Peter J. Gomatos

Reovirus Type 3: Physical Characteristics and Interaction with L Cells.

Abstract The physical characteristics of reovirus type 3 and its interaction with L cells have been investigated. After repeated passage of the Dearing strain in L cells, a variant emerged which was characterized by decreased sensitivity to specific antibody and by diminished release from cells. The original and variant strains were not significantly different in their antigenic constitution, size, or fine structure. The amount of each which adsorbed to L cells in 2 hours was approximately 80% of that adsorbed in 4 hours. The latent period was 7 hours and the maximal yields were reached at 15 hours with both the original and the variant strains. Both caused the development of a perinuclear inclusion in infected cells. The inclusion was Feulgen negative; it contained viral antigenic material. Both strains were insensitive to inhibition by 5-fluoro-2′-deoxyuridine and 5-bromo-2′-deoxyuridine, whereas vaccinia virus was markedly inhibited. Mitomycin C and actinomycin D inhibited reovirus to a lesser extent than vaccinia virus under most conditions used. However, after pretreatment of cells with actinomycin D, their ability to produce reo or vaccinia viruses was restricted to a similar degree. 5,6-Dibromo-1-β- d -ribofuranosylbenzimidazole and aminopterin inhibited the original Dearing strain and vaccinia virus to a similar degree. The variant strain was slightly less sensitive to the inhibitors than the original. The nucleic acid of reovirus 3 appears to be of the ribose type.

LPS-induced cytokine production in human dendritic cells is regulated by sialidase activity

Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS‐induced cytokine production in human monocyte‐derived DCs. Of the four genetically distinct sialidases (Neu1–4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up‐regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS‐ induced expression of IL‐6, IL‐12p40, and TNF‐α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1–/– and WT mice. We conclude that sialidase‐mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS‐induced cytokine production, thereby contributing to development of adaptive immune responses.

Reactive Sites of Reovirus Type 3 and their Interaction with Receptor Substances.

Abstract Reactions of reovirus type 3 with erythrocytes, serum components, ovomucin, and L cells were investigated. In most of the experiments, a variant of the Dearing strain was used which differs from the original only in a few properties. The variant, as well as the original Dearing strain, agglutinated ox erythrocytes. Treatment of erythrocytes with Vibrio cholerae filtrate rendered them inagglutinable by these viruses. One hemagglutinating unit of the variant strain corresponded to 6.2 × 10 6 PFU. Normal mouse, rabbit, rat, and calf sera, in order of decreasing activity, inhibited agglutination of ox erythrocytes by reovirus 3. Fetal calf serum showed no inhibitory activity. Treatment of normal mouse, rabbit, and rat sera with V. cholerae filtrate destroyed hemagglutination-inhibiting activity. Ovomucin also possessed hemagglutination-inhibiting activity which was eliminated by V. cholerae filtrate and Lee virus, but not by reovirus 3. Reovirus type 2, Amy strain, did not agglutinate ox erythrocytes, and agglutination of human O cells by the Amy strain was not inhibited by ovomucin. The inhibitory activity of normal animal sera on agglutination of human O cells by this virus was not affected by V. cholerae filtrate. Trypsin and p -chloromercuribenzoate (PCMB) reduced the hemagglutinating activity and infectivity of reovirus 3. PCMB did not disrupt virus particles as shown by negative staining. Clearly, reovirus 3-receptor interaction possesses characteristics which distinguish it from the interactions between receptors and reovirus 2, myxo, entero, and polyoma viruses.

Semliki Forest Virus Replication Complex Capable of Synthesizing 42S and 26S Nascent RNA Chains

A complex synthesizing Semliki Forest virus (SFV)-specific RNAs was purified from infected HeLa cells. During purification, the RNA-synthesizing complex was monitored by the presence of RNA chains synthesized during a 1 min pulse in vivo and the ability to synthesize 42S and 26S RNAs in vitro. Finally, the protein composition of the replication complex was analysed. Thirty to 40% of the pulse-labelled RNAs and 10 to 25% of the polymerase activity present in the postnuclear supernatant were recovered in smooth membranes. At this stage of purification single stranded 42S and 26S RNA were synthesized and released from the replication complex in vitro. After treatment of the smooth membrane fraction with Triton X-100 the replication complex was solubilized. When analysed by sucrose gradient centrifugation, the solubilized replication complex distributed heterogeneously. It had reduced RNA polymerase activity, but was still able to synthesize both 42S and 26S nascent RNA chains which were not released from RIs and RFs. The non-structural protein ns70 was the major virus-specified component associated with the replication complex.

Agglutination of Bovine Erythrocytes : a General Characteristic of Reovirus Type 3.

Summary 1. Reovirus type 3 strains, originally isolated from different animal species, all agglutinate bovine erythrocytes; only some agglutinate human erythrocytes. 2. None of the strains of reovirus types 1 and 2 agglutinate bovine erythrocytes; they all agglutinate human erythrocytes.

Base Composition of the RNA of a Reovirus Variant

A variant of reovirus 3, Dearing strain, isolated after repeated pdssage of the original Dearing virus in L cells, is similar to the parent virus in many ways. It is, however, less sensitive to specific antibodies and metabolic inhibitors, and is released from L cells to a lesser extent than the original virus. Calculated as moles per 100 moles of total base in RNA, the percentage of guanine is 20.2, adenine, 29.8, cytosine, 21.0, and uracil 29.1. These values closely approximate those reported previously for the parent virus.

Reovirus RNA-directed synthesis of DNA: I. The reaction catalyzed by DNA polymerase from Escherichia coli

Reovirus RNA is an effective template in DNA synthesis catalyzed by DNA polymerase from Escherichia coli B. The product has a base composition similar to that of the reovirus RNA primer and has nearest-neighbor sequence frequencies similar to those in the complementary RNA product of the reovirus RNA-primed RNA polymerase reaction. There is no evidence of deoxyribonucleotide homo-polymer synthesis in the DNA polymerase reaction. The DNA product collected after 30 minutes of incubation is largely double-stranded and maintains a denatured state after heating and rapid cooling. It has not yet been established whether there is first formed on the RNA template a single-stranded DNA, which on liberation from the double-stranded RNA template is preferentially copied to produce a fully denaturable double-stranded DNA, or whether the product formed on the double-stranded RNA template is initially double-stranded DNA. As is the case in DNA-primed reactions, actinomycin D inhibits reovirus RNA-primed incorporation of deoxyribonucleotides into DNA with DNA polymerase as enzyme to a lesser extent than the reovirus RNA-primed incorporation of ribonucleotides into RNA with RNA polymerase as enzyme. With ribonucleoside triphosphates as precursors, there is little or no RNA synthesis in the reovirus RNA-primed DNA polymerase reaction.

Enzymic synthesis of RNA with reovirus RNA as template: II. Nearest neighbor analysis of the products of the reaction catalyzed by the Escherichia coli RNA polymerase

Reovirus RNA is an effective template for the Escherichia coli RNA polymerase ( Gomatos, Krug & Tamm, 1964 ). The products of this reaction were characterized by nearest neighbor analysis. The base composition derived from the nearest neighbor analysis of the product obtained after four minutes of synthesis agrees with the base composition of the template, suggesting faithful copying of the nucleotide sequences of both strands of the template by the enzyme. The nearest neighbor sequence frequencies indicate that the chains in reovirus RNA are anti-parallel. These frequencies also show a deviation from randomness characteristic of DNA from animal and plant cells and not shown by DNA from bacteria and bacterial viruses. The nearest neighbor sequence frequencies of the products isolated after 120 minutes of incubation differ from those of the four-minute product. A is next to A and U is next to U more frequently at 120 minutes than at four minutes, whereas the neighbors next to G and to C do not change. Thus, at later times, there is continued synthesis of the product complementary to reovirus RNA, and also synthesis of polyadenylate and polyuridylate.