investigate the requirements for this event and map in detail the location of the signal. We have shown by deleting the region encoding the p62 signal and expressing the modified cDNA in COS cells that the p62 protein is not involved in the translocation of the E1 protein. The E1 signal was precisely mapped by progressive truncations of the 6 K peptide (located between p62 and E1 in the SFV polyprotein) and subsequent analysis in cell-free systems. A segment within the last 26 residues of the 6 K peptide was shown to be essential for translocation. This segment was then fused to the N-terminus of the chimpanzee alpha-globin and was shown to be sufficient for translocation. The E1 signal was cleaved efficiently even when attached to the alpha-globin protein. The activity of the signal was found to be SRP dependent in a wheat-germ cell-free system. We conclude that prior attachment of the ribosome to the membrane via the p62 signal peptide is not necessary for E1 translocation and that the reinitiation of translocation is mediated by an independent internal signal likely to be SRP dependent.     

The amphiphilic membrane glycoproteins of Semliki forest virus are attached to the lipid bilayer by their COOH-terminal ends

The membrane location of the Semliki Forest virus glycoproteins E1, E2 and E3 was studied by protease treatment of (1) virus particles and (2) rough micro somes from cells infected with SF virus2. Protease treatment of virus particles removes all but the membrane-associated segments of the glycoproteins. Analyses of protease-treated SF virus membranes in 15% to 22.5% gradient acrylamide gels demonstrate the presence of three distinct peptide species with apparent molecular weights of 9000, 6000 and 5500. The 9000 and the 5500 molecular weight peptides have been aligned to the COOH-terminal end of E2 and the 6000 molecular weight peptide to the COOH-terminal end of El. The mapping of the peptides was done in a “Dintzis”-type of experiment (Dintzis, 1961) where we labelled the proteins of the virus with a gradient of [³⁵S]methionine increasing towards their COOH-terminal end.Protease treatment of microsomes from cells infected with SF virus removes only those parts of the viral glycoproteins that are transversing the lipid bilayer. Analyses of such treated membranes in sodium dodecyl sulphate-containing gels show that a 3000 molecular weight piece is digested from the COOH-terminal end of p62, the cellular precursor of E2 and E3. The COOH-terminus of p62 is shown to be equivalent to that of E2. These results thus demonstrate that the two amphiphilic membrane proteins of SF virus, E1 and E2 (p62) are attached to the lipid bilayer by their COOH-terminal ends. The COOH-terminal end of p62 (E2) spans the microsomal membrane. The third membrane protein, E3, probably does not interact with membrane lipids but is bound to the virus on E1 and (or) E2.     

Effects of Different RNAs and Components of the Cell-Free System on In Vitro Synthesis of Sindbis Viral Proteins

Cell-free extracts from Krebs ascites cells and rabbit reticulocytes synthesized a variety of viral-specific proteins when programmed with several different kinds of Sindbis viral RNAs. The RNAs included purified virion RNA (42S) and two species (26S and "33S") of purified intracellular viral messenger RNAs from viral-infected BHK cells. Proteins formed in vitro were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rate-zonal centrifugation in urea-sucrose gradients, two-dimensional tryptic peptide fingerprints, and immunoprecipitation with rabbit anti-Sindbis virus serum. The only major identifiable protein formed in vitro was viral capsid, but the relative amount of capsid produced was determined by the mRNA, the source of cell-free extract, and the components of the cell-free system. Virion RNA directed synthesis of larger-molecular-weight proteins than did intracellular viral RNAs, and some of this protein was distinct from that formed by the smaller viral RNAs. Indirect evidence is presented for in vitro synthesis of viral envelope proteins.     

Defective mutant of Sindbis virus with a smaller-molecular-weight form of the E1 glycoprotein.

We have isolated from a single plaque a mutant of Sindbis virus characterized by an E1 glycoprotein with higher electrophoretic mobility. This higher mobility is not attributable to a different extent of glycosylation of the protein nor to an altered proteolytic maturation pathway of the polypeptide precursor, but is the result of a deletion occurring during the replication of the viral RNA. The 26S RNA (the messenger for the Sindbis structural proteins) extracted from cells infected with the mutant is about 0.75 x 10(5) daltons smaller than the 26S RNA from the parental strain. As a consequence, in cells infected with the mutant, an E1 glycoprotein is synthesized with a polypeptide chain about 70 amino acids shorter. The biological relevance of this naturally occurring deletion of the viral genome is discussed.     

Coronavirus multiplication: locations of genes for virion proteins on the avian infectious bronchitis virus genome.

Six overlapping viral RNAs are synthesized in cells infected with the avian coronavirus infectious bronchitis virus (IBV). These RNAs contain a 3'-coterminal nested sequence set and were assumed to be viral mRNAs. The seven major IBV virion proteins are all produced by processing of three polypeptides of ca. 23, 51, and 115 kilodaltons. These are the core polypeptides of the small membrane proteins, the nucleocapsid protein, and the 155-kilodalton precursor to the large membrane proteins GP90 and GP84, respectively. To determine which mRNAs specify these polypeptides, we isolated RNA from infected cells and translated it in a messenger-dependent rabbit reticulocyte lysate. Proteins of 23, 51, and 110 kilodaltons were produced. Two-dimensional tryptic peptide mapping demonstrated that these proteins were closely related to the major virion proteins. Fractionation of the RNA before cell-free translation permitted the correlation of messenger activities for synthesis of the proteins with the presence of specific mRNAs. We found that the smallest RNA, RNA A, directs the synthesis of P51, the nucleocapsid protein. RNA C, which contains the sequences of RNA A, directs the synthesis of the small membrane protein P23. RNA E directs the synthesis of the large virion glycoproteins. These results supported a model in which only the unique 5'-terminal domain of each IBV mRNA is active in translation and enabled us to localize genes for virion proteins on the IBV genome.     

Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor.

The proteolytic processes involved in the cotranslational production of the Semliki Forest virus proteins p62, 6K, and E1 from a common precursor polypeptide were analyzed by an in vitro translation-translocation assay. By studying the behavior of wild-type and mutant variants of the polyprotein, we show that the signal sequences responsible for membrane translocation of the 6K and E1 proteins reside in the C-terminal regions of p62 and 6K, respectively. We present evidence suggesting that the polyprotein is processed on the luminal side by signal peptidase at consensus cleavage sites immediately following the signal sequences. Our results also lead us to conclude that the 6K protein is a transmembrane polypeptide with its N terminus on the luminal side of the membrane (type I). Thus, the production of all three membrane proteins is directed by alternating signal and stop-transfer (anchor) sequences that function in translocation and cleavage of the virus precursor polyprotein. This also shows conclusively that internally located signal sequences can be cleaved by signal peptidase.     

In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro- synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl- [35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine- labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.     

How a single sindbis virus mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins

Previous work has shown that the 26S RNA found in Sindbis-infected chicken embryo fibroblasts encodes the three viral structural proteins, one internal protein, core, and two membrane glycoproteins, E₁ and E₂. This mRNA has one initiation site; core, E₁, and E₂ are derived by proteolytic cleavage. Here we show that during infection, the 26S RNA is found mainly in membrane-bound polysomes which synthesize all three virion structural proteins. These polysomes are released from the membrane upon treatment with puromycin and high salt. Newly synthesized core protein is localized on the cytoplasmic side of endoplasmic reticulum membranes, while newly synthesized envelope proteins are sequestered by the lipid bilayer. These results suggest that the nascent glycoproteins, presumably their amino termini, are of major importance in directing the binding of polysomes containing 26S mRNA to endoplasmic reticulum membranes and the subsequent transfer of glycoproteins into the bilayer.     

Inhibition of the membrane fusion machinery prevents exit from the TGN and proteolytic processing by furin

The Semliki Forest virus (SFV) glycoprotein precursor p62 is processed to the E2 and E3 during the transport from the trans-Golgi network (TGN) to the cell surface. We have studied the regulation of the membrane fusion machinery (Rab/N-ethylmaleimide (NEM)-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)-SNAP receptor) in this processing. Activation of the disassembly of this complex with recombinant NSF stimulated the cleavage of p62 in permeabilized cells. Inactivation of NSF with a mutant alpha-SNAP(L294A) or NEM treatment inhibited processing of p62. Rab GDP dissociation inhibitor inhibited the cleavage. Inactivation of NSF blocks the transport of SFV glycoproteins and vesicular stomatitis virus G-glycoprotein from the TGN membranes to the cell surface. The results support the conclusion that inhibition of membrane fusion arrests p62 in the TGN and prevents its processing by furin.     

Structural characterization of the proteins encoded by adenovirus early region 2A

Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus oocytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides, 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.