Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome.

Mouse hepatitis virus (MHV), a member of the Coronaviridae, contains a polyadenylated positive-sense single-stranded genomic RNA which is 31 kb long. MHV replication and transcription take place via the synthesis of negative-strand RNA intermediates from a positive-strand genomic template. A cis-acting element previously identified in the 3' untranslated region binds to trans-acting host factors from mouse fibroblasts and forms at least three RNA-protein complexes. The largest RNA-protein complex formed by the cis-acting element and the lysate from uninfected mouse fibroblasts has a molecular weight of about 200 kDa. The complex observed in gel shift assays has been resolved by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four proteins of approximately 90, 70, 58, and 40 kDa after RNase treatment. Specific RNA affinity chromatography also has revealed the presence of a 90-kDa protein associated with RNA containing the cis-acting element bound to magnetic beads. The 90-kDa protein has been purified from uninfected mouse fibroblast crude lysates. Protein microsequencing identified the 90-kDa protein as mitochondrial aconitase. Antibody raised against purified mitochondrial aconitase recognizes the RNA-protein complex and the 90-kDa protein, which can be released from the complex by RNase digestion. Furthermore, UV cross-linking studies indicate that highly purified mitochondrial aconitase binds specifically to the MHV 3' protein-binding element. Increasing the intracellular level of mitochondrial aconitase by iron supplementation resulted in increased RNA-binding activity in cell extracts and increased virus production as well as viral protein synthesis at early hours of infection. These results are particularly interesting in terms of identification of an RNA target for mitochondrial aconitase, which has a cytoplasmic homolog, cytoplasmic aconitase, also known as iron regulatory protein 1, a well-recognized RNA-binding protein. The binding properties of mitochondrial aconitase and the functional relevance of RNA binding appear to parallel those of cytoplasmic aconitase.

Phylogenetic analysis of two potyvirus pathogens of commercial cowpea lines: implications for obtaining pathogen-derived resistance.

As a prelude to developing engineered resistance to two important potyvirus pathogens of cowpea, a phylogenetic analysis of strains of Cowpea aphid-borne mosaic virus (CAbMV) and Bean common mosaic virus--blackeye cowpea strain (BCMV-B1C) was undertaken. Nucleotide sequences for the coat protein genes and 3'-untranslated regions of four CAbMV and one BCMV-B1C strains were determined and included in an analysis with published sequences. While all the newly sequenced viruses showed strong homology with the existing respective sequences in the database, the CAbMV group showed a divergence into two subgroups. These groups differed from each other by more than some CAbMV strains differed from the South African Passiflora virus (CAbMV-SAP), which has distinct biological characteristics. The implications of the sequence analyses are discussed with respect to a strategy for the generation of engineered resistance to both groups of viruses.

Preparation of GFLV CP-gene cDNA probe labeled by photosensitive biotin and its application in detection of GFLV.

The cDNA of the GFLV CP gene amplified from clone pGAB5 by PCR was labeled by photosensitive biotin. The probe sensitivity is 5 pg, and the detectable amount of unlabeled GFLV cDNA was 10 pg by Dot-blot hybridization when the biotin-labeled cDNA was used as a probe. The results of detection for GFLV by probe have positive correlation with those of ELISA. The minimum amount of grapevine tissue for detection by probe was 100 times lower than that by ELISA. The seasons and the position of grapevine leaves do not limit application of this method.

Detection of potato mop-top virus capsid readthrough protein in virus particles.

Potato mop-top furovirus (PMTV) RNA 3 encodes the 20 kDa coat protein and a larger readthrough protein of 67 kDa. The readthrough protein is expressed by suppression of the amber stop codon which terminates the coat protein gene. A 21 kDa C-terminal fragment of the readthrough protein was doned, fused to glutathione S-transferase and expressed in E. coli. An antiserum prepared against purified fusion protein was used in ELISA to detect the readthrough protein in extracts of PMTV-infected leaves. Immunogold labelling studies showed that the readthrough protein was located near one extremity of some of the virus particles.

Short and long distance spread of potato leafroll luteovirus: effects of host genes and transgenes conferring resistance to virus accumulation in potato.

Potato leafroll luteovirus (PLRV) movement through phloem of PLRV-resistant potato clones was examined in experiments in which stem pieces were grafted either between infected rootstocks and virus-free susceptible scions or between infected scions and virus-free susceptible rootstocks. These test plants permitted either upwards or downwards virus movement into the susceptible tissue. Resistant potato clones had either host gene-mediated resistance (H-MR) or transgene-mediated resistance (T-NR, conferred by transformation with the PLRV coat protein gene) to PLRV accumulation. The rate of PLRV movement was similar whether stem tissue was taken from H-MR, T-MR or susceptible potato clones. Virus movement through two graft unions began around 7 days after grafting and was generally complete by about 14 to 16 days. Virus movement occurred soon after acquiring functional phloem continuity across grafts as demonstrated by tracing with 6(5)-carboxyfluorescein, a phloem-mobile dye. Most of the delay in virus detection after grafting probably resulted from the time necessary to develop new phloem strands across graft unions; subsequent movement of PLRV was rapid suggesting a passive process. PLRV infection was largely excluded from external phloem bundles in stem tissue of clones with either H-MR or T-MR. This trait was less pronounced as tissue aged. The mechanism limiting PLRV invasion of external phloem bundles of the T-MR clones appears to be similar to that operating in the H-MR clones. Results are discussed in the context of a proposed model of PLRV movement.

Virion formation is required for the long-distance movement of red clover necrotic mosaic virus in movement protein transgenic plants.

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two single-stranded RNA species. The polycistronic RNA-1 encodes the viral RNA polymerase and capsid protein (CP) and the monocistronic RNA-2 encodes the 35 kDa cell-to-cell movement protein (MP). Nicotiana benthamiana plants transformed with the RCNMV MP gene were generated. When inoculated onto the MP transgenic plants, cell-to-cell movement of RNA-1 occurred at a rate similar to wild-type virus. However, long-distance (leaf-to-leaf) movement of RNA-1 was not observed. Neither CP nor virions were detected in the inoculated leaves of the MP transgenic plants. When RNA-1 was coinoculated with RNA-2 mutants, which do not express a functional MP, onto MP transgenic plants, CP and virions were readily detected and a systemic infection resulted. These results demonstrate that both RNA-1 and RNA-2 are necessary for the accumulation of both CP and virions. Furthermore, CP accumulation was found to be required for long-distance movement of RCNMV. Therefore, these data provide evidence that CP, in the form of virions, is necessary for the long-distance movement of RCNMV.

Expression of early and late adenoviral proteins in fatal adenovirus bronchopneumonia.

The localization and distribution of three adenoviral proteins, hexon, E1A, and 55-kDa E1B, in 16 cases of fatal adenovirus bronchopneumonia in infants and children, are described. The proteins were immunohistochemically demonstrated in paraffin sections using monoclonal antibodies followed by the avidin-biotin-peroxidase method. The hexon antigen was present in inclusion-bearing bronchial, bronchiolar, and alveolar cells, mainly in the so-called rosette cells, as well as in necrotic debris in necrotizing areas. E1A antigen was also recognized in cells with nuclear inclusions where the reaction decorated the inclusion, nuclear chromatin, and cytoplasm but distributed mainly in alveolar cells and to a lesser extent in bronchial and bronchiolar cells. The 55-kDa E1B protein was extensively present in "activated," reactive-appearing, nuclei of bronchial, bronchiolar, and alveolar epithelial cells and in the cytoplasm of rare cells having nuclear inclusions. These activated nuclei did not stain for the other two antigens. "Smudge" cells reacted poorly or not at all with any of the antibodies. The reactivity found produced a sort of complementary pattern between the hexon-positive, inclusion-containing cells and the 55-kDa E1B-positive, inclusion-noncontaining cells. The relationships of present findings and virologic data are discussed.

Capsid protein properties of cowpea aphid-borne mosaic virus and blackeye cowpea mosaic virus confirm the existence of two major subgroups of aphid-transmitted, legume-infecting potyviruses.

A study of the capsid proteins of different legume-infecting potyviruses using specific monoclonal antibodies on immunoblots of crude extracts from infected plants revealed that cowpea aphid-borne mosaic virus (CAMV) and blackeye cowpea mosaic virus (BICMV) have coat protein M(r) values of 32K and 35K, respectively. Immunoblot comparisons of BICMV, peanut stripe mosaic virus (PStV), bean common mosaic virus (BCMV) and azuki bean mosaic virus (AzMV) revealed equal reactivity of their 35K coat proteins. Similar comparisons between CAMV and the necrotic strain of BCMV (isolate NL3) showed a serological relationship between their 32K coat proteins, results providing the first evidence of a possible similarity between CAMV and BCMV NL3. Peptides from trypsin digests of the coat proteins of several of these legume-infecting potyviruses were analysed by HPLC. Comparison of the peptide profiles confirmed the serological results in distinguishing the two subgroups. Peptide profiles of coat protein from BICMV, PStV, AzMV and BCMV were almost identical, results suggesting that they could be considered as strains of one virus. In contrast, peptide profiles of various CAMV serotypes and BCMV NL3 were distinct from the first group and exhibited limited similarities to each other.

Preparation and specificity of antibodies against coat proteins of broad bean stain virus.

Mouse polyclonal antibodies were prepared against broad bean stain virus (BBSV, Comovirus group) and its coat protein subunits, large (L) and small (S) protein. These subunits were less immunogenic than native virus. Antibodies against L protein (Anti-L) and native virus (Anti-BBSV) did not react in immunoblots with S protein, but Anti-BBSV antibody reacted with S protein in plate-trapped antigen ELISA. Anti-S antibodies did not react with the related red clover mottle comovirus (RCMV), but Anti-L and Anti-BBSV antibodies reacted with RCMV similarly to BBSV. We assume that all the linear epitopes of the BBSV S protein are hidden in the native virions and the antigenic similarity between BBSV and RCMV is based mainly on their common linear L-specific epitopes.

Failure of long-distance movement of southern bean mosaic virus in a resistant host is correlated with lack of normal virion formation.

Sunn-hemp mosaic tobamovirus (SHMV) facilitated the spread of the cowpea strain of southern bean sobemovirus (SBMV-C) only in inoculated leaves of common bean (Phaseolus vulgaris L. cv. Bountiful), a resistant host for SBMV-C. Tissue prints of bean primary leaves doubly inoculated with SHMV and SBMV-C, developed by Western blotting, showed the presence of the SBMV-C capsid antigen in the mesophyll and epidermis, but no antigen was detected in the conducting bundles. Typical SBMV-C virions were not seen in electron micrographs of immunogold-labelled mesophyll cells; instead, specifically labelled, amorphous protein clumps were found in the vacuole. Particles of smaller diameter than that of typical SBMV-C virions were specifically trapped by SBMV antibodies following immunosorbent electron microscopy of extracts from doubly infected leaves. SBMV-C coat protein from infected Vigna unguiculata L. (cowpea) and bean plants showed no difference in its mobility following electrophoresis in denaturing SDS-polyacrylamide gels. Lack of efficient assembly of SBMV-C virions does not impede cell-to-cell movement of the virus in doubly infected leaves of bean, yet it is probably an important factor in determining the inability of SBMV-C to move into and/or through the vascular system of this host.

Strains of bean common mosaic virus consist of at least two distinct potyviruses.

Bean common mosaic virus (BCMV) consists of a large number of pathotypes and strains which have largely been identified by their characteristic interactions with a selected number of differential bean cultivars. The relationships among these strains and other potyviruses that infect legumes are complex, with indications that BCMV, blackeye cowpea mosaic virus (BlCMV) and azuki bean mosaic virus (AzMV) may be strains of the one virus. Using high performance liquid chromatographic peptide profiles of coat-protein digests, the NL3 and NY15 strains of BCMV were compared with each other, with the Type and W strains of BlCMV and with the mild mottle strain of peanut stripe virus (PStV). The results suggest that BCMV-NL3 and BCMV-NY15 are distinct potyviruses, not strains of the one virus, and that BCMV-NY15 is a strain of the same potyvirus that includes BlCMV, PStV, AzMV and three potyvirus isolates (74, PM, PN) from soybeans.

Characterization of bean mild mosaic virus: particle morphology, composition and RNA cell-free translation.

Virions of bean mild mosaic virus (BMMV) are built of 180 subunits of a single protein species of MW 40 x 10(3) [coat protein CP], packed into a T = 3 surface lattice. The capsomers on the five-fold symmetry axes protrude 2-3 nm from the particle surface. The virions encapsidate genome-size [approximately 4,200 nucleotides (nt)] as well as some heterogeneous RNAs of subgenomic size approximately 1,000-2,000 nt. In cell-free systems from Krebs-2 ascites cell extracts and rabbit reticulocyte lysates, genome-size RNA directed the synthesis predominantly of two polypeptides of MW 27 x 10(3) and 79 x 10(3) while the third major BMMV-specific polypeptide (MW 40 x 10(3), putative CP) seemed to be encoded by a shorter messenger RNA. The 'cap' analogue, m7GDP, partially inhibited BMMV RNA in vitro translation, suggesting that at least part of the BMMV-specific RNAs are capped. Oligo (dT)-cellulose column chromatography data suggested that poly(A)-tracts are absent from the BMMV genome. The data obtained confirm the previous classification of BMMV within the carmovirus group.

Novel virus-like particles containing circular single-stranded DNAs associated with subterranean clover stunt disease.

Novel virus-like particles, 17-19 nm in diameter, have been isolated from subterranean clover and pea plants infected with the pathogen of subterranean clover stunt disease (SCSD). The structure and genetic organization of these particles suggest that the pathogen of SCSD is representative of a new group of plant DNA viruses. SCS virus-like particles (SCSV) are isometric and band as a single component with buoyant densities of 1.24 g/ml in Cs2SO4 and 1.34 g/ml in CsCl. The A260 nm/A280 nm is about 1.35, which is consistent with an estimated nucleic acid content of 17%. Molecular calculations suggest that the particles have a T = 1 capsid structure containing 60 polypeptide subunits each with Mr of 19,000. Nucleic acid analysis including restriction enzyme digestions of double-stranded cDNAs suggests that SCSV have a divided genome composed of multiple species of circular, single-stranded DNA molecules each of approximately 850-880 nucleotides and that each is encapsidated in a separate particle. Linear and aggregated forms of these DNAs are also detected by gel electrophoresis. Evidence suggests that these virus-like particles are the pathogen of SCSD.

A beneficial effect of trypsin on the purification of turnip mosaic virus (TuMV) and other potyviruses.

The effects of treatment with trypsin during the purification of turnip mosaic virus (TuMV), on virus yield, infectivity and integrity of virus coat protein were examined. Trypsin increased yield markedly, and at a low concentration, increased infectivity. These effects were probably due to reduced aggregation of virus particles. At higher concentrations of trypsin, there was some degradation of virus coat protein, and infectivity was reduced. Treatment with trypsin at the optimum concentration can significantly improve purification of TuMV; more limited experiments suggest that it can also be applied to other potyviruses.

The isolation of the two electrophoretic forms of cowpea mosaic virus using fast protein liquid chromatography.

This report describes the first time entire viral capsids have been purified using fast protein liquid chromatography (FPLC) techniques. The FPLC is used here to separate the two electrophoretic forms of cowpea mosaic virus. The capsid forms are shown to be separated by the Mono-Q column without damaging the capsids.

Molecular cloning and sequence analysis of the human parainfluenza 3 virus RNA encoding the nucleocapsid protein.

The sequence of 1690 nucleotides from the 5' end of the viral complementary RNA for the human parainfluenza 3 virus was determined by molecular cloning. One large open reading frame consisting of 1548 nucleotides was demonstrated. The encoded protein, the nucleocapsid protein (NP), consists of 515 amino acids, and has a predicted molecular weight of 57,819. A noncoding 5' sequence of 51 nucleotides is present at the end of the NP-mRNA. Two consensus sequences were identified which are homologous with sequences found in Sendai virus. One of these sequences, AGGATTAAAG, was located at the 5' end of the nucleocapsid mRNA and may function in transcription initiation. The other consensus sequence, GTAAGGGAA, was found in the viral genomic leader sequence. The nucleocapsid protein amino acid sequence was compared to other members of the Paramyxoviridae family. The parainfluenza 3 virus protein nucleocapsid amino acid sequence demonstrated a high degree of homology with the Sendai virus nucleocapsid protein. Seventy percent of the first 387 amino acids from the amino termini were identical. Little homology was observed in the distal carboxy termini.

Hantaan virus: identification of virion proteins.

SDS-PAGE and immunoprecipitation analyses were carried out on the virion and cell-associated proteins of Hantaan virus, the causative agent of haemorrhagic fever with renal syndrome (HFRS). Purified virions have a density of 1.17 g/ml in sucrose, and contain four proteins with molecular weights of 45 000 (45K), 56K, 72K and 200K, confirming recent evidence that the virus is a member of the family Bunyaviridae. Detergent treatment of virions indicates that the 45K protein is the virus nucleoprotein. Both the 72K and the 56K proteins were labelled with [3H]glucosamine and were removed from virions by bromelain treatment, indicating that they are envelope glycoproteins. The 200K protein was found only in [35S]methionine-labelled preparations. By analogy to prototype viruses of the family Bunyaviridae, these proteins were designated N, G1, G2, and L respectively. Three virus-specific proteins (N, G1, G2) were detected in virus-infected cells. These proteins were precipitable by human convalescent serum and by serum of a Rattus norvegicus trapped in the United States. No additional virus proteins were detected in infected cells. These results confirm recent morphological and RNA studies that Hantaan virus is a member of the family Bunyaviridae. Our results also support the suggestion that Hantaan virus be placed in a new genus of Bunyaviridae.

Structural polypeptides of coronavirus IBV.

Avian infectious bronchitis virus (IBV) was grown and radiolabelled with 35S-methionine, 3H-leucine and 3H-glucosamine in de-embryonated chicken eggs. Approximately 12 different polypeptides were clearly detected by SDS-polyacrylamide gel electrophoresis of virus preparations. Growth of IBV in chorioallantoic membrane cells labelled with 35S-methionine indicated that most of these polypeptides, and additional ones, some of which were glycosylated, were host components. Five polypeptides appeared to be virus-coded, with apparent mol. wt. of 94 x 10(3), 84 x 10(3), 54 x 10(3), 30 x 10(3) and 28 x 10(3). Four of these, p94, p84, p30 and p28, were glycosylated. The virion spikes appeared to be composed of p94 and p84, while p30 and p28 were partially embedded in the virion membrane. By analogy with other reports, p54 is the nucleocapsid polypeptide.