The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.     

Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes.

The nonhistone proteins sedimenting in low-salt glycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradients as the first dimension and sodium dodecyl sulfate-gel electrophoresis as the second dimension. By densitometric quantitation of the radiolabeled proteins present in each fraction of the gradients, it was possible to identify sedimenting with all or a fraction of the simian virus 40 chromosomes. VP-1 sedimented with simian virus 40 chromosomes; additional evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI 6.0; Mr 32,000, pI 7.2; Mr 35,000, pI 8.5; and Mr 80,000, pI 7.2) sedimented with specific subsets of chromosomes.     

Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.     

Complete nucleotide sequence of tobacco streak virus RNA 3

Double-stranded cDNA of in vitro polyadenylated tobacco streak virus (TSV) RNA 3 has been cloned and sequenced. The complete primary structure of 2,205 nucleotides reveals two open reading frames flanked by a leader sequence of 210 bases, an intercistronic region of 123 nucleotides and a 3'-extracistronic sequence of 288 nucleotides. The 5'-terminal open reading frame codes for a Mr 31,742 protein, which probably corresponds to the only in vitro translation product of TSV RNA 3. The 3'-terminal coding region predicts a Mr 26,346 protein, probably the viral coat protein, which is the translation product of the subgenomic messenger, RNA 4. Although the coat proteins of alfalfa mosaic virus (A1MV) and TSV are functionally equivalent in activating their own and each others genomes, no homology between the primary structures of those two proteins is detectable.     

Release of a virus coded glycoprotein from herpes simplex virus type 1 infected cells

HEp-2 cells, which were infected with HSV-1, excrete besides other proteins a soluble glycoprotein (Mr 125000–130000) related to the virus protein gC. The excretion of the glycoprotein and the production of extracellular virus particles is reduced to a similar extent when the cells were treated with monensin. Possible consequences of the excretion of soluble viral proteins to a modulation of the immune response are discussed.Abbreviations HSV-1 Herpes simplex virus type 1 - PAGE Polyacrylamide gel electrophoresis - SDS Sodium dodecylsulfate     

Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity.

A cDNA fragment covering the genomic region that encodes the structural proteins of hog cholera virus (HCV) was inserted into the tk gene of vaccinia virus. Expression studies with vaccinia virus/HCV recombinants led to identification of HCV-specific proteins. The putative HCV core protein p23 was demonstrated for the first time by using an antiserum against a bacterial fusion protein. The glycoproteins expressed by vaccinia virus/HCV recombinant migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. A disulfide-linked heterodimer between gp55 and gp33 previously detected in HCV-infected cells was also demonstrated after infection with the recombinant virus. The vaccinia virus system allowed us to identify, in addition to the heterodimer, a disulfide-linked homodimer of HCV gp55. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.     

Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus.

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated. Three forms of CM2 with different electrophoretic mobilities (CM2(0), CM2a, and CM2b) were detected in infected cells by immunoprecipitation with antiserum to the glutathione S-transferase (GST)-CM2 fusion protein. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with endoglycosidase H or peptide-N-glycosidase F suggested that a mannose-rich oligosaccharide core is added to unglycosylated CM2(0) (Mr, approximately 16,000) to form CM2a (Mr, approximately 18,000) and that the processing of the carbohydrate chain from the high-mannose type to the complex type converts CM2a into CM2b, which is heterogeneous in electrophoretic mobility (Mr, approximately 22,000 to 30,000). Labeling of infected cells with [3H]palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. The CM2 protein was also found to form disulfide-linked dimers and tetramers on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Trypsin treatment of infected cell surfaces as well as of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this C-terminal domain is intracellular and exposed to the cytoplasms of microsomes. Furthermore, evidence that a small amount of CM2 is incorporated into progeny virus particles was obtained by Western blot analysis. These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.     

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.     

Solubilization of the membrane proteins from Semliki Forest virus with Triton X100

Increasing concentrations of Triton X100 have been found to cause stepwise dissociation of the membrane of Semliki Forest virus. The final stage of the breakdown process leads to solubilization of the membrane proteins which can be separated from the membrane lipids and the viral nucleocapsid by density gradient centrifugation in the presence of 0.05% Triton X100. Two different forms of Semliki Forest virus protein have been observed with sedimentation coefficients of approximately 4 S and 23 S. The 4 S aggregate appears to consist of two polypeptide chains complexed with about 75 molecules of Triton X100. The 23 S form is a rosette-like aggregate containing about 16 polypeptide chains and about 260 molecules of Triton X100. Sucrose alters the equilibrium between the 4 S and 23 S forms: removal of sucrose leads to association of the 4 S form to the 23 S form and addition of sucrose to dissociation.A scheme for the dissociation of the Semliki Forest virus membrane is presented which is discussed with reference to other biological membranes. It is suggested that Triton X100 and deoxycholate solubilize amphipathic membrane proteins by binding to the hydrophobic segments of these proteins.     

In vitro expression of belladonna mottle virus genome.

In vitro translation of belladonna mottle virus BDMV(I) genomic RNA in a rabbit reticulocyte lysate system produced proteins of Mr 210,000, 150,000 and 78,000 which form the non-structural proteins. The coat protein, on the other hand, was expressed from a subgenomic RNA which was found to be encapsidated in the empty capsids forming the top component viral particles. The implications of subgenomic RNA encapsidation in viral replication and assembly are discussed.     

Simian virus 40-transformed cells express new species of proteins precipitable by anti-simian virus 40 tumor serum.

In addition to the virus-coded large-T and small-t antigens, two new classes of proteins were immunoprecipitated by anti-simian virus 40 (SV40) tumor serum from extracts of various SV40-transformed cell lines. These were as follows: (i) proteins (termed "super-T proteins") with an Mr higher than that of large-T antigen (86,000), which were found in many SV40-transformed cell lines derived from mouse and rat cells (super-T proteins and large-T antigen appeared to have closely related structures as judged by the Chromobead elution patterns of their methionine-labeled tryptic peptides); (ii) proteins (termed "55K proteins") with an Mr ranging from 50,000 to 60,000, which were present in all SV40-transformed cell lines examined so far, including those obtained by chromosome-mediated gene transfer. The 55K proteins were not structurally related to large-T antigens, as judged by the Chromobead elution patterns of their methionine-labeled tryptic peptides. Our data are compatible with the assumption that the 55K proteins are largely or totally cell coded.     

Complete amino acid sequence of human T-cell leukemia virus structural protein p15

The complete amino acid sequence of human T-cell leukemia virus (HTLV) structural protein p15 has been determined. The intact protein and peptides generated by enzymatic digestion and acid cleavage were purified by reversed-phase liquid chromatography and subjected to semi-automated Edman degradation. HTLV p15 is a basic linear polypeptide composed of 85 amino acids with Mr 9458. The primary structure indicates that HTLV p15 is homologous to the nucleic acid binding proteins of other type-C retroviruses and especially related to bovine leukemia virus p12.     

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.     

Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein.

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.     

Neoplastic transformation by E26 leukemia virus is mediated by a single protein containing domains of gag and myb genes.

The genome of avian leukemia virus E26 shares homology with v-myb, the oncogene of avian myeloblastosis virus, and encodes a protein with an Mr of 135,000. Analyses of tryptic oligopeptides show that this protein is related to the proteins encoded by gag (Pr76gag) as well as v-myb (p45v-myb[AMV] ) and c-myb (p75c-myb). We found no evidence for the existence of additional myb-related proteins or subgenomic species of myb-related RNA in myeloblasts transformed by strain E26.     

Molecular interactions of Epstein-Barr virus capsid proteins

The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S-transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Structural proteins of La Crosse virus.

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).