Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.     

Interaction of calmodulin with the particulate fraction of cardiac muscle

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.     

Phosphorylation site of eukaryotic initiation factor 4E

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) was labeled in situ with [32P]orthophosphate in cultured HeLa cells and rabbit reticulocytes and purified by affinity chromatography. Tryptic digestion yielded one labeled peptide which contained predominantly serine and lysine. After treatment of the protein with citraconic anhydride to block epsilon-amino groups of lysyl residues, tryptic digestion yielded a labeled peptide whose composition was consistent with the structure Trp-Ala-Leu-Trp-Phe-Phe-Lys-Asn-Asp-Lys-Ser(P)-Lys-Thr-Trp-Gln-Ala-Asn-L eu-Arg, one of the arginyl peptides predicted from the human eIF-4E cDNA sequence. The only serine in this peptide is located at position 53 of eIF-4E. Thus, it is concluded that eIF-4E contains a single site of phosphorylation for an endogenous protein kinase, which is Ser-53 in the human eIF-4E sequence.     

Demonstration of immunoreactive forms of erythrocyte protein 4.2 in nonerythroid cells and tissues

Protein 4.2 is a major component of the erythrocyte membrane cytoskeleton. Here we show that immunoreactive forms of human (Mr 72,000) and pig (Mr 75,000) protein 4.2 are also associated with the plasma membrane of various nonerythroid cells and tissues, such as platelets, brain, and kidney. Protein 4.2 can be extracted from platelet membranes under the same conditions (pH 11, 1 M KI, 1 M urea) which are required to extract protein 4.2 from the erythrocyte plasma membrane. The demonstration of protein 4.2 in nucleated cells that contain also several other proteins of the erythrocyte membrane cytoskeleton indicates some general principles underlying the molecular construction of the plasma membrane in erythrocytes and nonerythroid cells.     

Joining of linear plasmid DNA is reduced and error-prone in Bloom''s syndrome cells.

A linearized, replicating, shuttle vector plasmid, pZ189, was used to measure in vivo DNA joining ability of cells from patients with the cancer-prone, immunodeficient, chromosome breakage disorder, Bloom's syndrome (BS). The BS cell lines we studied were reported to contain reduced in vitro activity of DNA ligase I. We assessed in vivo joining ability by transfecting linear plasmids with overlapping or blunt ends (produced by EcoRI or StuI) into BS and normal fibroblast or lymphoblast host cells and measuring the amount of re-joined, replicated plasmids by their ability to transform bacteria. With plasmids having either overlapping or blunt ends we found a 1.3- to 3-fold lower (P less than 0.05) joining efficiency in BS cells than in the normal cells. The mutation frequency of the recovered plasmids was measured by screening for function of the suppressor tRNA contained in pZ189, for plasmid size, for presence of restriction sites, or by DNA sequencing. The spontaneous mutation frequency with the circular plasmid was 0.05-0.08% with both BS cell lines, values 2- to 21-fold higher (P less than 0.03) than with the normal cell lines. The mutation frequency with the linear plasmid passaged through both BS cell lines was 21-52%, values 1.4- to 5.4-fold higher (P less than 0.001) than with the normal lines. Detailed analysis of 210 recovered plasmids revealed an increase (P less than or equal to 0.001) in deletions, insertions or complex mutations at the joining sites, and in point mutations with the EcoRI cut plasmid with the BS cells in comparison to the normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA.

A method is presented for choosing optimal oligodeoxyribonucleotides as probes for filter hybridization, primers for sequencing, or primers for DNA amplification. Three main factors that determine the quality of a probe are considered: stability of the duplex formed between the probe and target nucleic acid, specificity of the probe for the intended target sequence, and self-complementarity. DNA duplex stability calculations are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. [Proc. Natl. Acad. Sci. U.S.A. (1986), 83: 3746]. Temperatures of duplex dissociation predicted by the method described here were within 0.4 degrees C of the values obtained experimentally for ten oligonucleotides. Calculations for specificity of the probe and its self-complementarity are based on a simple dynamic algorithm.     

Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)