Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.     

Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif.

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.     

Sensitivity of some common grain fungi to irradiation on grain and in phosphate-buffered saline

The sensitivity of nine cereal fungi to irradiation on grain and in phosphate-buffered saline (PBS) was investigated. Species of Fusarium and Alternaria were more resistant to irradiation (higher D ₁₀ value) than Aspergillus spp. and Penicillium spp. Generally D ₁₀ values determined on grain were lower than the corresponding values measured in PBS. The plating medium did not significantly affect the D ₁₀ values.     

A calmodulin and alpha-subunit binding domain in human erythrocyte spectrin

Human erythrocyte spectrin binds calmodulin weakly under native conditions. This binding is enhanced in the presence of urea. The site responsible for this enhanced binding in urea has now been shown to reside in a specific region of the spectrin beta-subunit. Cleavage of spectrin with trypsin, cyanogen bromide or 2-nitro-5-thiocyanobenzoic acid generates fragments of the molecule which retain the ability to bind calmodulin under denaturing conditions. The origin of these fragments, identified by two-dimensional peptide mapping, is the terminal region of the spectrin beta-IV domain. The smallest peptide active in calmodulin binding is a 10 000 Mr fragment generated by cyanogen bromide cleavage. Only the intact 74 000 Mr fragment generated by trypsin (the complete beta-IV domain) retains the capacity to reassociate with the isolated alpha-subunit of spectrin. The position of a putative calmodulin binding site near a site for subunit-subunit association and protein 4.1 and actin binding suggests a possible role in vivo for calmodulin regulation of the spectrin-actin membrane skeleton or for regulation of subunit-subunit associations. This beta-subunit binding site in erythrocyte spectrin is found in a region near the NH2-terminus at a position analogous to the alpha-subunit calmodulin binding site previously identified in a non-erythroid spectrin by ultrastructural studies.     

Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.     

Pathogenesis of the delayed phase of Rauscher virus-induced thrombocytopenia

BALB/c (H-2d) mice infected with Rauscher murine leukemia virus (RMuLV) developed two phases of thrombocytopenia: an acute phase, probably due to direct virus-platelet interactions, and a delayed phase, starting 2 to 3 wk after virus injection, which was associated with the infection of megakaryocytes by RMuLV and with the expression of RMuLV gp70 and p30 antigens on platelet membranes. This study was concerned with the pathogenesis of this second phase of thrombocytopenia. During this period, the number of marrow megakaryocytes was increased. A peripheral platelet destruction was further indicated by reduced platelet life span. It was shown that radiolabeled platelets, either normal or infected, were submitted to a more rapid clearance in infected recipients than in normal recipients. This might be due to the splenomegaly observed in infected recipients. However, the immediate clearance of gp70+ platelets was more accelerated in infected recipients with high titers of serum anti-gp70 antibodies than in infected recipients without detectable serum anti-gp70 antibodies. In addition, the passive transfer of anti-RMuLV serum to normal BALB/c mice induced a rapid and specific clearance of previously injected radiolabeled platelets expressing RMuLV antigens. In H-2d mice, viral gp70 antigen expression on platelets correlated with the development of delayed thrombocytopenia; but H-2k strains of mice, although susceptible to RMuLV and expressing RMuLV-related antigens on their platelets, did not develop any anti-RMuLV antibodies nor any delayed thrombocytopenia. These results suggest that specific clearance of gp70+ platelets in the presence of significant amounts of serum antiviral antibodies and nonspecific hypersplenism play a role in the development of delayed thrombocytopenia in RMuLV-infected mice.     

Structure of the murine serum amyloid A gene family. Gene conversion

Serum amyloid A (SAA) is an apolipoprotein produced by the liver in response to inflammation; the levels of SAA mRNA and SAA protein increase at least 500-fold within 24 h. We have obtained clones of all three genes and pseudogene that make up the murine SAA gene family. Two of the genes have 96% sequence homology over their entire length, including introns and flanking sequences 288 base pairs (bp) 5' and 443 bp 3' to the genes: an overall length of 3215 bp. The sharp boundaries between homologous and nonhomologous sequences and the absence of interspersed repeated sequences there suggest that conversion has occurred between these two genes. The homologous regions are bounded by short inverted repeats containing alternating purine and pyrimidine residues, as described for other gene conversion units. The third SAA gene has evolved separately, although all are closely linked on chromosome 7. Comparison of the upstream regions of the SAA genes with those of the rat fibrinogen genes, whose expression is also induced by inflammation, reveals sequences common to all six genes which are very improbable on a random basis.     

A chimaeric hygromycin resistance gene as a selectable marker in plant cells

Summary We show here that plant cells are sensitive to the antibiotic hygromycin-B⁴. We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.