Topographic analysis of tobacco etch virus capsid protein epitopes

Monoclonal antibodies have been prepared, which react with capsid protein of an aphid-transmitted isolate of tobacco etch virus (TEV). Ten different monoclonal antibodies were characterized with reference to (1) antibody class, (2) reactivity with different plant virus antigens, (3) the spatial relationship between epitopes, and (4) whether these epitopes were located on the exterior surface of the virion. Three monoclonal antibodies were specific for TEV isolates. These monoclonal antibodies reacted with epitopes exposed on the external surface of the TEV particle. Seven monoclonal antibodies reacted with a variety of different potyviruses including TEV, potato virus Y, tobacco vein mottling virus, pepper mottle virus, watermelon mosaic virus II, and maize dwarf mosaic virus. In general, these seven monoclonal antibodies defined epitopes not readily accessible on the virion surface.     

Nucleotide sequence at the 3' terminus of pepper mottle virus genomic RNA: evidence for an alternative mode of potyvirus capsid protein gene organization

The sequence of the 3'-terminal 1481 nucleotides of the pepper mottle virus (PeMV) genome has been determined. The sequence was determined by dideoxy nucleotide sequencing of complementary DNA which had been inserted into M13 bacteriophage cloning vectors and was confirmed by sequencing selected regions of PeMV RNA. A discrete open reading frame of 993 nucleotides, ending 333 nucleotides from the 3'-terminal polyadenylate tract, was identified that potentially encoded a 37,669-MW protein. The amino acids predicted at positions 64 through 84 of this putative polypeptide were identical to the amino-terminal 21 amino acids of the PeMV capsid protein ascertained by chemical sequencing. These combined nucleotide and amino acid sequence data suggest that the PeMV capsid protein is encoded by the 3'-most cistron on the genomic RNA and that it may be expressed as a precursor that is proteolytically processed to produce the mature capsid protein.     

Analytical centrifugation of the disk aggregates of tobacco rattle virus protein.

Oligomeric protein (>7 S) isolated from purified preparations of the CAM and PRN strains of tobacco rattle virus (TRV) polymerized into disk-like aggregates of about 38 S at pH values above 5 and ionic strength of about 0.1. At ionic strengths of 0.25–1.0, the proportion of oligomeric protein detectable at pH 6.7 and 8 increased with increasing ionic strength. Adjusting protein solutions from 4 to 20° increased reversibly the proportion sedimenting at about 38 S. Adding 0.4 M urea at pH 4.7 and 5 decreased the proportion of 38 S component. It is suggested that, in addition to electrostatic interactions, hydrophobic bonding contributes to protein-protein interactions in the disk aggregate. In solutions of pH 7 and I = 0.1, increasing the protein concentrations from 3 to 10 mg/ml decresed S, and such data gave estimates of s_20,w⁰, (CAM) = 40 S, and s_20,w⁰ (PRN) = 39 S. Below 1 mg/ml, decreasing the concentration decreased S, suggesting a reversible equilibrium between 39–40 S component and lower molecular weight material. Treatment with 0.2% formaldehyde at pH 7 prevented this dissociation. The molecular weight of formaldehyde-treated 40 S disk aggregates of PRN protein was estimated to be 2.3 × 10⁶ based on sedimentation and diffusion data in one experiment. More precise estimates of 2.0 × 10⁶ were obtained from sedimentation equilibrium experiments with fixed disk aggregates of protein of both strains. There are estimated to be 96 ± 13 subunits in TRV disk aggregates, which therefore seem to comprise between three and four layers of protein.     

RNA and capsid accumulation in cowpea protoplasts that are resistant to cowpea mosaic virus strain SB

Leaf protoplasts from the Arlington line of cowpea (Vigna unguiculata) support only a limited increase of cowpea mosaic virus strain SB (CPMV-SB), whereas cowpea severe mosaic virus, another member of the comovirus group, replicates efficiently in Arlington cowpea protoplasts. CPMV-SB replicates efficiently in protoplasts of cowpea line Blackeye 5. Some characteristics of the virus-specific resistance of Arlington protoplasts to CPMV-SB are reported. Differences between progeny CPMV-SB from Arlington and Blackeye 5 protoplasts were not detected. Inoculation with CPMV-SB RNA, rather than virions, did not make Arlington protoplasts fully susceptible. These results favor, for likely involvement in the CPMV-SB restriction phenomenon, events in the virus life cycle that occur after exposure of virion RNA to the cytoplasm and before assembly of particles is completed. The accumulation of CPMV-SB RNAs of both polarities was found to be depressed in inoculated Arlington protoplasts. However, (+)RNA (virion RNA polarity) accumulated to no lesser extent, per unit of (?)RNA, in Arlington protoplasts than in, Blackeye 5 protoplasts. Capsid antigen accumulation, per unit of (+)RNA, was reduced in Arlington protoplasts as compared to Blackeye 5 protoplasts. A working hypothesis consistent with the above and other observations is that Arlington protoplasts have an inhibitory substance that interferes with the production or/and function of CPMV-SB specified proteins.     

Cloning of bovine rotavirus (RF strain): nucleotide sequence of the gene coding for the major capsid protein

The genes of the RF strain of bovine rotavirus have been cloned into pBR 322 following the synthesis and hybridization of cDNA transcribed from both strands of in vitro polyadenylated genomic RNA. Cloned rotavirus DNAs were assigned to most of the 11 genomic RNA segments by Northern blot hybridization. The complete sequence of gene 6 that codes for the major inner capsid protein has been determined. The gene is 1356 nucleotides long and possesses an unique long open reading frame that could encode a protein (397 amino acids) of similar size to the known gene 6 product. Comparison of the RF bovine rotavirus gene 6 sequence with the sequence of the simian rotavirus gene 6, showed these genes to be very similar in nucleotide sequence (87% homology). Most of the base changes are silent and the predicted amino acid sequences are almost identical (97% homology).     

The four C-terminal amino acids of the v-erbA polypeptide are encoded by an intronic sequence of the v-erbB oncogene

The genome of avian erythroblastosis virus (AEV), a defective acute leukemia retrovirus, carries two distinct cell-derived oncogenes in the structure 5' delta gag-erbA-erbB-delta env3'. The nucleotide sequence of the v-erbA gene was recently reported. In order to determine the boundary between the two adjacent oncogenes, the sequence of the v-erbA/v-erbB junction of AEV was compared to that of a recombinant lambda phage containing a chicken cellular sequence representing the 5' part of c-erbB. The four C-terminal amino acids of v-erbA are in fact encoded by a c-erbB intron-derived sequence thus demonstrating that the virus acquired a truncated c-erbA gene. Furthermore the 7 to 10 amino acid residues upstream from the 4 C-terminal amino acids mentioned above appeared to be derived from env-related sequences. The splice acceptor site at the beginning of the only open reading frame for v-erbB is also present and functional in c-erbB when expressed to generate a truncated EGF (epidermal growth factor) receptor. Thus AEV joins a truncated erbA gene to a truncated erbB gene through env-derived sequences and intronic sequences from c-erbB.     

The 126,000 molecular weight protein of tobacco mosaic virus is associated with host chromatin in mosaic-diseased tobacco plants

The infection-related polypeptide of apparent molecular weight (MW) 116,000, which previously was shown to be associated with host chromatin from mosaic-diseased leaves of tobacco mosaic virus (TMV)-infected tobacco, comigrated during electrophoresis in polyacrylamide gels with the 126,000 molecular weight protein translated from TMV RNA in vitro. Limited proteolysis of the 116,000 MW polypeptide and of the 126,000 MW translational product with protease V8 generated the same peptides, indicating that the proteins were very similar or identical. Thus, the virally coded 126,000 MW protein is associated with host chromatin. The possibility that viruses may perturb host metabolism at the genome level is discussed.     

Amino-terminal amino acid sequence and carboxyl-terminal analysis of Rauscher murine leukemia virus glycoproteins

The two glycoproteins of Rauscher murine leukemia virus, gp70 and gp45, were found to have the following identical NH₂-terminal amino acid sequences: Ala-Ala-Pro-Gly-Ser-Pro-His-Gln-Val-Tyr-X-Ile-Thr-X-Glu-Val-(X - unidentified). The COOH-terminal amino acid of gp70 is tyrosine and that of gp45 is leucine. Computer-analyzed amino acid compositional data indicated that the protein moiety of gp45 is smaller than that of gp70. These results are compatible with the suggestion that gp45 is derived from gp70 by proteolytic cleavage.     

Varicella-zoster virus envelope glycoproteins: Biochemical characterization and identification in clinical material

Varicella-zoster virus (VZV)-infected human foreskin fibroblasts synthesize viral glycoproteins of 125,000 (gp125), 118,000 (gp118), 92,000 (gp92), 63,000 (gp63), 59,000 (gp59), and 47,000 (gp47) Da. In biochemical studies, all of these VZV glycoproteins were shown to contain asparagine-linked (N-linked) oligosaccharide chains and, except for gp125 and gp47, to be sialoglycoproteins. Experiments with endo-β-N-acetylglucos-aminidase H (endo H) demonstrated that gp92 contained only complex type (endo H-resistant) N-linked glycosyl chains, while the other mature glycoproteins contained both high-mannose (endo H-sensitive) and complex-type oligosaccharides. Monoclonal antibodies recognizing multiple glycoproteins, gp63/gp125 or gp92/gp59/gp47, neutralized virus infection, suggesting the glycoproteins were important components of the virus envelope. This was confirmed for gp92/gp59/gp47 by immunoelectron microscopy, which revealed dense staining localized exclusively to the virion envelope and to the plasma membrane of virus-producer cells. The mature forms of all of these glycoproteins were also present in viral material isolated from vesicles of varicella and zoster patients, indicating that in infected individuals the viral glycoproteins are synthesized and processed in a manner similar to that in tissue culture cells.     

Immunoprecipitation analysis of potyviral in vitro translation products using antisera to helper component of tobacco vein mottling virus and potato virus Y

The serological relationships of the products of in vitro translation of the RNA of various potyviruses were analyzed by using antisera to helper component (HC) from tobacco plants infected with either tobacco vein mottling virus (TVMV) or potato virus Y (PVY). The PVY-HC antiserum immunoprecipitated a specific PVY-RNA translation product; this product was not reactive with antisera to PVY-induced cylindrical inclusion protein or capsid protein or to the two tobacco etch virus nuclear inclusion proteins. The antiserum to PVY-HC did not immunoprecipitate significant amounts of any translation products of 16 other potyviruses including TVMV. In contrast the antiserum to TVMV-HC efficiently immunoprecipitated a specific product(s) of four different potyviruses, some isolates of which are poorly transmitted or nontransmissible by aphids, and less efficiently a product(s) from 12 other potyviruses, including PVY. Distinct serotypes were resolved among the major in vitro translation products of 17 different potyviral RNAs by the antisera to TVMV-HC and PVY-HC. There appears not to be a correlation between the serological reactivities of HC-related polypeptides and the ability of different HC-virus combinations to effect aphid transmission of the virus.     

Herpes simplex virus and protein transport are associated with the cytoskeletal framework and the nuclear matrix in infected BSC-1 cells

Triton cytoskeletons and nuclear matrices were prepared from herpes simplex virus (HSV)-infected cells by a sequential fractionation scheme. Electron microscopic studies revealed the association of mature HSV with the filamentous network of the nuclear matrix. Indirect immunofluorescence assays with monoclonal antibodies revealed that ICP5, the major capsid protein, accumulated on the nuclear matrix while ICP8, the major viral DNA binding protein, accumulates in the chromatin fraction that can be separated from the nuclear matrix by extraction with DNase and salt. Pulse-chase experiments confirmed the kinetics studies of D. M. Knipe and A. E. Spang (J. Virol.43, 314–324, 1982) and showed that ICP5 is transported after a lag from the cytoplasmic framework to the nuclear matrix, while ICP8 is transported faster to the chromatin fraction.     

Biochemical and biological characteristics of epitopes on the E1 glycoprotein of western equine encephalitis virus

Antigenic determinants identified by monoclonal antibodies (Mabs) on the E1 glycoprotein of western equine encephalitis (WEE) virus have been characterized by their serological activity, requirements for secondary structure, expression on the mature virion, and their role in protecting animals from WEE virus challenge. On the basis of a cross-reactivity enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition assay, eight antigenic determinants (epitopes) on the E1 glycoprotein have been identified, ranging in reactivity from WEE-specific to alphavirus group reactive. No neutralization of virus infectivity was demonstrable with any of the Mabs. An alphavirus group-reactive hemagglutination (HA) site, a WEE complex-reactive HA site, and a WEE virus-specific HA site were identified. Spatial arrangement of these epitopes was determined by a competitive binding ELISA. Four competition groups defining three distinct antigenic domains were identified. Antibodies directed against four E1 epitopes were capable of precipitating the E1/E2 heterodimer from infected cells or purified virus disrupted with nonionic detergents. These same antibodies precipitated only E1 in the presence of 0.1% SDS. That E1 conformation was important was shown by the inability of antibodies specific for seven of the epitopes to bind to virus denatured in 0.5% SDS. As determined by equilibrium gradient analysis of virus-antibody mixtures, four epitopes were found to be fully accessible on the mature virion, three epitopes were inaccessible, and one epitope was partially accessible to antibody binding. Antibodies specific for three epitopes were able to passively protect mice from WEE virus challenge.     

Biochemical characterization of two Epstein-Barr virus early antigen-associated phosphopolypeptides

Epstein-Barr virus (EBV) nonproducer cells NC37 induced to viral early antigen (EA) synthesis by the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) were labeled at Day 4 after induction with 32P, and were analyzed by immunoprecipitation with human EA-positive sera. By employing this method the appearance of two phosphopolypeptides of 50 and 58K (pp50 and pp58) was well correlated with EA complex. Partial V8 protease digestion and two-dimensional peptide analysis revealed that the polypeptides pp50 and pp58 are related. The analysis of phosphoamino acids indicated that pp58 contains phosphoserine and phosphothreonine to the same percentage, whereas in pp50 only phosphoserine was found. The analysis of the subcellular distribution revealed that pp50 and pp58 are located in the chromatin. Both phosphopolypeptides exhibit DNA-binding activity, and are recognized by two monoclonal antibodies described recently (R3 and 1108-1).     

Location of the gene specifying the smaller protein of the cowpea mosaic virus capsid

Cowpea mosaic virus (CPMV) preparations contain two centrifugal nucleoprotein components, middle and bottom, both of which are required for infection. The capsid of the virus is constructed from equal numbers of large and small protein subunits. A mutant isolate, designated G3, was derived from nitrous acid-treated virus. Mutant G3 demonstrated three electrophoretic forms in contrast to the two electrophoretic forms found in wild-type preparations. As with wild-type virus, the slower forms of G3 were found to undergo an apparent in vivo conversion to the fast form. The conversion could be simulated in vitro by the action of chymotrypsin. This suggests that the G3 mutant carries an altered electronic charge on the labile carboxy-terminal portion of the smaller capsid protein. Examination of separated proteins by gel electrophoresis verified that wild-type and G3 capsids differed only in their smaller proteins. Complementation experiments with separated centrifugal components demonstrated that the recovery of G3 type progeny is dependent upon the presence of G3 middle component in the inoculum. This study indicates that the gene specifying the smaller capsid subunit is located in the middle component RNA of CPMV.     

Human T-cell leukemia virus type II: primary structure analysis of the major internal protein, p24 and the nucleic acid binding protein, p15

The 24,000-molecular-weight major internal protein (p24) and the 15,000-molecular-weight nucleic acid binding protein (p15) of human T-cell leukemia virus type II (HTLV-II) were subjected to amino acid composition and amino-terminal amino acid sequence analysis. A comparison of amino acid composition of p24 and p15 of HTLV-II with those of the analogous proteins of HTLV-I revealed that these two proteins share overall similarity. Further, alignment of the amino-terminal amino acid sequence for the first 27 residues of p24 and 34 residues of p15 from HTLV-II showed extensive sequence homology with analogous proteins of HTLV-I. These data suggest that although disease associated with HTLV-I is malignant T-cell leukemia and that associated with HTLV-II is a relatively benign variant of hairy-cell leukemia, HTLV-I and HTLV-II are closely related to each other, at least in their gag-gene-encoded sequences.     

Diaphorase activity located in inclusions induced by soil-borne wheat mosaic virus.

Extracts of rye leaves infected with soil-borne wheat mosaic virus, isolate US-B, were examined with the electron microscope. They contained inclusions consisting of membrane bound tubules. Partially purified inclusions had the same fine structures as that observed in thin sections of leaf cells. Inclusions in epidermal strips and partially purified preparations were examined cytochemically. They had an intense diaphorase activity (NADH or NADPH nitrobluetetrazolium oxidoreductase). The activity was lost after heating at 55° for 5 min, fixation with ethanol or glutaraldehyde, and incubation with 5 × 10^?5M parachloromercuribenzoate. The activity was intense when NADH was used as a hydrogen donor, but was weak with NADPH. The cytoplasm of leaf cells also had similar diaphorase activity although the activity was weak. When diaphorase activity in various cellular fractions was assayed, about 60–70% of the activity was in the soluble fraction, 13–14% was in the 15,000 g pellet, and 1–3% in the microsomal fraction. The ratio of specific activity of NADH diaphorase to NADPH diaphorase was about 2 in the microsomal fractions but was 0.6–0.9 in the other fractions. Microsomal fractions from infected leaves contained fragments of inclusions and had higher ratios of NADH to NADPH diaphorase activity than those from uninfected leaves. Partially purified inclusions had higher NADH diaphorase activity than NADPH diaphorase. These results indicated that diaphorase activity was bounded to inclusion tubules and that the activity had properties in common with endoplasmic reticulum.     

Subcellular localization of the v-erb-B protein,the product of a transforming gene of avian erythroblastosis virus

Avian erythroblastosis virus (AEV) is an oncogenic retrovirus capable of transforming both fibroblasts and immature erythroid cells. The v-erb-B locus within the AEV genome encodes a glycosylated protein, expression of which is required for oncogenic transformation of either cell type. Subcellular localization of the v-erb-B glycoprotein in AEV-transformed cells is reported here. Results indicate that the v-erb-B protein is synthesized on dense membrane fractions and appears to possess the properties of an integral membrane protein. The bulk of the v-erb-B protein remains with dense membranes after synthesis, although a small quantity may slowly become associated with the plasma membrane. The biogenesis and subcellular location of the v-erb-B protein are thus quite different from those of the transforming proteins that display protein kinase activity. These differences are especially provocative because the amino acid sequences of the v-erb-B protein and the protein kinases are closely related to one another.     

Enhanced tobacco mosaic virus production and suppressed synthesis of a virus inhibitor in protoplasts exposed to antibiotics

Actinomycin D and chloramphenicol, when added up to 24 hr after inoculation, markedly increased tobacco mosaic virus (TMV) replication in protoplasts of Samsun NN, a cultivar in which the infection in the intact plant is localized. No increase was observed when TMV-infected protoplasts of Samsun, a systemic-responding cultivar, were incubated in the presence of these antimetabolites. Concomitant with the increase in virus replication in protoplasts of Samsun NN, production of the inhibitor of virus replication (IVR) (G. Loebenstein and A. Gera, Virology, 114, 132-139, 1981) from these protoplasts was suppressed almost completely. These results strengthen the suggestion that IVR is associated with the localizing mechanism by suppressing virus replication.