Characterization of a small, nonstructural viral polypeptide present late during infection of BHK cells by Semliki Forest virus.

BHK cells, late in infection with Semliki Forest virus, were found to contain a small virus-specific polypeptide not found in the mature virion. This polypeptide had an apparent molecular weight of 6,000 and is referred to here as the 6K protein. No [2-3H]mannose was incorporated into 6K, and hence it does not appear to be a glycoprotein. This protein appears to be a primary translation product of the subgenomic 26S mRNA, which encodes the viral structural proteins. The genes encoding the viral structural proteins are arranged on the message in the order of 5'-C-E3-E2-E1-3'. We have found that the gene coding for 6K is located to the 3' side of the gene encoding E2. Subcellular fractionation of pulse-labeled cells infected with Semliki Forest virus demonstrated that 6K, like the viral glycoproteins p62 and E1, was present predominantly in the rough microsomal membrane fraction. 6K appears to be analogous, therefore, to the nonstructural 4.2K protein present in cells infected with Sindbis virus.     

Replication of Sindbis Virus V. Polyribosomes and mRNA in Infected Cells

Cells infected with wild-type Sindbis virus contain at least two forms of mRNA, 26S and 49S RNA. Sindbis 26S RNA (molecular weight 1.6 x 10(6)) constitutes 90% by weight of the mRNA in infected cells, and is thought to specify the structural proteins of the virus. Sindbis 49S RNA, the viral genome (molecular weight 4.3 x 10(6)), constitutes approximately 10% of the mRNA in infected cells and is thought to supply the remaining viral functions. In cells infected with ts2, a temperature-sensitive mutant of Sindbis virus, the messenger forms also include a third species of RNA with a sedimentation coefficient of 33S and an apparent molecular weight of 2.3 x 10(6). Hybridization-competition experiments showed that 90% of the base sequences in 33S RNA from these cells are also present in 26S RNA. Sindbis 33S RNA was also isolated from cells infected with wild-type virus. After reaction with formaldehyde, this species of 33S RNA appeared to be completely converted to 26S RNA. These results indicate that 33S RNA isolated from cells infected with either wild-type Sindbis or ts2 is not a unique and separate form of Sindbis RNA.     

Membrane integration and intracellular transport of the coronavirus glycoprotein E1, a class III membrane glycoprotein

The E1-glycoprotein (Mr = 26,014; 228 amino acids) of mouse hepatitis virus A59 is a class III membrane glycoprotein which has been used in this study as a model system in the study of membrane integration and protein transport. The protein lacks an NH2-terminal cleavable signal sequence and spans the viral membrane three times. Hydrophobic domains I and III could serve as signal sequences for cotranslational membrane integration. Domain I alone was sufficient to translocate the hydrophilic NH2 terminus of E1 across the membranes as evidenced by glycosylation of a newly introduced N-glycosylation site. The COOH-terminal part of E1 involving amino acids Leu124 to Thr228 was found to associate tightly with membranes at the post-translational level, although this part of the molecule lacks pronounced hydrophobic sequences. Membrane protection assays with proteinase K showed that a 2-kDa hydrophilic fragment was removed from the COOH terminus of E1 indicating that the protein is largely embedded into the membrane. Microinjection of in vitro transcribed capped and polyadenylated mRNA into CV-1 cells or into secretory AtT20 pituitary tumor cells showed that the E1-protein accumulated in the Golgi but was not detectable at the plasma membrane or in secretory granules. The 28 NH2-terminal hydrophilic amino acid residues play no role in membrane assembly or in intracellular targeting. Various NH2-terminal portions of E1 were fused to Ile145 of the cytoplasmic N-protein of mouse hepatitis virus. The resulting hybrid proteins were shown to assemble into membranes in vitro and were detected either in the rough endoplasmic reticulum or transient vesicles of microinjected cells.     

Basis for Variable Response of Arboviruses to Guanidine Treatment

The effect of guanidine on the replication of the group A arboviruses, Sindbis virus, and Semliki Forest virus (SFV) was studied. Guanidine rapidly, but reversibly, inhibited SFV ribonucleic acid (RNA) synthesis. The synthesis of all species of viral RNA was inhibited, but that of ribonuclease-resistant forms was least affected. This inhibition occurred when the drug was added at any point during the log phase of virus growth. The growth of SFV was also markedly inhibited, but Sindbis virus growth was unimpaired. Infection of guanidine-treated cells with the viruses together resulted in a significant inhibition of the yields of both. It appears that, in the case of Sindbis virus, viral RNA is ordinarily produced in such excess that inhibition of its synthesis does not reduce virus yields. In the case of SFV, guanidine also markedly distorts the pattern of RNA synthesis by greatly decreasing the production of the 26S interjacent RNA form. This may account for the observed inhibition of SFV growth in the presence of guanidine.     

Assembly of the semliki forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro

A cell-free system has been constructed to study the mechanism by which a single messenger RNA directs the synthesis of proteins destined for two different cellular locations. The Semliki Forest virus (SFV) 26 S mRNA codes for the viral capsid protein (C protein) and the membrane proteins p62 and E1. The three virus proteins are read in this order from the messenger RNA using one initiation site. The C protein is left on the cytoplasmic side and the p62 and the El proteins are inserted into the endoplasmic reticulum membrane. Translation of 26 S mRNA in a HeLa cell-free system in the presence of microsomes from dog pancreas reproduced the segregation, and proteolytic processing and glycosylation observed in infected cells. The signal for membrane binding was in the amino-terminal end of p62. The results indicate that the membrane proteins become inserted in the nascent state. The cleavage between p62 and El was coupled to membrane insertion. If the membranes were added after a period corresponding to the synthesis of about 100 amino acids of the p62 protein, segregation, glycosylation and cleavage between p62 and E1 failed to occur.     

Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat.     

The primary structure of rat ribosomal proteins: the amino acid sequences of L27a and L28 and corrections in the sequences of S4 and S12

The amino acid sequences of rat ribosomal proteins L27a and L28 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed from the NH2-terminal amino acid sequences of the proteins. L27a contains 147 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 16 476. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 18-22 copies of the L27a gene. The mRNA for the protein is about 600 nucleotides in length. L27a is homologous to mouse L27a (there are 3 amino acid changes) and to yeast L29. Rat ribosomal protein L28 has 136 amino acids (its NH2-terminal methionine is also processed after translation) and has a molecular weight of 15 707. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 9 or 10 copies of the L28 gene. The mRNA for the protein is about 640 nucleotides in length. L28 contains a possible internal duplication of 9 residues. Corrections are recorded in the sequences reported before for rat ribosomal proteins S4 and S12.     

Absence of a cleavable signal sequence in Sindbis virus glycoprotein PE2.

Partial NH2-terminal sequence analysis has been performed on some products that result from the translation of 26 S mRNA of Sindbis virus either in vivo or in vitro. In vivo products were obtained after pulse-labeling of virus-infected cells. In vitro products were obtained after cell-free translation either in the absence or presence of microsomal membrane vesicles from dog pancreas. The sequence data indicate that the selective translocation across the microsomal membrane required for a distinct portion of one of the integral viral envelope proteins (PE2) is not accompanied by cleavage of its putative signal sequence. Furthermore, the NH2-terminal sequence of a proteolytic derivative (PE'2) that contains the bulk of PE2 and that is generated after exposure of the microsomal vesicles to proteolytic enzymes is identical to that of intact PE2, strongly suggesting that only a COOH-terminal portion of PE2 is excluded from translocation across the microsomal membrane.     

The Transmembrane Domains of Sindbis Virus Envelope Glycoproteins Induce Cell Death

Sindbis virus, the prototype alphavirus, kills cells by inducing apoptosis. To investigate potential mechanisms by which Sindbis virus induces apoptosis, we examined whether specific viral gene products were able to induce cell death. Genes encoding the three structural proteins—capsid, the precursor E1 (6K plus E1), and the precursor E2 (P62 or E3 plus E2)—were cotransfected with a β-galactosidase reporter plasmid in transient-transfection assays in rat prostate adenocarcinoma AT3 cells. Cell death, as determined by measuring the loss of blue cells, was observed in AT3 cells transfected with 6K plus E1 and with P62 but not in cells transfected with capsid. Deletion mutagenesis of P62 indicated that large regions of the cytoplasmic domain and extracellular domain were not essential for the induction of cell death. However, constructs containing the minimal E3 signal sequence fused to the E2 transmembrane domain and the minimal E3 signal sequence fused to the E1 transmembrane domain induced death as efficiently as full-length P62 and 6K plus E1, whereas no cell death was observed after transfection with a control construct containing the E3 signal sequence linked to the transmembrane domain of murine CD4. These data demonstrate that intracellular expression of the transmembrane domains of the Sindbis virus envelope glycoproteins can kill AT3 cells.     

Formation of a Sindbis virus nonstructural protein and its relation of 42S mRNA function.

Chicken embryo fibroblasts infected with an RNA- temperature-sensitive mutant (ts24) of Sindbis virus accumulated a large-molecular-weight protein (p200) when cells were shifted from the permissive to nonpermissive temperature. Appearance of p200 was accompanied by a decrease in the synthesis of viral structural proteins, but [35S]methionine tryptic peptides from p200 were different from those derived from a 140,000-molecular-weight polypeptide that contains the amino acid sequences of viral structural proteins. Among three other RNA- ts mutants that were tested for p200 formation, only one (ts21) produced this protein. The accumulation of p200 in ts24- and ts21-infected cells could be correlated with a shift in the formation of 42S and 26S viral RNA that led to an increase in the relative amounts of 42S RNA. These data indicate that p200 is translated from the nonstructural genes of the virion 42S RNA and further suggest that this RNA does not function effectively in vivo as an mRNA for the Sindbis virus structural proteins.     

一株辛德毕斯病毒的基因组序列测定及分析

目的：对引进的一株辛德毕斯病毒的基因组序列进行测定,阐明其与已报道毒株序列的关系。方法：对辛德毕斯病毒基因组编码区进行分段RT-PCR扩增,对非编码区采用RACE法进行扩增,将扩增产物直接进行测序,应用DNAStar软件将测序结果拼接得到基因组序列,采用MEGA3.1软件对9株辛德毕斯病毒基因组序列进行系统进化发生树的构建。结果与结论：此株辛德毕斯病毒基因组共11663nt,编码3745个氨基酸残基,其中5＇端的2/3基因组编码4种非结构蛋白NSp1、NSp2、NSp3和NSp4,3＇端的1/3基因组编码5种结构蛋白E1、E2、E3、6K和C;结构基因和非结构基因之间有48nt的连接区为非翻译区;病毒基因组5＇末端和3＇末端分别有59、318nt的非编码区;序列同源性分析结果表明,此株病毒与S.A.AR86株的同源性最高,两者核苷酸序列的同源性为99.7%,氨基酸序列的同源性为99.6%,而与本室保存的另一辛德毕斯病毒MEI株的遗传进化关系稍远,系统进化发生树处于不同分支上。     

Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types.     

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.     

Subgenomic mRNA of Aura alphavirus is packaged into virions.

Purified virions of Aura virus, a South American alphavirus related to Sindbis virus, were found to contain two RNA species, one of 12 kb and the other of 4.2 kb. Northern (RNA) blot analysis, primer extension analysis, and limited sequencing showed that the 12-kb RNA was the viral genomic RNA, whereas the 4.2-kb RNA present in virus preparations was identical to the 26S subgenomic RNA present in infected cells. The subgenomic RNA is the messenger for translation of the viral structural proteins, and its synthesis is absolutely required for replication of the virus. Although 26S RNA is present in the cytosol of all cells infected by alphaviruses, this is the first report of incorporation of the subgenomic RNA into alphavirus particles. Packaging of the Aura virus subgenomic mRNA occurred following infection of mosquito (Aedes albopictus C6/36), hamster (BHK-21), or monkey (Vero) cells. Quantitation of the amounts of genomic and subgenomic RNA both in virions and in infected cells showed that the ratio of genomic to subgenomic RNA was 3- to 10-fold higher in Aura virions than in infected cells. Thus, although the subgenomic RNA is packaged efficiently, the genomic RNA has a selective advantage during packaging. In contrast, in parallel experiments with Sindbis virus, packaging of subgenomic RNA was not detectable. We also found that subgenomic RNA was present in about threefold-greater amounts relative to genomic RNA in cells infected by Aura virus than in cells infected by Sindbis virus. Packaging of the Aura virus subgenomic RNA, but not those of other alphaviruses, suggests that Aura virus 26S RNA contains a packaging signal for incorporation into virions. The importance of the packaging of this RNA into virions in the natural history of the virus remains to be determined.     

Analysis of alphavirus polypeptides by two dimensional polyacrylamide gel electrophoresis

Semliki Forest, Sindbis and Chikungunya viruses were grown and radio-labeled with [³H]-amino acids in Vero cells. Analysis of virus infected cell lysates by two dimensional polyacrylamide gel electrophoresis resulted in detection of polypeptides of molecular, weights corresponding to those of E1, P62, ns60, ns70/72 for Semliki Forest virus, the C, E1, 6K, 14K, PE2, P97, ns60, ns82 for Sindbis virus and E1. P62, P97, ns70/72 for Chikungunya virus. Charge and molecular weight heterogeneity in the precursor polypeptide P62 of Semliki Forest virus was detected. Structural poly-peptides e.g. E1 and E2 of Semliki Forest virus and C, E1, E2 of Sindbis virus and E1 of Chikungunya virus were detected when purified radiolabeled virus preparations were analyzed by two dimensional polyacrylamide gel-electrophoresis. Membrane glycoprotein E1 and E2 of Semliki Forest and E1 of Sindbis and Chikungunya viruses exhibited charge heterogeneity. In contrast to the marked difference in isoelectric points of E1 and E2 of Sindbis virus; E1 and E2 of Semliki Forest virus had almost identical isoelectric points.     

Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus

The modification of viral glycoproteins through the covalent attachment of fatty acids was studied in baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV). Comparative pulse-chase experiments with [3H]palmitic acid and [35S]methionine revealed that a precursor polypeptide, designated p62, of the structural SFV glycoprotein and E1 serve as the primary acceptors of acyl chains. Acylation of p62 occurs immediately prior to its proteolytical cleavage to E2 and E3 emphasizing the post-translational and specific nature of this hydrophobic modification. To trace the acyl donor(s) for protein acylation the covalent attachment of fatty acids to p62 was studied after extremely short labeling periods with [3H]palmitic acid and correlated to the metabolism of the exogenous tritiated fatty acid. The shortest possible labeling time, a 10 s pulse with [3H]palmitic acid, was sufficient to acylate SFV p62. Analysis of the labeled lipids extracted from the same cells revealed that palmitoyl-CoA and phosphatidic acid showed the highest specific radioactivity among the tritiated lipid species. Out of these lipid species palmitoyl-CoA was identified as the functional acyl donor lipid in a cell-free system for the acylation of polypeptides.     

Amino acid composition and NH2-terminal amino acid sequence of human phospholipase A2 purified from rheumatoid synovial fluid

The amino acid composition and partial NH2-terminal amino acid sequence of an extracellular phospholipase A2 in human rheumatoid synovial fluid were determined. The predominant amino acids in the phospholipase A2 were cysteine, glycine, arginine, and lysine, suggesting that it is a basic one. The NH2-terminal 34 amino acids were found to be as follows: Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-Gly-Lys-Glu-Ala-Ala-Leu- Ser-Tyr-Gly-Phe-Tyr-Gly-Cys-X-Cys-Gly-Val-Gly-Gly-Arg-Gly The enzyme contains Phe-5, Met-8, Ile-9, Tyr-24, Gly-25, Cys-26, Cys-28, Gly-29, Gly-31, Gly-32, and Gly-34 residues, all of which are conserved in most of the sequenced phospholipase A2. The remarkable feature of this enzyme was the absence of Cys-11, which is conserved in the "Group I" enzyme family. This is the first report concerning partial amino acid sequences of human non-pancreatic phospholipase A2.     

Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.