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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.

Structural studies of tomato bushy stunt virus and Sindbis virus are discussed in terms of the information they provide about specificity and control in virus and membrane assembly.

The carbohydrate content of Sindbis virus was determined by gas chromatographic analysis. The two viral glycoproteins were found to be approximately 8% carbohydrate by weight. Mannose is the sugar present in the largest amount. Smaller amounts of glucosamine, galactose, sialic acid, and fucose were also detected. Each of the two viral glycoproteins appears to contain two structurally unrelated oligosaccharides. Two of the three Sindbis-specific glycoproteins found in infected chick cells were shown to contain short, unfinished oligosaccharides.

Neoplastic transformation of fibroblasts results in widely different cell morphologies. We have attempted to correlate cell morphology with cytoskeletal organization and fibronectin expression in murine and avian fibroblasts transformed by a diverse group of viral and chemical agents. The distribution of vinculin, alpha-actinin, actin, and surface fibronectin was studied, and, where appropriate, also the extent of phosphotyrosine modification of vinculin. Irrespective of the transforming agent we found that increased cell rounding was generally correlated with a reduction in vinculin-containing focal adhesions, a dissolution of microfilament bundles, and a reduction of extracellular fibronectin. In contrast, spindle-shaped fibroblasts expressed relatively high levels of surface fibronectin. Reorganization of vinculin, actin, and alpha-actinin into rosette-like structures was observed in polygonal or rounded cells transformed by viruses encoding tyrosine kinases, but was not seen in fibroblasts transformed by agents without associated tyrosine kinase activity or in spindle-shaped cells. No correlation was found between the extent of phosphotyrosine modification of vinculin and the extent of cell rounding. Irrespective of cell morphology, the extent of tyrosine phosphorylation of vinculin was high in all cells transformed by viruses carrying the src gene, but low in those transformed by viruses expressing the fps gene. Our results indicate that the morphology of a transformed cell is determined by a combination of several factors which are affected to different extents by different transforming agents.

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.

p56lck, a member of the src family of cytoplasmic tyrosine protein kinases, is expressed primarily in lymphoid cells. Previous RNase protection data demonstrated the existence of at least two lck mRNAs (type I and type II) with different 5' untranslated regions in most T cells. These have been found here to arise from two separate promoters. S1 nuclease analysis and primer extension were used to locate the site of initiation of type I lck mRNA. The nucleotide sequence of the region upstream of this start site contains no classical promoter motifs. A cDNA clone of type II lck mRNA was isolated. The promoter of this mRNA must be more than 10 kilobases upstream of the type I promoter region. In two murine thymoma cell lines, LSTRA and Thy19, lck is expressed at elevated levels as a result of Moloney murine leukemia virus retrovirus promoter insertion. p56lck is encoded in these cells by a hybrid virus-lck mRNA containing the 5' untranslated region of Moloney virus mRNA. The structures and the sites of integration of the proviruses upstream of lck in these cells were examined by molecular cloning and Southern analysis. A truncated and rearranged provirus, flanked by 554 nucleotides (nt) of duplicated cellular sequences, was found 962 nt upstream of the start site for type I lck mRNA in LSTRA cells. What appears to be a Moloney mink cytopathic focus-forming provirus was found between 584 to 794 nt upstream of the start site for type I lck mRNA in Thy19 cells. Thus in both tumor cell lines, viral DNA is present between the promoters for type I and type II lck mRNAs. Comparison of the sequences of the 5' ends of the lck and c-src genes suggests that divergence of these two genes involved exon shuffling and that a homolog of the neuronal c-src(+) exon is not present in lck.

p60src, the transforming protein kinase of Rous sarcoma virus, contains the 14-carbon saturated fatty acid, myristic acid, linked through an amide bond to the alpha-amino group of its NH2-terminal glycine residue. Myristic acid is known to be attached to four other eukaryotic proteins. In each case the fatty acid is also linked through an amide bond to an NH2-terminal glycine. We have used oligonucleotide-directed mutagenesis to examine the amino acid specificity of the enzyme that myristoylates the NH2 terminus of these proteins. Replacement of the NH2-terminal glycine in p60src with either alanine or glutamic acid prevented myristoylation completely. This indicates that the myristoylating enzyme may have an absolute specificity for glycine. Strikingly, neither nonmyristoylated mutant src protein induced morphological transformation of infected cells, even though wild-type levels of phosphorylation of cellular proteins on tyrosine were observed in these cells. Since conversion of the NH2-terminal residue from glycine to alanine should have little effect on the conformation of p60src, the inability of this mutant p60src protein to induce morphological transformation suggests that the myristoyl moiety is essential for the transforming activity of the protein.

Myristic acid, a minor component of cellular fatty acids, has been shown previously to be covalently bound to most molecules of p60src, the transforming protein of Rous sarcoma virus. We have now determined at what time during the life cycle of p60src, and where within the cell, this lipid becomes attached to the protein. p60src was found to acquire myristic acid at only one time, during or immediately after its synthesis. p60src is known to be synthesized on free polysomes and appears at the cytoplasmic face of the plasma membrane after a lag of 10 min. The addition of myristic acid to p60src therefore precedes the binding of the protein to the plasma membrane. The lipid attached to p60src is a permanent, metabolically stable part of the protein; we found no evidence for turnover of the myristyl moiety. However, we did find myristate attached to various soluble forms of p60src and to a large number of cytosolic cellular proteins as well. This demonstrates that the attachment of myristic acid to a protein is not in itself sufficient to convert a soluble protein into a membrane-bound protein.

p60src, the transforming protein of Rous sarcoma virus, was found to contain 0.5 to 0.9 mol of total phosphate per mol of polypeptide. The protein is known to be phosphorylated at two sites, a serine in the amino-terminal domain and a tyrosine in the carboxy-terminal domain. Because our indirect analysis suggests that the serine is phosphorylated to approximately twice the extent of the tyrosine, we estimate that p60src contains approximately 0.3 to 0.6 mol of phosphoserine and 0.2 to 0.3 mol of phosphotyrosine per mol of polypeptide. p60src was found to represent approximately 0.02% of the total incorporated radioactivity in Rous sarcoma virus-transformed chick cells labeled with [35S]methionine for 48 h. This corresponds to approximately 500,000 molecules of p60src per cell. Pulse-chase experiments revealed that the half-life of p60src ranged from 2 to 7 h, depending on the strain of virus examined. The P60src of the Schmidt-Ruppin strain was significantly more stable than that of the Prague strain.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.

Fibroblasts transformed by Abelson murine leukemia virus differ from normal fibroblasts in that they contain several cellular proteins, including one of 29 and one of 36 kilodaltons, which are phosphorylated at tyrosine residues. Since it has been shown before that these proteins also become phosphorylated at tyrosine after transformation of fibroblasts by a number of other retroviruses, their phosphorylation may play an important role in the transformation of these cells. In contrast, the 36-kilodalton phosphoprotein was not detectable in three of the four lines of Abelson virus-transformed B lymphoma cell lines studied here. These three cell lines, RAW307.1.1, 18-48, and 18-81, and a B lymphoma induced by mineral oil, WEHI 279, were all found to lack both the phosphorylated and unphosphorylated forms of the 36-kilodalton protein. It thus appears that expression of this major cell protein is not essential for the survival of B lymphoma cells in culture and that the phosphorylation of the 36-kilodalton protein at tyrosine is not essential for transformation of pre-B lymphocytes by Abelson virus.

We have found that the transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus all contain tightly bound lipid. This modification could play a role in the binding of these proteins to cellular membranes. The lipid associated with p60src, the transforming protein of Rous sarcoma virus, is located in the NH2-terminal domain of the polypeptide. This is the region of the protein that has been shown previously to participate in binding the protein to membranes. Two mature forms of p21, the transforming protein of Harvey sarcoma virus, contain lipid. Lipid is not, however, associated with newly synthesized p21. While mature p60src and p21 are bound to cellular membranes, the newly synthesized forms of these proteins are not. The posttranslational addition of lipid may therefore be the means by which these proteins acquire an affinity for membranes.

We provide direct evidence that serine 17 is the major site of serine phosphorylation in p60v-src, the transforming protein of Rous sarcoma virus, and in its cellular homolog, p60c-src. The amino acid composition of the tryptic peptide containing the major site of serine phosphorylation in p60v-src was deduced by peptide map analysis of the protein labeled biosynthetically with a variety of radioactive amino acids. Manual Edman degradation revealed that the phosphorylated serine in this peptide was the amino terminal residue. These data are consistent only with the phosphorylation of serine 17. The major site of serine phosphorylation in chicken p60c-src, the cellular homolog of p60v-src, is contained in a tryptic peptide identical to that containing serine 17 in p60v-src of Schmidt Ruppin Rous sarcoma virus of subgroup A. Serine 17 is therefore also phosphorylated in p60c-src. The p60v-src protein encoded by Prague Rous sarcoma virus was found to contain two sites of tyrosine phosphorylation. The previously unrecognized site of tyrosine phosphorylation may be tyrosine 205 or possibly tyrosine 208. Treatment of Prague Rous sarcoma virus-infected cells with vanadyl ions stimulated the protein kinase activity of p60v-src and increased the phosphorylation of tyrosine 416 but not the phosphorylation of the additional site of tyrosine phosphorylation.

Immobilon, a membrane of polyvinylidene difluoride to which gel-fractionated proteins can be transferred electrophoretically, was found to be an excellent matrix for the analysis of the phosphoamino acid content of phosphoproteins. Hydrolysis of 32P-labeled proteins bound to Immobilon with 5.7 N HCl resulted in the release of 90% of the 32P in the form of Pi, phosphoamino acids, and phosphopeptides. Two-dimensional electrophoretic analysis of the released phosphoamino acids yielded undistorted patterns. Because direct hydrolysis of proteins transferred to Immobilon eliminated the need for both preparative extraction of proteins from a gel and recovery by precipitation, analysis was rapid and yields of phosphoamino acids were extremely consistent. The yield of phosphoamino acids from proteins bound to Immobilon, unlike that from proteins eluted from gels, was independent of the size of the protein. The detection of 32P-labeled, phosphotyrosine-containing proteins in sodium dodecyl sulfate-polyacrylamide gels has been shown to be substantially improved by incubation of the gel in 1.0 N KOH for 2 h at 55 degrees C. Base hydrolysis of proteins bound to Immobilon proved to be faster and more sensitive than hydrolysis of proteins in gels. Less than 10% of bound protein was lost from Immobilon during the 2-h incubation at 55 degrees C in 1.0 N KOH. The autoradiographic image after alkaline hydrolysis of proteins on Immobilon was sharper than that obtained after hydrolysis of proteins in the gel. In addition, unlike base-treated gels, the dimensions of the Immobilon filter were unaffected by treatment with base.(ABSTRACT TRUNCATED AT 250 WORDS)

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.

p60src, the transforming protein of Rous sarcoma virus1 (RSV), is a protein kinase2 that has a strict specificity for tyrosine3. The phosphorylation of cellular proteins by p60src (ref. 4) results in transformation. Recently, Barker and Dayhoff5 discovered that residues 259–485 of p60src have 22% sequence identity with residues 33–258 of the catalytic subunit of cyclic AMP-dependent protein kinase, an enzyme that has a specificity for serine. Because it was necessary to introduce eight gaps to align the two proteins, the question remained as to whether this apparent homology reflected a common evolutionary origin. We demonstrate here that the ATP analogue p-fluorosulphonylbenzoyl 5′-adenosine (FSBA) inactivates the tyrosine protein kinase activity of p60src by reacting with lysine 295. When aligned for maximum sequence identity, lysine 295 of p60src and the lysine in the catalytic subunit which also reacts specifically with FSBA are superimposed precisely. This functional homology is strong evidence that the protein kinases, irrespective of amino acid substrate specificity, comprise a single divergent gene family.

All vertebrate cells have been shown to contain a gene, sarc , that has some homology with the transforming gene of Rous sarcoma virus, src . We have compared the polypeptide products of the sarc gene, p60 sarc , of human, mouse, and chicken cells with the polymorphic polypeptide product of the src gene, p60 src , of several strains of Rous sarcoma virus by two-dimensional peptide mapping. p60 sarc from chicken cells was clearly related to every viral p60 src . Eleven of its 13 methionine-containing tryptic peptides were present in some viral p60 src . Conversely, the other two peptides were not present in any p60 src we have examined so far. The 11 peptides from p60 sarc of chickens that were shared with viral p60 src , however, were not all present in any single viral p60 src . These 11 peptides most closely resemble those in the p60 src s of B77 virus and the Prague strain of Rous sarcoma virus. These data are consistent with the hypothesis that cellular sarc is the progenitor of viral src . The p60 sarc s of human, mouse, and chicken cells were so similar in tryptic peptide composition that they were more closely related to each other than were some viral p60 src s. The two mammalian p60 sarc s differed from avian p60 sarc most notably in that they lacked a peptide that chicken p60 sarc shares with all the viral p60 src s. The similarity of these maps suggests that the sequence of the p60 sarc polypeptide has diverged very little during evolution. This may imply that p60 sarc is an essential cellular component.

The mechanisms by which the membrane proteins of Sindbis virus are initially glycosylated during growth of the virus in chick cells was studied. The experiments suggest strongly that the two viral glycoproteins are glycosylated before release from the polysome, and that this glycosylation involves transfer of a large 1800 dalton oligosaccharide to the polypeptide chains. The donor of the oligosaccharide is most probably a lipid.

The rate of glucose transport in sparse, rapidly growing chick-embryo fibroblasts is much greater than that in density-inhibited cells. The addition of fresh chicken serum or trypsin to the medium of density-inhibited cells causes a large increase in the rate of glucose transport that is detectable 15 min after addition. The increase in glucose transport precedes the increase in DNA synthesis by 5-6 hr. Only small changes in rates of transport are seen with nucleosides or a nonmetabolizable amino acid. The increase in glucose transport requires protein synthesis but not RNA or DNA synthesis.

Sindbis virus was iodinated by using the enzyme lactoperoxidase, an iodination technique which labels only surface proteins. By this technique, the two viral glycoproteins are labeled, and the internal viral protein is not. The two glycoproteins are iodinated to strikingly different extents. This difference in susceptibility to iodination apparently is due to the position or conformation of the glycoproteins in the envelope spikes of the virion and not to differing contents of tyrosine, the amino acid substrate of lactoperoxidase. Both viral glycoproteins are iodinated by lactoperoxidase on the surface of Sindbis-infected chicken cells. Here, as in the virion, the glycoproteins are iodinated unequally, with the smaller glycoprotein again being preferentially iodinated. Another virus-specific protein found in large amounts in infected cells, and from which the preferentially iodinated virion glycoprotein is produced by a proteolytic cleavage, is not iodinated by lactoperoxidase. Thus it appears that the viral glycoproteins are present on the cell surface and that the precursor protein is not.

At least half of the more than 20 known proto-oncogenes encode proteins which phosphorylate polypeptide substrates. This suggests that protein phosphorylation plays a central role inthe regulation of cellular multiplication, glucose metabolism and cell shape and that a surprisingly large number of protein kinases exert control over these processes in normal cells and can perturb them in malignant cells.

An adolescent girl, devoid of detectable humoral immunity to measles virus despite two inoculations of live, attenuated vaccine, developed an exaggerated atypical rubeola illness following exposure to wild virus. This observation suggests that attenuated virus may be capable of immunologically sensitizing some individuals as has been described with killed virus vaccine.

p56lck, a cellular tyrosine protein kinase (EC 2.7.1.112) of the src family, is expressed in essentially all T cells and in some B cells. Expression in nonlymphoid cells is observed only rarely. We have found that mutation of a carboxyl-terminal phosphorylation site, tyrosine-505, reveals an oncogenic activity of this protein. Infection of fibroblasts with a retrovirus encoding wild-type p56lck is without consequence. In contrast, infection with a virus encoding the mutant protein leads to greatly increased phosphorylation of cellular proteins on tyrosine, morphological transformation, and anchorage-independent growth. This suggests that the tyrosine protein kinase activity and the oncogenic potential of p56lck are normally suppressed in vivo by phosphorylation of tyrosine-505. Since similar results were obtained previously with an analogous mutant of c-src, our results suggest that the protein kinase activity of all members of the src family of cytoplasmic tyrosine protein kinases will prove to be regulated by tyrosine phosphorylation at a conserved residue near the carboxyl terminus. Because p56lck is normally expressed only in lymphoid cells, it was possible that p56lck would be without effect in other tissues. The transformation of fibroblasts by mutant p56lck shows that this lymphoid protein can interact productively with nonlymphoid polypeptide substrates.

Incubation of quiescent chicken embryo cells with platelet-derived growth factor, epidermal growth factor, or serum was found to stimulate phosphorylation of two proteins of ca. 42,000 daltons on tyrosine. These proteins are structurally related to each other and to two proteins phosphorylated on tyrosine under similar conditions in mitogen-treated mouse fibroblasts. Three other very different mitogenic agents, the protease trypsin and the chemically unrelated tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin, stimulated phosphorylation of the same proteins. In all cases, phosphotyrosine was detected in these phosphoproteins. Although additional changes in protein phosphorylation were evident, no other proteins were observed by two-dimensional gel electrophoresis which contained increased amounts of phosphotyrosine in mitogen-treated chicken embryo cells. One of these 42,000-dalton proteins was shown previously to be phosphorylated on tyrosine in chicken embryo cells transformed with various retroviruses whose transforming proteins possess tyrosine protein kinase activity. Phosphorylation of the 42,000-dalton proteins could be important in the regulation of cell division.

All cells transformed by Rous sarcoma virus contain levels of phosphotyrosine in protein which are 6–10 fold greater than the very low levels present in uninfected cells. The increase is due largely to modification of cellular polypeptides. The abundance of phosphorylated tyrosines in protein in cells infected with tsLA29, a mutant of Rous sarcoma virus which is temperature-sensitive for cellular transformation, increases to 60% of maximum within 60 min of a shift to the permissive temperature and drops to a level close to that in uninfected cells within 60 min of a shift to the restrictive temperature. In light of the fact that pp60 src phosphorylates tyrosine in vitro, these results suggest strongly that the modification of one or more cellular polypeptides by way of pp60 src is critical for cellular transformation by Rous sarcoma virus. There is, however, no increase in the abundance of phosphotyrosine in protein in mouse cells transformed by Kirsten sarcoma virus, Moloney sarcoma virus, or SV40 virus, in chick embryo cells infected with avian myelocytomatosis virus MC29, and in rat and hamster cells transformed by polyoma virus. Thus increased phosphorylation of tyrosine is neither a universal mechanism of transformation nor an inevitable secondary cellular response to transformation.

The oligosaccharides of the membrane glycoproteins of Sindbis virus, vesicular stomatitis virus, and Rous sarcoma virus were compared on the basis of apparent size and sugar composition. It appears that each virus acquires a different set of oligosaccharides during growth in a single type of cell.

The exchange of the cholesterol in the membranes of two enveloped viruses, Sindbis virus and vesicular stomatitis virus, with cholesterol present in lipid vesicles and in serum was measured. Biosynthetically labeled viral cholesterol underwent spontaneous and complete transfer to both lipid vesicles and to serum. The rate with which and the extent to which this process occurred were very similar for these two viruses. During incubation with lipid vesicles in excess, half of the viral cholesterol underwent transfer in approximately 4 h and more than 90% underwent transfer in 24h at 37 degrees C. Similar rates and extents of movement of viral cholesterol were observed when incubations were carried out with vesicles which contained cholesterol and phospholipid in the same molar ratio as in the virus or with egg lecithin vesicles which contained no cholesterol. When labeled cholesterol was present initially in the lipid vesicles, movement of cholesterol from the vesicles to the virus was observed. One implication of the fact that viral cholesterol undergoes extensive exchange with serum cholesterol is that cellular cholesterol is in equilibrium with that in the extracellular fluid.

We have investigated how the information contained within the large RNA genome of IBV is expressed. We began by examining the structure of IBV-specified RNAs. Infected chicken embryo kidney (CEK) cells contain at least 6 IBV RNA species. These consist of the viral genome, RNA F, with an estimated complexity of 23 kb1, and subgenomic RNAs A, B, C, D, and E, which range in size from 2.4 kb to 7.9 kb2. We have recently identified an additional IBV-specified RNA species, RNA M, by Northern blot analysis using a hybridization probe containing cloned IBV cDNA sequences (see below). RNA M is intermediate in size between RNAs B and C (Table 1). Structural analysis of RNAs A, B, C, D, E, and F by ribonuclease T1 fingerprinting revealed that they comprise a 3’ coterminal nested set3. Synthesis of 3’ coterminal RNAs is now known to be a characteristic feature of Coronavirus multiplication4,5.

The sequence in which sugars are added to the Sindbis virus glycoproteins was studied. Infected cells contain three glycosylated virus-specific proteins: the two virion glycoproteins and the immediate precursor to the smaller virion glycoprotein. Larger Sindbis-specific proteins are not glycosylated. The cell-associated forms of both of the virion glycoproteins contain glucosamine, mannose, galactose, and fucose. The glycosylated precursor contains only glucosamine, mannose, and some galactose. The conversion of precursor to virion protein involves both the addition of galactose and fucose and the loss of mannose. The apparent extent of glycosylation of each virus-specific protein is not influenced by the host cell.

The CD4 and CD8 T cell receptor accessory molecules can both be isolated from T lymphocytes in association with p56lck, a membrane-associated, cytoplasmic tyrosine protein kinase that is expressed exclusively in lymphoid cells. The enzymatic activity of p56lck may therefore be regulated by CD4 and CD8 and be important in antigen-induced T cell activation. Exposure of human T cells and some mouse T cells to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, caused the dissociation of p56lck and CD4. Activation of protein kinase C may therefore interrupt regulation of p56lck by CD4 and alter the ability of p56lck to interact with polypeptide substrates. In contrast, exposure of cells to TPA did not cause dissociation of p56lck and CD8. Regulation of p56lck by CD4 may therefore differ from regulation by CD8.