Herbert K. Oie

Reovirus Type 2: Induction of Viral Resistance and Interferon Production in Fathead Minnow Cells

Summary Reovirus type 2 was found to adsorb to cells derived from a poikilothermic animal, the fathead minnow. Although this interaction between cell and virus did not result in the production of infectious virus, viral antigen, or viral inclusion, a state of viral resistance was developed and an antiviral substance was produced. This viral inhibitor was shown to be interferon based on the possession of common characteristics.

Role of lysine in the replication of reovirus

Omission of lysine from the extracellular medium of HeLa cells infected with reovirus type 2 results in the production of virus structures which could be separated by isopycnic sedimentation in CsCl into a top band of empty particles (L-T) and a bottom band (L-B) containing a mixture of defective and complete virus structures. The L-T and L-B particles were found to be similar to the complete virus in the following properties: adsorption characteristics to HeLa cells, RNA base composition, and qualitative distribution of the double-stranded viral RNA segments and structural viral proteins. Polyacrylamide gel electrophoresis analyses of the capsid proteins of L-T and L-B particles indicated a quantitative alteration in the distribution of their protein components. In addition, L-T particles contained markedly reduced amounts of the single-stranded adenine-rich RNA component. The synthesis of viral double-stranded RNA and cytoplasmic proteins were both reduced in lysine-deficient cell cultures. In contrast, the synthesis of single-stranded RNA remained unaltered. Addition of increasing concentrations of lysine to lysine-deficient cultures resulted in increasing yields of infectious virus.

Reovirus Type 2 Production of and Sensitivity to Interferon in Human Amnion Cells (RA)

Summary The antiviral inhibitor substance produced by reovirus type 2 in a stable human amnion cell line (RA) was found to have several properties similar to interferon. It inhibited the growth of inducing virion as well as of heterologous virions, was not sedimentable at 45,000 rpm for 1.5 hours, was not neutralized by reovirus antiserum, was not toxic to cells, was stable upon treatment at pH 2, was inactivated by trypsin, and was ineffective in cells of unrelated species (mouse). These results indicate that the reovirus can induce interferon formation in cells and, like other virions, are also susceptible to the action of the inhibitor.

GROWTH CHARACTERISTICS OF REOVIRUS TYPE 2: ULTRAVIOLET LIGHT INACTIVATED VIRION PREPARATIONS AND CELL DEATH.

Ultraviolet light inactivated preparations of purified reovirus type 2 when added to monolayers of HeLa cells have been found to cause a cytotoxic effect (CTE). At multiplicities of 80–100 infectious units per cell (IU/cell) cellular death due to CTE was initially observed at 3–4 hours post infection and reached maximum 9–10 hours after infection. The cell survival curves suggest that the rate of cell killing was dependent upon the multiplicity of infection and exhibited multiple hit type of kinetics. The CTE was preceded by marked alterations in the synthesis of cellular RNA, DNA, and protein, and not produced by UV-irradiated preparations of uninfected HeLa cell homogenates nor by UV-R2 preparations heated at 56°C for 30 minutes. When UV-R2 preparations were subjected to ultracentrifugation (45,000 r.p.m., 1.5 hours), activity was restricted to the sediment only. In addition, CTE could not be serially transferred and could be prevented by addition of reovirus type 2 antiserum. The data strongly suggest that the toxic factor is an intimate part of the UV-inactivated viral particle.

Growth characteristics and immunocytochemical studies of reovirus type 2 in a line of human amnion cells.

The intracellular development of reovirus type 2 and its interaction with an established human amnion cell line (RA) have been investigated. Adsorption studies indicate that maximal adsorption of the virus to the RA cell was attained within 75–90 minutes after exposure. Single cycle growth studies revealed an eclipse period of 9 hours, and maximal yields were reached at 36 hours post-infection (p.i.). In comparison, the eclipse period for reovirus type 3 in the same cell line was 6 hours, with maximal yields attained at approximately 26 hours p.i. Immunofluorescent and cytochemical (acridine orange) examination of virus-induced alterations in the cell during a single growth cycle of reovirus type 2 revealed the following: a) appearance of virus antigen in the perinuclear region as early as 4 hours p.i. and a maximum number of cells containing antigen by 10 hours p.i.; b) appearance of orthochromatically green-staining perinuclear inclusions at 6 hours p.i. These inclusions were resistant to digestion by ribonuclease and deoxyribonuclease. Metachromatically red-staining inclusions sensitive to ribonuclease were observed to appear in a few cells late in the infectious cycle.

Growth characteristics of reovirus type 2: The intracellular fate of viral RNA

Tritium-labeled reovirus type 2 was adsorbed rapidly and irreversibly to HeLa cells at 37°C. However, if adsorption was carried out at 4°C and the virus-cell complex subsequently incubated at 37° C, more than 25% of the labeled particles was rapidly eluted in 5 minutes. The adsorbed virus remaining at 37°C was stable and no further elution occurred. The eluted virions were found to be biologically and physically unaltered and the elution phenomenon was determined to be dependent on the multiplicity of virus used. In following the fate of the non-eluted adsorbed virion, labeled viral UNA analyzed in sucrose gradients was found to retain its structural integrity throughout the eclipse period. Loss of radioactivity of the viral genome did not occur until about 12 hours after infection, which was several hours after the first appearance of newly-formed progeny.

Studies on reovirus type 2: Viral interference with UV-inactived virus

Ultraviolet light inactivated reovirus type 2 was found to cause cell death by cytotoxicity. The surviving cells developed resistance to virus challenge and produced interferon. Studies using actinomycin D to inhibit interferon production and the development of resitsance suggest that interferon may not play a primary role in the acquisition of resistance by cells exposed to inactivated reovirus type 2.

Synthesis of Reovirus Structural Proteins

Summary A study of the synthesis of structural viral proteins revealed that the majority of the major proteins were synthesized early during the infectious cycle. An exception was major component IIIb which was not made until 4 hr after infection. Analysis of the “soluble“ supernatants from the cytoplasm of infected cells revealed several new nonstructural viral components. This investigation was supported in part by Grant AI 07647 from Natl. Inst. of Allergy and Infect. Dis. and by the Brown-Hazen Fund of the Research Corporation.