Structural Proteins of Rabies Virus

Purified rabies virions, unlabeled or labeled with radioactive amino acids or d-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electroiphoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecular weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein component (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons).     

Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

Iodination of reovirus, cytoplasmic polyhedrosis virus (CPV), and wound tumor virus (WTV), and their respective subviral forms, followed by analysis of the labeled polypeptides by using polyacrylamide gel electrophoresis, has been used to compare the protein contents of these three diplornaviruses. This approach, when combined with electron microscopy and buoyant density determinations, appears capable of localizing individual polypeptides in some of the viral and subviral forms. CPV (p = 1.435 g/cm(3)) seems to resemble reovirus cores (p = 1.440 g/cm(3)) in both ultrastructure and polypeptide composition. CPV is composed of five polypeptides with molecular weights of about 151,000, 142,000, 130,000, 67,000, and 33,000. The polyhedral matrix, which in nature encapsulates the virions, is, in turn, composed mainly of two polypeptide species with molecular weights of about 30,000 and 20,000, and several minor proteins. The proteins of WTV consist mainly of four species of polypeptide with molecular weights of about 156,000, 122,000, 63,000, and 44,000, and several minor components. These molecular weight determinations are consistent with the hypothesis that, as has been suggested for reovirus, the viral proteins of CPV and WTV seem to be coded for by monocistronic mes senger RNA molecules transcribed from distinct segments of the double-stranded RNA viral genomes.     

Different Polypeptides Are Rapidly Transported in Auditory and Optic Neurons

Abstract: Rapidly transported proteins and glycoproteins in the auditory and optic nerves of the guinea pig were analyzed by electrophoresis and two-dimensional electrofocusing/electrophoresis. Proteins transported in the auditory nerve were analyzed in the cochlear nucleus 3 h after cochlear injection of radioactive precursor, and proteins transported in the optic nerve were analyzed in the superior colliculus 6 h after intraocular injection of radioactive precursor. Two-dimensional analysis showed that several rapidly transported polypeptides were present in one system, but not in the other. By use of [³H]fucose as a precursor or by separating [³⁵S]methionine-labeled polypeptides on immobilized concanavalin A or wheat germ agglutinin, it was shown that most of the proteins transported in only one system are glycoproteins. As previously reported a polypeptide of molecular weight 140,000 was a major labeled species in the auditory nerve. This polypeptide was also found in the optic nerve, but only as a minor species. Two other polypeptides with molecular weights and isoelectric points similar to those of the 140,000 molecular weight polypeptide were present in both systems, but were much more abundant in the optic nerve. The major labeled polypeptide in both systems had a molecular weight of 25,000.     

Synthesis of Vaccinia Viral Proteins in Cytoplasmic Extracts: I. Incorporation of Radioactively Labeled Amino Acids into Polypeptides

Polypeptide synthesis has been studied in cell-free systems prepared from vaccinia virus-infected and uninfected HeLa cells. Cytoplasmic extracts containing endogenous messenger ribonucleic acid were used. Amino acid incorporation into hot trichloroacetic acid-precipitable material was linear for 15 to 20 min at 37 C. The initial rate of protein synthesis was approximately 15% of the rate in intact cells. Optimal conditions for polypeptide synthesis were similar in cell-free systems prepared from infected or uninfected cells. Requirements for an energy source and Mg(++) were demonstrated. The optimal Mg(++) concentration was 4 to 5 mm. Ribonuclease, puromycin, and cycloheximide were inhibitory. The molecular weights of the polypeptides labeled in the cell-free systems, as determined by gel filtration in 5 m guanidine hydrochloride, ranged from 16,000 to above 68,000. Polyacrylamide gel electrophoresis indicated that the polypeptides labeled in cell-free extracts of uninfected and infected cells were different. The latter closely corresponded in electrophoretic mobility with the viral polypeptides made in intact, infected cells.     

Proteins of hepatitis B surface antigen.

Purified 22-nm forms of hepatitis B surface antigen (Hbsag) representing the three major antigenic subtypes (adw, ayw, and adr) were analyzed for their constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No consistent difference in either the number or relative distributions of the polypeptides was observed for the various subtypes. Seven polypeptides were designated as P-1 through P-7 in order of their decreasing mobilities. By comparison with protein standards, their molecular weights were estimated as 23, 29.5, 36, 41.5, 53.5, 72, and 97 thousand. The P-1 and P-2 components represented the major polypeptides; P-2 and P-5 might by glycoproteins, based on their reaction with periodic acid-Shiff reagent. Each polypeptide contains cysteine residues. HBSAg was radiolabeled with 3H or 14C by reductive methylation or iodinated with 125I by the chloramine-T or lactoperoxidase procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled HBSAg yielded patterns identical to those obtained with protein stain. Comparison of HBSAg labeled by the chloramine-T and lactoperoxide procedures indicated that there was no distinction between internal or external components within the 22-nm structure.     

Monoclonal antibodies to two glycoproteins of herpes simplex virus type 2.

Monoclonal antibodies to herpes simplex virus type 2 were found to precipitate different numbers of radiolabeled polypeptides from lysates of virus-infected cells. Antibodies directed against two viral glycoproteins were characterized. Antibodies from hybridoma 17 alpha A2 precipitated a 60,000-molecular-weight polypeptide which chased into a 66,000- and 79,000-molecular-weight polypeptide. All three polypeptides labeled in the presence of [3H]glucosamine and had similar tryptic digest maps. The 60,000-molecular-weight polypeptide also chased into a 31,000-molecular-weight species which did not label with [3H]glucosamine. Antibodies from hybridoma 17 beta C2 precipitated a 50,000-molecular-weight polypeptide which chased into a 56,000- and 80,000-molecular weight polypeptide. These polypeptides also shared a similar tryptic digest map and labeled with [3H]glucosamine. Both monoclonal antibodies were herpes simplex virus type 2 specific. The viral proteins precipitated by 17 alpha A2 antibodies had characteristics similar to those reported for glycoprotein E, whereas the proteins precipitated by 17 beta C2 antibodies appeared to represent a glycoprotein not previously described. This glycoprotein should be tentatively designated glycoprotein F.     

Structural Polypeptides of the Granulosis Virus of Plodia interpunctella

Techniques were developed for the isolation and purification of three structural components of Plodia interpunctella granulosis virus: granulin, enveloped nucleocapsids, and nucleocapsids. The polypeptide composition and distribution of protein in each viral component were determined by sodium dodecyl sulfate discontinuous and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of 15 structural proteins ranging in molecular weight from 12,600 to 97,300. Five of these proteins, having approximate molecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surface radioiodination of the enveloped nucleocapsids. Present in purified nucleocapsids were eight polypeptides. The predominant proteins in this structural component had molecular weights of 12,500 and 31,000. Whereas no evidence of polypeptide glycosylation was obtained, six of the viral proteins were observed to be phosphorylated.     

Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins

Nuclear membrane fractions were prepared by two procedures from KB cells pulse labeled with [(3)H]thymidine for 5 min late after infection with adenovirus 2: (i) the M-band technique, which yields a sharp peak containing most of the newly synthesized viral DNA, and (ii) the discontinuous sucrose gradient method, which yields three membrane fractions, one which bands at the interface between sucrose layers at density 1.18 and 1.20 g/ml and contains most of the newly synthesized viral DNA. Studies using cycloheximide to inhibit protein synthesis showed that proteins whose synthesis begins early after infection and occurs in the absence of viral DNA replication are required for viral DNA synthesis late after infection. To study the nature of these proteins, nuclear membrane fractions were isolated from cells labeled with [(3)H]leucine from 6 to 24 h postinfection in the presence of arabinosyl cytosine to block viral DNA replication, and were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Two proteins of molecular weights 75,000 and 45,000 were the major labeled polypeptides in the nuclear membrane fractions prepared from infected cells both by the M-band and the discontinuous sucrose gradient methods. These two proteins were not found in nuclear membrane fractions from uninfected cells. It is suggested that the 75,000 and 45,000 proteins may be early viral gene products that may play a role in the viral DNA replication.     

Structural Proteins of Simian Virus 40: Phosphoproteins

All five structural polypeptides of infectious simian virus 40 grown in African green monkey kidney cells were found to be phosphorylated. The polypeptides with the largest and smallest molecular weights are phosphorylated to a somewhat lower extent than the other polypeptides. The protein moiety of "empty" virus, which is essentially devoid of deoxyribonucleic acid, exhibited a degree of phosphorylation similar to that of infectious virus. In the major polypeptide (molecular weight: 49,000), the phosphate appears to be bound to the seryl or threonyl residues, or both. The nature of the phosphate-polypeptide bond in the other viral polypeptides remains obscure.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes

Sindbis virus-specific polypeptides were synthesized in lysates of rabbit reticulocytes in response to added 26 S or 49 S RNA. Sindbis 26 S RNA was translated into as many as three polypeptides which co-migrate in acrylamide gels with proteins found in infected cells.Wild type 26 S RNA was translated primarily into two polypeptides, which appear to be the Sindbis nucleocapsid protein (mol. wt 30,000) and the precursor of the two glycoproteins of the virion (mol. wt 100,000). A larger polypeptide (mol. wt 130,000) was synthesized in response to ts2 26 S RNA, a species of RNA which was isolated from cells infected with the ts2 mutant of Sindbis virus. This large polypeptide is apparently the protein which accumulates in cells infected with the mutant virus and which is thought to be a precursor of all three viral structural proteins.These results support the hypothesis that 26 S RNA is the messenger for the three structural proteins of the virion and that the RNA codes for one large polypeptide precursor. The precursor may then be cleaved at a specific site to yield the nucleocapsid protein and a second polypeptide which, in infected cells, is cleaved in a series of steps to yield the two glycoproteins of the virion.Sindbis 49 S RNA was translated into eight or nine polypeptides ranging from 60,000 to 180,000 molecular weights. The viral structural proteins, as such, were not synthesized in response to the added 49 S RNA.     

Characterization of cricket paralysis virus-induced polypeptides in Drosophila cells

Cricket paralysis virus purified from Galleria mellonella larvae was shown to be similar to virus purified from Drosophila melanogaster cells. Cricket paralysis virus contained three major structural polypeptides of similar molecular weight (around 30,000), had a buoyant density of 1.344 g/ml, and had a capsid diameter of 27 nm. Twenty virus-induced polypeptides could be detected in CrPV-infected Drosophila cells. Two major polypeptides found in the infected cells corresponded to two structural viral polypeptides (VP1 and VP3), whereas the third major intracellular polypeptide was the apparent precursor of the third viral structural polypeptide (VP2). Three of the primary virus-induced polypeptides had molecular weights of 144,000, 124,000, and 115,000. These and other polypeptides were chased into lower-molecular-weight proteins when excess cold methionine was added after a short [³⁵S]methionine pulse. Although cricket paralysis virus has a number of characteristics in common with the mammalian enteroviruses, the extremely fast processing of high-molecular-weight polypeptides into viral proteins seems atypical. Also, no VP4 (8,000 to 10,000 molecular weight) has been found in the virus particles.     

Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus.

Purified virions or radiolabeled lysates of infected MA104 cells were used to characterize the structural and nonstructural polypeptides of a porcine group C rotavirus. At least six structural proteins were identified from purified group C rotavirus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these, two (37,000- and 33,000-molecular-weight polypeptides) were associated with the outer shell, as demonstrated by the ability of EDTA to remove them from the purified virion. The other four polypeptides (molecular weights, 125,000, 93,000, 74,000, and 41,000) were located in the inner shell. The structural or nonstructural nature of a 25,000-molecular-weight protein identified in our studies was unclear. Glycosylation inhibition studies with tunicamycin in infected cells demonstrated that the 37,000- and 25,000-molecular-weight proteins were glycosylated and contained mannose-rich oligosaccharides identified by radiolabeling of the infected cells with [3H]mannose. The 37,000-molecular-weight outer shell glycoprotein was shown by pulse-chase experiments to be posttranslationally processed. The kinetics of viral polypeptide synthesis in infected cells were also studied, and maximal synthesis occurred at 6 to 9 h postinfection. The 41,000-molecular-weight inner capsid polypeptide was the most abundant and was the subunit structure of a 165,000-molecular-weight protein aggregate. Two polypeptides (molecular weights, 39,000 and 35,000) appeared to be nonstructural, as determined by comparison of the protein pattern of radiolabeled infected cell lysates with that of purified virions. Radioimmunoprecipitation was used to examine the serologic cross-reactions between the viral polypeptides of a group C rotavirus with those of a group A rotavirus. No serologic cross-reactivities were detected. The polypeptides of group A and C rotaviruses are compared and discussed.     

Purification of measles virus and characterization of subviral components.

Purified measles virus was obtained from [35S]methionine-labeled cells infected at 33 degrees C and maintained in the absence of fetal calf serum. The pellet that was produced by a single high-speed ultracentrifuge spin of culture medium contained virus of purity sufficient for structural analysis. Purified virions contain seven polypeptides with estimated molecular weights of: L, 200,000; G, 80,000; P2, 70,000; NP, 60,000; A, 43,000; F1, 41,000; and M, 37,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Treatment of virions with 0.25% trypsin resulted in a less dense particle which lacked polypeptides G and F1. Solubilization of the viral membrane with the detergent Triton X-100 in low-salt buffer resulted in the loss of the G polypeptide, whereas in the presence of 1 M KCl, Triton X-100 also removed most of the M polypeptide. The nucleocapsids (p = 1.3) obtained from virions treated with Triton X-100 and 1 M KCl contained the L, P2, NP, and M polypeptides. Nucleocapsids isolated from the cytoplasm of infected cells were predominantly composed of the NP polypeptide with smaller amounts of either polypeptide P2 or novel polypeptides, related to NP, with estimated molecular weights of 56,000 to 58,000 and 45,000 to 46,000. A significant amount of polypeptide L was always found in association with nucleocapsids isolated either from virions or from the cytoplasm of infected cells. A membrane component containing the viral membrane polypeptides G, F1, and M was also isolated from infected cells. The data presented here thus suggest that L is an integral part of the nucleocapsid complex. In addition, 37,000-molecular-weight polypeptide (M) appears to have the function described for the matrix proteins of other paramyxoviruses.     

Identification of Virus-Induced Proteins in Cells Productively Infected with Simian Virus 40

The number and molecular weight of the structural polypeptides of highly purified simian virus 40 (SV40) were determined by polyacrylamide gel electrophoresis. Six different polypeptides were found, two of which (VP1 and VP2) comprise the bulk of the viral capsid proteins. The pattern of protein synthesis in productively infected CV-1 cells was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Identification of virus-induced proteins in the infected CV-1 cells was achieved in double-labeling experiments by electrophoresis with purified labeled SV40 capsid proteins. Four of these proteins (VP1 and VP4) could be classified as components of the virion because their synthesis occurred after the onset of viral deoxyribonucleic acid (DNA) replication and because they were inhibited by arabinofuranosylcytosine (ara-C). Appearance of two other virus-induced proteins was not prevented by ara-C; one of them did not comigrate in the electrophoresis with purified virion polypeptides, and both could be detected before the onset of viral DNA synthesis. These latter two proteins were classified on the basis of these criteria as nonvirion capsid proteins (NCVP1 and NCVP2).     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.     

Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses: I. Avian Leukemia-Sarcoma Viruses

Gel filtration of avian tumor virus proteins in 6 m guanidine hydrochloride clearly resolved seven major protein species. The antigenic activity of these proteins was recovered in good yield after removal of the denaturing solvent, permitting a correlation of specific polypeptides with the principal antigens of the virion. Two of the proteins, of molecular weights 70,000 and 32,000, contain carbohydrate and are situated on the viral membrane, as shown by their being accessible in the intact virus to specific antibodies. Four proteins, with molecular weights (in guanidine) of 27,000, 19,000, 15,000, and 12,000, have different group-specific (gs) antigens and are enclosed within the viral membrane. The smallest protein, with a molecular weight of 10,000, has not previously been described; it is not detectable with antisera and possesses a mobility identical to that of one gs protein when subjected to electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. Of the proteins lacking carbohydrate, three are present in the virion in a molecular ratio of 2:2:1, and the two others, present in almost equal amount, are rich in lysine and arginine.     

Immunological Studies on Viral Polypeptide Synthesis in Cells Replicating Murine Sarcoma-Leukemia Virus

Antibodies to disrupted murine sarcoma-leukemia virus (MSV[MLV]) were used to study the synthesis of viral polypeptides in the transformed, virus-producing rat cell line 78A1. When cultures were labeled for 10 min with radioactive amino acids, about 9% of the total labeled proteins were precipitated with antiserum against purified MSV(MLV), and 3 to 4% were precipitated with the same antiserum after it had been absorbed with an extract from uninfected rat cells. The difference is due to the presence in the unabsorbed antiserum of antibodies to cellular proteins that are present in purified virus preparations. Intracellular viral proteins labeled with radioactive amino acids were isolated by immunoprecipitation and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The mobilities of intracellular viral polypeptides were identical to those of the purified virion. However, labeled polypeptides having electrophoretic mobilities lower than that of the major virion polypeptide, the group-specific antigen of molecular weight 31,000, were present in higher proportion in the total cell extract and in the membrane fraction than in the virion. These polypeptides appear to be of cellular origin for they were present only in minute amounts in the immunoprecipitates obtained with the absorbed serum. After a 10-min labeling period, radioactive proteins were assembled into extracellular virions rapidly for the first 4 hr followed by a slower rate. More than 2% of the total proteins of the cell labeled in a 10-min pulse were assembled into virions at the completion of a 24-hr chase. The high-molecular-weight polypeptides with the same mobilities as those detected in the immunoprecipitate of intracellular proteins were found in virions released from cells after a 10-min pulse. A larger proportion of these high-molecular-weight proteins was detected in virions released after short chase periods (30-120 min) than after longer chase periods (6-24 hr). Two possible interpretations of these data are that the high-molecular-weight cell-derived polypeptides (i) have a turnover rate higher than that of the major virion polypeptides or (ii) are cleaved proteolytically from the virions during long incubation in the culture media.     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.