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1.
Preparations of La Crosse virus, a member of the California encephalitis group of bunyaviruses, were found to possess three major virion proteins. Two of the proteins were glycosylated (G1 and G2) and were located on the surface of the virus particles. These two glycoproteins were present in equimolar amounts and possessed apparent molecular weights of 120 X 10(3) and 34 X 10(3). Virion nucleocapsids, isolated by a nonionic detergent and salt treatment, contained another major protein, N (molecular weight = 23 X 10(3)). A large, but minor, protein species L (molecular weight = 180 X 10(3)) was also found in virus preparations. The approximate number of protein molecules per virion has been determined. Electron microscopy of purified La Crosse virus indicated that the virus particle (mean diameter, 91 nm) is enveloped and possesses irregular surface projections (length, 10 nm). 相似文献
2.
Further morphological characterization and structural proteins of infectious bursal disease virus.
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An outer layer surrounding the capsid of infectious bursal disease virus was evident from electron micrographs of intact virus particles having diameters of 62 to 63 nm. The capsid was found to be composed of large morphological units or capsomeres, measuring about 12 nm in diameter. The architecture of the capsid appears to be that of T = 3 symmetry, with a probable 32 morphological units by rotational enhancement of image detail. Structural proteins of infectious bursal disease virus consist of seven species, two major and five minor polypeptides. These are P1 to P7, with molecular weights of 133 x 10(3), 124 x 10(3), 98 x 10(3), 51 x 10(3), 33 x 10(3), 26 x 10(3), and 23 x 10(3), respectively. 相似文献
3.
In the present study we investigated the functional properties of the three recombinant hyaluronan synthases (HAS proteins) HAS1, HAS2, and HAS3. HAS3-transfected CHO clones exhibited the highest hyaluronan polymerization rate followed by HAS2 transfectants which were more catalytically active than HAS1 transfectants. In living cells all three HAS proteins synthesized hyaluronan chains of high molecular weight (larger than 3.9 x 10(6)). In vitro, the HAS2 isoform produced hyaluronan chains of a molecular weight larger than 3.9 x 10(6), whereas HAS3 produced polydisperse hyaluronan (molecular weight 0.12-1 x 10(6)), and HAS1 synthesized much shorter chains of an average molecular weight of 0.12 x 10(6). Thus, each HAS protein may interact with different cytoplasmic proteins which may influence their catalytic activity. CHO transfectants with the ability to synthesize about 1 microgram hyaluronan/1 x 10 (5) cells/24 h were surrounded by hyaluronan-containing coats, whereas transfectants generating about 4-fold lower amounts of hyaluronan formed coats only in the presence of chondroitin sulfate proteoglycan. An inverse correlation between hyaluronan production on the one hand and cell migration and cell surface CD44 expression on the other was found; a 4-fold lower migration and a 2-fold decrease of cell surface CD44 receptors was seen when hyaluronan production increased 1000-fold over the level in the untransfected cells. The inverse relationships between hyaluronan production and migration and CD44 expression of cells are of importance for the regulation of cell-extracellular matrix interactions. 相似文献
4.
Structural characterization of virion proteins and genomic RNA of human parainfluenza virus 3 总被引:14,自引:11,他引:3
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The virion proteins and genomic RNA of human parainfluenza virus 3 have been characterized. The virion contains seven major and two minor proteins. Three proteins of 195 X 10(3) molecular weight (195K), 87K, and 67K are associated with the nucleocapsid of the virion and have been designated L, P, and NP, respectively. Three proteins can be labeled with [14C]glucosamine and have molecular weights of 69K, 60K, and 46K. We have designated these proteins as HN, F0, and F1, respectively. HN protein has interchain disulfide bonds, but does not participate in disulfide bonding to form homomultimeric forms. F1 appears to be derived from a complex, F1,2, that has an electrophoretic mobility similar to that of F0 under nonreducing conditions. A protein of 35K is associated with the envelope components of the virion and aggregates under low-salt conditions; this protein has been designated M. The genome of human parainfluenza virus 3 is a linear RNA molecule with a molecular weight of approximately 4.6 X 10(6). 相似文献
5.
Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins 总被引:6,自引:0,他引:6
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We are studying the structural proteins and molecular interactions required for formation and release of influenza virus-like particles (VLPs) from the cell surface. To investigate these events, we generated a quadruple baculovirus recombinant that simultaneously expresses in Sf9 cells the hemagglutinin (HA), neuraminidase (NA), matrix (M1), and M2 proteins of influenza virus A/Udorn/72 (H3N2). Using this quadruple recombinant, we have been able to demonstrate by double-labeling immunofluorescence that matrix protein (M1) localizes in nuclei as well as at discrete areas of the plasma membrane where HA and NA colocalize at the cell surface. Western blot analysis of cell supernatant showed that M1, HA, and NA were secreted into the culture medium. Furthermore, these proteins comigrated in similar fractions when concentrated supernatant was subjected to differential centrifugation. Electron microscopic examination (EM) of these fractions revealed influenza VLPs bearing surface projections that closely resemble those of wild-type influenza virus. Immunogold labeling and EM demonstrated that the HA and NA were present on the surface of the VLPs. We further investigated the minimal number of structural proteins necessary for VLP assembly and release using single-gene baculovirus recombinants. Expression of M1 protein alone led to the release of vesicular particles, which in gradient centrifugation analysis migrated in a similar pattern to that of the VLPs. Immunoprecipitation of M1 protein from purified M1 vesicles, VLPs, or influenza virus showed that the relative amount of M1 protein associated with M1 vesicles or VLPs was higher than that associated with virions, suggesting that particle formation and budding is a very frequent event. Finally, the HA gene within the quadruple recombinant was replaced either by a gene encoding the G protein of vesicular stomatitis virus or by a hybrid gene containing the cytoplasmic tail and transmembrane domain of the HA and the ectodomain of the G protein. Each of these constructs was able to drive the assembly and release of VLPs, although enhanced recruitment of the G glycoprotein onto the surface of the particle was observed with the recombinant carrying a G/HA chimeric gene. The described approach to assembly of wild-type and chimeric influenza VLPs may provide a valuable tool for further investigation of viral morphogenesis and genome packaging as well as for the development of novel vaccines. 相似文献
6.
Analyses of the virion polypeptides and genomes of several Phlebotomus fever group viruses, Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses, have established that they are biochemically similar to the accepted members of the Bunyaviridae family. Like snowshoe hare virus (a member of the California serogroup of the Bunyavirus genus of the Bunyaviridae family), Karimabad, Punta Toro, Chagres, and the sandfly fever Sicilian serotype viruses all have three viral RNA species, designated large (L), medium (M), and small (S). Oligonucleotide fingerprint analyses of Karimabad and Punta Toro virus RNA species indicated that their L, M, and S RNA species are unique. By polyacrylamide gel electrophoresis it was determined for Karimabad virus that the apparent molecular weights of its L, M, and S RNA species are 2.6 X 10(6), 2.2 X 10(6), and 0.8 X 10(6), respectively. For Punta Toro virus, the apparent molecular weights of its L, M, and S RNA species are 2.8 X 10(6), 1.8 X 10(6), and 0.75 X 10(6), respectively. The major internal nucleocapsid (N) protein of Karimabad virus was found to have a molecular weight of 21 X 10(3). A similar polypeptide size class was identified in preparations of sandfly fever Sicilian serotype, Chagres, and Punta Toro viruses. The Karimabad virus glycoproteins formed the external surface projections on virus particles and could be removed from virus preparations by protease treatment. The glycoproteins in an unreduced sample could be resolved into two size classes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They had apparent molecular weights of 62 X 10(3) and 50 X 10(3) in continuous polyacrylamide gels. When Karimabad virus preparations were reduced with 1% beta-mercaptoethanol, prior to resolution by continuous polyacrylamide gel electrophoresis, all the viral glycoprotein was recovered in a single size class, having an apparent molecular weight of 62 X 10(3). Two or three major virion polypeptides have been identified in preparations of Punta Toro, Chagres, and sandfly fever Sicilian serotype viruses. 相似文献
7.
Characterization of bovine parainfluenza virus type 3. 总被引:2,自引:0,他引:2
Bovine parainfluenza virus type 3 (PIV-3) has a buoyant density of 1.197. The RNA of PIV-3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self-anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 X 10(6), being slightly smaller than Sendai virus RNA (47S, 5.3 X 10(6)). PIV-3 has 5 main structural proteins, of which 2 are glycoproteins. The molecular weights of protein 1, protein 2, protein 3, glycoprotein 1, and glycoprotein 2 were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000, respectively. Protein 2 was suggested to be nucleocapsid protein. 相似文献
8.
A new Sendai virus vector deficient in the matrix gene does not form virus particles and shows extensive cell-to-cell spreading
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Inoue M Tokusumi Y Ban H Kanaya T Shirakura M Tokusumi T Hirata T Nagai Y Iida A Hasegawa M 《Journal of virology》2003,77(11):6419-6429
A new recombinant Sendai virus vector (SeV/DeltaM), in which the gene encoding matrix (M) protein was deleted, was recovered from cDNA and propagated in a packaging cell line expressing M protein by using a Cre/loxP induction system. The titer of SeV/DeltaM carrying the enhanced green fluorescent protein gene in place of the M gene was 7 x 10(7) cell infectious units/ml or more. The new vector showed high levels of infectivity and gene expression, similar to those of wild-type SeV vector, in vitro and in vivo. Virus maturation into a particle was almost completely abolished in cells infected with SeV/DeltaM. Instead, SeV/DeltaM infection brought about a significant increase of syncytium formation under conditions in which the fusion protein was proteolytically cleaved and activated by trypsin-like protease. This shows that SeV/DeltaM spreads markedly to neighboring cells in a cell-to-cell manner, because both hemagglutinin-neuraminidase and active fusion proteins are present at very high levels on the surface of cells infected with SeV/DeltaM. Thus, SeV/DeltaM is a novel type of vector with the characteristic features of loss of virus particle formation and gain of cell-to-cell spreading via a mechanism dependent on the activation of the fusion protein. 相似文献
9.
Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5.
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The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins. 相似文献
10.
11.
Stephen C. Graham René Assenberg Olivier Delmas Anil Verma Alireza Gholami Chiraz Talbi Raymond J. Owens David I. Stuart Jonathan M. Grimes Hervé Bourhy 《PLoS pathogens》2008,4(12)
The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins. 相似文献
12.
Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus.
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We examined the human cytotoxic T-cell repertoire of nine adults to 9 of the 10 proteins of respiratory syncytial (RS) virus. Peripheral blood mononuclear cells from normal adults were stimulated with RS virus in vitro. The resulting polyclonal cultures were tested for lysis of B-lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing each of nine individual RS virus proteins. The use of peripheral blood dendritic cells to present antigen gave more easily reproducible results over a shorter culture period than conventional methods. The six RS virus proteins most strongly recognized were the nucleoprotein N (nine of nine donors with greater than 10% above background lysis; P = 0.0004), the surface proteins SH (six of nine donors; P = 0.002) and F (five of nine donors; P = 0.008), the matrix proteins M (five of nine donors; P = 0.004) and 22K (three of nine donors; P = 0.01) and the nonstructural protein 1b (six of nine donors; P = 0.004). There was no significant recognition of the major surface glycoprotein G (two of nine donors), the internal phosphoprotein P (one of nine donors), or the nonstructural protein 1c (one of nine donors). Recognition was major histocompatibility complex class I restricted, but no association between major histocompatibility complex phenotype and protein specificity of T cells was seen. Recognition of F and 22K appeared to be associated with recent infection indicated by increased levels of anti-RS virus immunoglobulin G antibody in serum measured by enzyme-linked immunosorbent assay. Since cytotoxic T-cell recognition of RS virus proteins has been demonstrated to be important in the clearance of virus from infected hosts, the N, M, SH, 1b, F, and 22K proteins should be considered potential vaccine components. 相似文献
13.
Comparison of the Structure and Polypeptide Composition of Three Double-Stranded Ribonucleic Acid-Containing Viruses (Diplornaviruses): Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus 总被引:5,自引:4,他引:1
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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. 相似文献
14.
Feline leukemia virus: biochemical and immunological characterization of gag gene-coded structural proteins. 总被引:18,自引:12,他引:6
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The major non-glycosylated structural proteins of feline leukemia virus have been isolated, and competition immunoassays have been developed for each. These proteins include the 27,000- to 30,000-molecular-weight major internal antigen designated p30, a 15,000-molecular-weight protein (p15), an acidic protein of 12,000 molecular weight (p12), and a highly basic 10,000-molecular-weight protein (p10). Immunologically and biochemically corresponding proteins of feline and murine leukemia viruses have been identified. and, on the basis of analogy to the known sequence of a prototype type C virus of mouse origin, the map order of the gag region of the feline type C viral genome has been tentatively deduced as NH2-p15-p12-p10-COOH. The demonstration of two feline leukemia virus gag gene-coded proteins, p15 and p12, expressed in the form of an uncleaved precursor in a mink cell line nonproductively transformed by feline sarcoma virus provides indirect support for the proposed sequence. 相似文献
15.
Physical and Chemical Properties of an Oncornavirus Associated with a Murine Adrenal Carcinoma Cell Line 总被引:1,自引:1,他引:0
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A type C oncornavirus has been isolated from a continuous cell line of murine adrenal carcinoma in culture. The particles have a buoyant density of 1.165 g/cm(3), exhibit typical type C morphology by electron microscopy, possess an RNA-dependent DNA polymerase, and have a high molecular weight RNA (6.1 x 10(6)) which can be denatured to a homogeneous lower molecular weight species (3.2 x 10(6)) when extracted from rapidly harvested "immature" virions. The virus is related antigenically to other mammalian oncornaviruses and exhibits a similar, although much more complex, sodium dodecyl sulfate gel electrophoretic profile of virion proteins when compared to the profiles of other type C RNA tumor viruses. 相似文献
16.
Identification and characterization of viral structural proteins of infectious salmon anemia virus
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Infectious salmon anemia virus (ISAV) is an unclassified Orthomyxovirus that has been shown to contain a segmented genome with eight single-stranded RNA species coding for 10 viral proteins. Four major structural proteins were characterized in the present study: two glycosylated proteins with estimated molecular masses of 42 and 50 kDa, one 66-kDa phosphoprotein, and one 22-kDa protein. Examination of lysed virions revealed the two glycoproteins and the 22-kDa protein in the soluble fraction, while the 66-kDa phosphoprotein and a minor part of the 22-kDa protein were found in the pelleted fraction. Immunofluorescence staining of infected cells demonstrated that the 22-kDa protein was a late protein accumulating in the nucleus. We conclude that the 66-kDa protein is the nucleoprotein, the 22-kDa protein is the matrix protein, and the 42- and 50-kDa proteins are the surface proteins. Radioimmunoprecipitation analysis of the 42-kDa glycoprotein, which was previously shown to represent the ISAV hemagglutinin, indicated that this protein exists at least as dimers. Further, by labeling of purified ISAV with [1,3-(3)H]diisopropyl fluorophosphate, it was also demonstrated that the viral esterase is located with the hemagglutinin. This finding was confirmed by demonstration of acetylesterase activity in affinity-purified hemagglutinin preparations. Finally, the active-site serine residue could be tentatively identified at position 32 within the amino acid sequence of the hemagglutinin of ISAV strain Glesvaer/2/90. It is proposed that the ISAV vp66 protein be termed nucleoprotein, the gp42 protein be termed HE protein, and the vp22 protein be termed matrix protein. 相似文献
17.
Purification, Characterization, and Attempts at Isotopic Labeling of Mouse Interferon 总被引:8,自引:3,他引:5
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Under optimal conditions which minimized the accumulation of extraneous proteins, interferon preparations were obtained in L cells containing from 2 x 10(4) to 5 x 10(4) units/mg of protein. The radiolabeled proteins were liberated simultaneously with interferon from cultures exposed to tritiated amino acids after viral stimulation and from corresponding controls, and were subsequently purified with the following results. Chromatography of interferon on carboxymethyl-Sephadex C-25 eliminated selectively unlabeled or poorly labeled proteins, resulting in a greater than sixfold increase in counts per minute per milligram of protein. Similarly purified control material harbored at least 12 times less tritium per milligram of protein than interferon, and the label was more diversely distributed among proteins of different molecular weights. On electrophoresis of interferon in polyacrylamide gels, labeled proteins were reduced further by a factor of at least 10 without loss in titer. Final purification was estimated at greater than 280-fold, representing a calculated specific activity of at least 1.4 x 10(7) units of interferon per milligram of protein. 相似文献
18.
A polypeptide with molecular mass of 17 kDa has been partially purified and identified as a major secretory glycoprotein in the rat epididymis. It is phosphorylated and contains high mannose-type oligosaccharides with 5 and 6 mannose units predominantly. These sugar residues are sufficiently exposed in the molecule to be released by endo-beta-N-acetylglucosaminidase H without prior denaturation or protease digestion. Specific binding of the glycoprotein to testicular spermatozoa was demonstrated with Ka 0.2 x 10(9) M-1 and 17,200 sites per cell, while no binding to epididymal spermatozoa was detectable. Direct labeling of surface proteins on cauda epididymis spermatozoa revealed the presence of a major band of 16.2 kDa, which may be equivalent to GP17. The interaction of the epididymal secretory protein with sperm suggests a possible role in the maturation process. 相似文献
19.
Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells 总被引:1,自引:0,他引:1
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Shmuel Rozenblatt Marian Gorecki Helen Shure Carol L. Prives 《Journal of virology》1979,29(3):1099-1106
Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE. 相似文献
20.
Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 x 10(6) (RNA 1) and 0.5 x 10(6) (RNA 2), respectively. Virions contained two proteins, beta with a molecular weight of 43,000 (43K) and gamma (5K), and traces of a third protein, alpha (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein alpha. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A, alpha, beta, gamma, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [(35)S]methionine-labeled cultures. All but protein gamma could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein alpha and the coordinate appearance of proteins beta and gamma, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor alpha. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and alpha, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor alpha peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly. 相似文献