Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cellular proteins. Ten gene products (P1–P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non-producing mutants. N-terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two-dimensional gels. Protein P8 was identified as l -histidine amino-transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligo-nucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb Bam HI fragment and a 6.5 kb Pvu II fragment, respectively, from the genome of Streptomyces tendae Tü901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb Bam HI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene-disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes Involved in nikkomycin production.     

Deletion mutants of Moloney murine leukemia virus which lack glycosylated gag protein are replication competent

A series of deletion mutations localized near the 5' end of the Moloney murine leukemia virus genome was generated by site-specific mutagenesis of cloned viral DNA. The mutants recovered from such deleted DNAs failed to synthesize the normal glycosylated gag protein gPr80gag. Two of the mutants made no detectable protein, and a third mutant, containing a 66-base pair deletion, synthesized an altered gag protein which was not glycosylated. All the mutants made normal amounts of the internal Pr65gag protein. The viruses were XC positive and replicated normally in NIH/3T3 cells as well as in lymphoid cell lines. These results indicate that the additional peptides of the glycosylated gag protein are encoded near the 5' end, that the glycosylated and internal gag proteins are synthesized independently, and that the glycosylated gag protein is not required during the normal replication cycle. In addition, the region deleted in these mutants apparently encodes no cis-acting function needed for replication. Thus, all essential sequences, including those for packaging viral RNA, must lie outside this area.     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral El A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant d1312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1 B genes.     

Genetic and molecular analyses of Escherichia coli K1 antigen genes

The plasmid pSR23, composed of a 34-kilobase E. coli chromosomal fragment inserted into the BamHI site of the pHC79 cosmid cloning vector, contains genes encoding biosynthesis of the K1 capsular polysaccharide. Deletions, subclones, and Tn5 insertion mutants were used to localize the K1 genes on pSR23. The only deletion derivative of pSR23 that retained the K1 phenotype lacked a 2.7-kilobase EcoRI fragment. Subclones containing HindIII and EcoRI fragments of pSR23 did not produce K1. Cells harboring pSR27, a subclone containing a 23-kilobase BamHI fragment, synthesized K1 that was not detectable extracellularly. Six acapsular Tn5 insertion mutants of three phenotypic classes were observed. Class I mutants synthesized K1 only when N-acetylneuraminic acid (NANA) was provided in the medium. Reduced amounts of K1 were detectable in cell extracts of class II mutants. Class III mutants did not produce detectable K1 in either extracts or when cells were provided exogenous NANA. All mutants had sialyltransferase activity. Analysis in the E. coli minicell system of proteins expressed by derivatives of pSR23 identified a minimum of 12 polypeptides, ranging in size from 18,000 to 80,000 daltons, involved in K1 biosynthesis. The 16-kilobase coding capacity required for the proteins was located in three gene clusters designated A, B, and C. We propose that the A cluster contains a NANA operon of two genes that code for proteins with apparent molecular weights of 45,000 and 50,000. The A region also includes a 2-kilobase segment involved in regulation of K1 synthesis. The B region encoding five protein species appears responsible for the translocation of the polymer from its site of synthesis on the cytoplasmic membrane to the cell surface. The C region encodes four protein species. Since the three gene clusters appear to be coordinately regulated. we propose that they constitute a kps regulon.     

Detection of CaMV gene I and gene VI protein products in vivo using antisera raised to COOH-terminal β-galactosidase fusion proteins

Specific antisera were prepared to the inclusion body protein (gene VI product) and the gene I product of cauliflower mosaic virus (CaMV). Translational fusions between the lacZ gene and gene VI or gene I were constructed by cloning the relevant DNA fragments into the expression vectors pUR290, pUR291 or pUR292. Large amounts of fusion protein were synthesized when the inserted DNA fragment was in frame with the lacZ gene of the expression vector. These fusion proteins were used to raise specific antisera to gene VI and gene I proteins of CaMV. Antiserum to the gene VI product detected a range of proteins in crude extracts and in a subcellular fraction enriched for virus inclusion bodies. This range of proteins was further shown to be related to gene VI by Staphylococcus aureus V8 partial proteolysis. Antiserum to the gene I product detected viral specific proteins of 46, 42 and 38 K in preparations of CaMV replication complexes from infected plants but not in any other subcellular fraction.     

Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.     

Envelopment of Sindbis Virus: Synthesis and Organization of Proteins in Cells Infected with Wild Type and Maturation-Defective Mutants

The synthesis and organization of Sindbis virus structural proteins was investigated in BHK cells infected with wild-type virus (SVHR) or temperature-sensitive (ts) mutants defective in maturation. Cells infected with ts-23 or ts-20 (complementation groups D and E) were similar in the polypeptides synthesized at the nonpermissive temperature and differed from SVHR-infected cells in that the envelope protein E2 was not cleaved from the PE2 precursor. Data from experiments utilizing pulse-chase procedures or protein synthesis inhibitors indicated that although infectious virions were released from cells infected with these mutants in shift-down experiments, the particles were produced almost exclusively from proteins synthesized after the return to permissive temperature. This suggests that a stable complex may be formed among the structural proteins before budding. A membrane fraction isolated from cells infected with either ts mutants or SVHR contained the PE2, E1, and C polypeptides, whereas E2 was restricted to fractions obtained from SVHR-infected cells. Although equivalent amounts of virus-specific protein were synthesized in cells infected with either mutant and the cells contained qualitatively the same proteins in the isolated membranes, cells infected with ts-23 did not have virus-specific proteins exposed on their surface that could be detected by ferritin-conjugated antibody-labeling procedures or lactoperoxidase-mediated iodination. In contrast, ts-20-infected cells had significant amounts of viral protein, mainly E1, that could be detected on the plasma membrane by either procedure. Iodine was incorporated into E1 and E2 on the surface of SVHR-infected cells in the same relative amounts as seen in iodinated virions. PE2, however, although present in membranes, could not be iodinated on the surface of infected cells under any of the conditions used in this study. We also monitored the relative efficiency with which these viral proteins could be removed from intact cells by dilute solutions of nonionic detergents. The results indicated that E2 was most efficiently removed, followed by E1. PE2 (the precursor to E2) and C remained associated with the cell and could be subsequently isolated in the membrane fraction.     

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses

Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.     

Envelope Proteins of Vesicular Stomatitis Virus: Effect of Temperature-Sensitive Mutations in Complementation Groups III and V

All five major viral proteins were synthesized in chicken embryo cells infected with vesicular stomatitis virus temperature-sensitive (ts) mutants of complementation groups III and V and maintained at the nonpermissive temperature. The distribution of these proteins among cytoplasmic cellular fractions separated on discontinuous sucrose gradients was identical for wild-type and tsIII-infected cells. Strikingly different patterns were observed for the G protein in gradients from cells infected by tsV mutants; very little, if any, G protein was found in the lightest fraction. Pulse and chase experiments with wild-type, virus-infected cells showed that protein G moves from the heaviest to the lightest fraction before being incorporated into the virion. After shift down to the permissive temperature (30 C), G protein synthesized at 39.6 C in tsV-infected cells became associated with the lightest cellular fraction and later with the released virions. In contrast, M protein, synthesized at 39.6 C in tsIII-infected cells, was not incorporated into the virions after shift down. These data strongly suggest, first, that M protein is encoded by the vesicular stomatitis gene III, and second, that incorporation of G protein in the lightest cellular fraction is a necessary step of vesicular stomatitis maturation. This step is impaired by tsV mutations.     

The relationship between group C adenovirus tumor antigen and the adenovirus single-strand DNA-binding protein

The group C adenoviruses code for a single-strand specific DNA-binding protein of molecular weight 72,000 daltons which is synthesized at early times after productive viral infection. Experiments were designed to determine whether this single-strand specific DNA-binding protein was expressed in adenovirus tumors and transformed cells.Two independently derived preparations of antisera from hamsters bearing group C adenovirus tumors were tested for antibody against the single-strand DNA-binding proteins. One antiserum contained antibodies that reacted with these DNA-binding proteins, while the second antiserum did not contain detectable levels of antibody. Five adenovirus type 2 transformed rat cell lines were tested for the presence of the single-strand specific DNA-binding proteins. Two of the five transformed cells expressed detectable levels of this protein. These results indicate that the group C adenovirus single-strand specific DNA-binding proteins are expressed in some, but not all, adenovirus tumors and transformed cell lines.Those transformed cell lines (type 2) containing a portion of the adenovirus genome designated by the Eco R-I-B restriction enzyme fragment express the single-strand specific DNA-binding proteins. Those cell lines missing this Eco R-I-B fragment do not contain this viral protein. Other experiments have located the structural gene of the single-strand specific DNA-binding protein in the Eco R-I-B DNA fragment, indicating that when this gene is present in a transformed cell, it is expressed.     

Association between flower stalk elongation,an Arabidopsis developmental trait,and the subcellular location and movement dynamics of the nonstructural protein P3 of Turnip mosaic virus

Virus infections affect plant developmental traits but this aspect of the interaction has not been extensively studied so far. Two strains of Turnip mosaic virus differentially affect Arabidopsis development, especially flower stalk elongation, which allowed phenotypical, cellular, and molecular characterization of the viral determinant, the P3 protein. Transiently expressed wild-type green fluorescent protein-tagged P3 proteins of both strains and selected mutants of them revealed important differences in their behaviour as endoplasmic reticulum (ER)-associated peripheral proteins flowing along the reticulum, forming punctate accumulations. Three-dimensional (3D) model structures of all expressed P3 proteins were computationally constructed through I-TASSER protein structure predictions, which were used to compute protein surfaces and map electrostatic potentials to characterize the effect of amino acid changes on features related to protein interactions and to phenotypical and subcellular results. The amino acid at position 279 was the main determinant affecting stalk development. It also determined the speed of ER-flow of the expressed proteins and their final location. A marked change in the protein surface electrostatic potential correlated with changes in subcellular location. One single amino acid in the P3 viral protein determines all the analysed differential characteristics between strains differentially affecting flower stalk development. A model proposing a role of the protein in the intracellular movement of the viral replication complex, in association with the viral 6K2 protein, is proposed. The type of association between both viral proteins could differ between the strains.     

The region of human transferrin involved in binding to bacterial transferrin receptors is iocalized in the C-lobe

Iron-saturated human transferrin was digested with either chymotrypsin or trypsin to produce C-lobe and N-lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C-lobe and N-lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C-lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transterrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophlius influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin-binding proteins (Tbps), we demonstrated that both transferrin-binding proteins were able to bind to the C-lobe fragment of human transferrin.     

Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides.

At least 10 distinct early virus-induced polypeptides were synthesized within 0 to 6 h after infection of permissive cells with cytomegalovirus. These virus-induced polypeptides were synthesized before and independently of viral DNA replication. A majority of these early virus-induced polypeptides were also synthesized in nonpermissive cells, which do not permit viral DNA replication. The virus-induced polypeptides synthesized before viral DNA replication were hypothesized to be nonstructural proteins coded for by the cytomegalovirus genome. Their synthesis was found to be a sequential process, since three proteins preceded the synthesis of the others. Synthesis of all early cytomegalovirus-induced proteins was a transient process; the proteins reached their highest molar ratios before the onset of viral DNA replication. Late viral proteins were synthesized at the time of the onset of viral DNA replication, which was approximately 15 h after infection. Their synthesis was continuous and increased in molar ratios with the accumulation of newly synthesized viral DNA in the cells. The presence of the amino acid analog canavanine or azetadine during the early stage of infection suppressed viral DNA replication. The amount of viral DNA synthesis was directly correlated to the relative amount of late viral protein synthesis. Because synthesis of late viral proteins depended upon viral DNA replication, the proteins were not detected in permissive cells treated with an inhibitor of viral DNA synthesis or in nonpermissive cells that are restrictive for cytomegalovirus DNA replication.     

Amino acid replacements in the Serratia marcescens haemolysin ShlA define sites involved in activation and secretion

The haemolysin of Serratia marcescens (ShlA) is translocated through the cytoplasmic membrane by the signal peptide-dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShlA is haemolytic. ShIB also converts in vitro inactive ShlA (ShlA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShlA involved in both processes, ShlA derivatives were isolated and tested for secretion and activation. Analysis of C-terminally truncated proteins (ShlA) assigned the secretion signal to the amino-terminal 238 residues of ShlA. Trypsin cleavage of a secreted ShlA' derivative yielded a 15kDa N-terminal fragment, by which a haemolytically inactive ShlA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N-terminal fragment. Replacement of asparagine-69 and asparagine-109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala-Asn-Pro-Asn, which are critical to both activation and secretion. These point mutants as well as N-terminal deletion derivatives which were not activated by ShIB were activated by adding a non-haemolytic N-terminal fragment synthesized in an ShIB⁺ strain (complementation). Apparently the activated N-terminal fragment substituted for the missing activation of the ShlA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.