Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants

Tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (TEV-GUS) was used for direct observation and quantitation of virus translocation in plants. Four TEV-GUS mutants were generated containing capsid proteins (CPs) with single amino acid substitutions (R154D and D198R), a double substitution (DR), or a deletion of part of the N-terminal domain (delta N). Each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell movement through inoculated leaves. The R154D, D198R and DR mutants were restricted essentially to single, initially infected cells. The delta N variant exhibited slow cell-to-cell movement in inoculated leaves, but was unable to move systemically due to a lack of entry into or replication in vascular-associated cells. Both cell-to-cell and systemic movement defects of each mutant were rescued in transgenic plants expressing wild-type TEV CP. Cell-to-cell movement, but not systemic movement, of the DR mutant was rescued partially in transgenic plants expressing TEV CP lacking the C-terminal domain, and in plants expressing CP from the heterologous potyvirus, potato virus Y. Despite comparable levels of accumulation of parental virus and each mutant in symptomatic tissue of TEV CP-expressing transgenic plants, virions were detected only in parental virus- and delta N mutant-infected plants, as revealed using three independent assays. These data suggest that the potyvirus CP possesses distinct, separable activities required for virion assembly, cell-to-cell movement and long-distance transport.     

Evaluation of a recombinant 27-kDa outer membrane protein of Coxiella burnetii as an immunodiagnostic reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Serological characterization of the 3'-proximal encoded proteins of beet yellows closterovirus

The 3'-proximal open reading frames (ORFs) of beet yellows closterovirus, California isolate (BYV-CA), were sequenced and the expression of the corresponding proteins analyzed. The nucleotide sequence of ORF 5 (coding for p24) was most conserved compared with ORF 7 (coding for p20) and ORF 8 (coding for p21) among the isolates analyzed. Polyclonal antisera were produced to GST fusion proteins of p24, p20, and p21. Accumulation of p24, CP, p20 and p21 was studied in infected Tetragonia expansa plants and Chenopodium quinoa protoplasts. All four proteins were expressed in all tissues (old leaves, young leaves and stems), and most abundantly in young leaves. The subcellular localization of each protein in different tissues showed that compared with p24, CP and p21, p20 accumulated less in transfected protoplasts. Immunogold labeling in sugarbeet with p24 and CP antisera demonstrated co-localization of p24 and CP in vascular petiole tissues. In infectivity neutralization tests, antisera against p24 and CP greatly reduced transmission of BYV by viruliferous aphids compared with viruliferous aphids fed on preimmune serum or antiserum to p21.     

The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.     

Separation used for purification of recombinant proteins

The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.     

Molecular cloning and functional expression of a water-soluble chlorophyll protein, a putative carrier of chlorophyll molecules in cauliflower

A cDNA for a water-soluble chlorophyll (Chl) protein (WSCP) from cauliflower (Brassica oleracea L. var botrys) was cloned and sequenced. The cDNA contained an open reading frame encoding 19 residues for a signal peptide and 199 residues for the mature form of WSCP. The sequence showed extensive homology to drought-stress-related, 22-kDa proteins in some Brassicaceae plants. Functional WSCP was expressed in Escherichia coli as a fusion protein with a maltose-binding protein (MBP). When the recombinant MBP-WSCP was incubated with thylakoid membranes, the MBP-WSCP removed Chls from these membranes. During this process, the monomer of the apo-MBP-WSCP successfully bound Chls and was converted into tetrameric holo-MBP-WSCP. The reconstituted MBP-WSCP exhibited absorption and fluorescent spectra identical to those of the native WSCP purified from cauliflower leaves. The Chl a/b ratio in native WSCP indicates a high content of Chl a, which was mainly due to the higher affinity of MBP-WSCP for Chl a. WSCP is the first example of a hydrophilic protein that can transfer Chls from thylakoid hydrophobic proteins. Possible functions of WSCP are discussed.     

The adenylyl and guanylyl cyclase superfamily

Antibodies and their recombinant fragments have enormous potential for therapy of malignant and other diseases, but there can be problems associated with their production and purification in the quantities required for therapeutic use. We investigated the use of gene therapy for the production of such recombinant antibody fragments in vivo. We generated two recombinant adenoviruses expressing the single chain Fvs (scFvs) fused to murine GM-CSF (mGM-CSF). The scFvs used are MFE-23 which binds to a human tumour-associated marker carcino-embryonic antigen (CEA) and B1.8 which binds the hapten 4-hydroxy-3-nitro-5-iodo-phenylacetyl (NIP). Using scFvs to target GM-CSF to tumour cells should reduce the systemic toxicity of GM-CSF but retain its ability as a cytokine to induce systemic immune responses to tumour targets. Cell lines infected with the recombinant adenoviruses in vitro express and secrete high levels of the scFv.mGM-CSF fusion proteins. The scFv retains specificity while the mGM-CSF portion is fully bioactive and there is no detectable degradation of the fusion product. We also demonstrated effective in vivo expression of the scFv.mGM-CSF proteins. C57BI/6 mice injected intravenously with the adenovirus encoding the MFE-23.mGM-CSF fusion produce high levels of the fusion protein by 2 days after infection. The scFv.mGM-CSF protein can be detected in the serum, at biologically active levels, for at least 20 days and the level of protein produced is related to the amount of adenovirus injected. This approach has the potential to streamline the testing of the many therapeutic strategies based on recombinant scFvs and we are currently testing these constructs in an animal model for antitumour activity.     

Purification and characterization of cysteine proteinase from a baculovirus gene

To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.     

Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.     

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).     

Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.     

The relative biological effectiveness of accelerated carbon ions with different LET for inducing mitotic crossing over and intragenic reversion of the white-ivory allele in Drosophila larvae

We previously described an expression cassette that relies on the tobacco etch virus (TEV) nuclear inclusion a (NIa) protease and leads to the coordinated accumulation of multiple proteins through self processing of a polyprotein [21]. However, low levels of proteins accumulated when the full-length protease was encoded within the polyprotein [22]. Studies were conducted to evaluate whether the disruption of NIa nuclear localization would affect the levels of proteins produced via the cassette. Modifications comprised either removal of its nuclear localization signals (NLSs), removal of the VPg domain (which includes the NLSs), and fusion to the 6 kDa protein, previously demonstrated to be a viral cytoplasmic anchor [28]. In in vitro translation reactions and in vivo protoplast experiments the modified NIa retained sequence-specific proteolysis. Moreover, the removal of the NLSs correlated with an increase in GUS reporter accumulation. The modified cassette, pPRO10, led to the synthesis of up to three viral coat protein (CPs) in addition to NIa. However, the accumulation of proteins in protoplasts depended upon the position of the CP coding sequence within the cassette as well as on the stability of the protein.     

Cloning and characterization of multigenes encoding the immunodominant 30-kilodalton major outer membrane proteins of Ehrlichia canis and application of the recombinant protein for serodiagnosis

A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp. , p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.     

Application and evaluation of male-specific bacteriophage as a process integrity or faecal contamination indicator in a pork slaughterhouse environment

Epitope tagging simplifies detection, characterization and purification of proteins. Gene fusion to combine the coding region of a well-characterized epitope with the coding region for a protein of interest generally requires several subcloning steps. Alternatively, a PCR strategy can be used to generate such a chimeric gene. In addition to its simplicity, this approach allows one to limit the size of the multiple cloning sites present in conventional expression vectors, thus reducing the introduction of artifactual amino-acid sequences into the fused protein. In this communication, we describe new vectors that allow PCR cloning and selection of chimeric genes coding for N- or C-terminal His-tagged proteins. These vectors are based on the control of cell death CcdB direct selection technology and are well adapted to the cloning of blunt-ended PCR products that were generated by using thermostable polymerases that provide proofreading activity.     

Clinical gait analysis in a rehabilitation context: some controversial issues

The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin alpha-sarcin to be used for immunoconjugate production. alpha-Sarcin cDNA was isolated from Aspergillus giganteus strain MDH 18,894 and its expression in Escherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant alpha-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernatant. A variant form of alpha-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.     

Assembly and movement of a plant virus carrying a green fluorescent protein overcoat

Potato virus X (PVX) is a filamentous plant virus infecting many members of the family Solanaceae. A modified form of PVX, PVX.GFP-CP which expressed a chimeric gene encoding a fusion between the 27-kDa Aequorea victoria green fluorescent protein and the amino terminus of the 25-kDa PVX coat protein, assembled into virions and moved both locally and systemically. The PVX.GFP-CP virions were over twice the diameter of wild-type PVX virions. Assembly of PVX.GFP-CP virions required the presence of free coat protein subunits in addition to the fusion protein subunits. PVX.GFP-CP virions accumulated as paracrystalline arrays in infected cells similar to those seen in cells infected with wild-type PVX The formation of virions carrying large superficial fusions illustrates a novel approach for production of high levels of foreign proteins in plants. Aggregates of PVX.GFP-CP particles were fluorescent, emitting green light when excited with ultraviolet light and could be imaged using confocal laser scanning microscopy. The detection of virus particles in infected tissue demonstrates the potential of fusions between the green fluorescent protein and virus coat protein for the non-invasive study of virus multiplication and spread.     

Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: use in differential diagnosis between vaccinated and infected animals

Vesicular stomatitis is a viral disease of cattle, pigs, and horses. The disease is characterized by vesicular lesions on the epithelium of the mouth, feet, and teats. The pathological lesions are virtually indistinguishable from that of foot-and-mouth disease. We have developed a recombinant baculovirus that expresses the nucleocapsid (N) protein of the New Jersey serotype of vesicular stomatitis virus (VSVNJ) in insect cells (Sf9) and larvae (Spodoptera exigua). The gene was expressed under control of the polyhedrin promoter as a fusion or nonfusion protein. The recombinant N protein expressed in insect cells could not be distinguished from N protein produced in VSVNJ-infected CHO cells by immunological and biochemical analyses. The level of expression of N as a percentage of the total protein in Sf9 cells was 41% for the fusion and 60% for the nonfusion protein. Higher level (68%) of expression of the nonfusion N protein was obtained in larvae. Recombinant N protein was used in an ELISA to distinguish animals vaccinated with a recombinant VSV glycoprotein from those exposed to the whole virus by infection or classical vaccine. Lysate of a single infected larva (0.2-0.3 g) was adequate for coating ELISA plates to perform 10,000 serum assays in duplicate.     

GFP:HIV-1 protease production and packaging with a T4 phage expression-packaging processing system

A bacteriophage T4-derived protein expression, packaging and processing system was used to create recombinant phage that encode, produce and package a protein composed of human HIV-1 protease fused to green fluorescent protein (GFP). The fusion protein is targeted within the phage capsid by an N-terminal capsid targeting sequence (CTS), which is cleaved through proteolysis by the viral scaffold protease P21. The fusion protein is designated CTS [symbol see text] GFP:PR. The [symbol see text] symbol indicates the linkage peptide sequence leu(ile)-N-glu that is cleaved by the T4 head morphogenetic proteinase gp21 during head maturation. The fusion protein is fluorescent and has protease activity as detected by the appearance of the expected substrate cleavage product on a Western blot. CTS [symbol see text] GFP:PR packaging occurs at about 200 molecules per phage particle. The CTS [symbol see text] GFP:PR fusion protein, when protected within the phage capsid, has been maintained stably for over 16 months at 4 degrees C. Production and storage of fusion protein within the phage circumvents problems of toxicity and solubility encountered with E. coli expression systems. Because recombinant phage inhibit host proteolytic enzymes, foreign proteins are stabilized. This phage system packages and processes the fusion protein by means of the CTS. Proteins can be purified from the phage to give high yields of soluble, proteolytically processed protein. The T4 phage packaging system provides a novel means of identification, purification and long-term storage of toxic proteins whose folding and DNA-directed activities can be studied readily in vivo.     

Expression of measles virus V protein is associated with pathogenicity and control of viral RNA synthesis

Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314-322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V-), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats (Sigmodon hispidus) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.