Interference with Virus Infection Induced by RNA in Chick Embryo Cells

Abstract

Some single-stranded RNA's, notably RNA from E. coli bacteriophage MS2, were found to induce interference with viral growth (Sindbis, vesicular stomatitis, and vaccinia viruses) in chick embryo cell culture. Oligonucleotides appear to have a more essential role for induction of interference than high molecular weight RNA, since (a) phage RNA preparations containing degraded components are more consistently effective than preparations consisting essentially of intact molecules, (b) chick embryo RNA (homologous to the cells), ineffective in itself, becomes strongly effective after digestion with T1 RNase, and (c) calf liver RNA, which gives variable results, becomes reproducibly effective after T1 RNase digestion. Certain structural specificities of oligonucleotides are required, since (a) digests by pancreatic RNase, (b) Ti RNase digests of E. coli and yeast RNA's, and (c) mononucleotides, are all ineffective or only slightly effective. Various characteristics of the interference observed indicate that the RNA does not directly inactivate free virus but inhibits viral replication, most probably by inducing some cellular processes unfavorable for viral growth. Release of interferon into the culture medium during or after RNA treatment was examined, but in many cases only low titers or none of interferon activity were observed. Experiments on Newcastle disease virus, a virus relatively insensitive to interferon, yielded variable results as to its sensitivity to RNA-induced interference. Thus the role of interferon in the RNA-induced interference described here remains unsettled.

RNA‐Induced Interference with Animal Virus Infection

“…We have independently observed that, not only double-stranded RNAs, but also some kinds of single-stranded RNAs, and oligonucleotides may also interfere with virus multiplication in chick embryo cell cultures [4,5]. The relationship between our findings and those of other workers on doublestranded RNAs is not yet clear, though they may possibly be closely related, and we have investigated the further characterization of the interference phenomenon caused by single-and double-stranded RNAs in chick cells.…”

“…Interferon induced in chick cell cultures by infection with SbV was used. The details have been described in our previous paper [4].…”

“…Poly C and poly I were purchased from Sigma Chemical Co., and were used as an equimolar mixture. For more details, see [4]. Chemicals.…”

“…Confluent cell sheets were treated at 37 C for various periods with RNA or interferon in serum-free Eagle's medium, without streptomycin, containing double strength vitamins and amino acids, in the presence of protamine 10 g/m1 [4]. The degree of cellular resistance to virus was determined either by plaque reduction as described previously [4], or by reduction in virus yield after one-step growth.…”

“…Confluent cell sheets were treated at 37 C for various periods with RNA or interferon in serum-free Eagle's medium, without streptomycin, containing double strength vitamins and amino acids, in the presence of protamine 10 g/m1 [4]. The degree of cellular resistance to virus was determined either by plaque reduction as described previously [4], or by reduction in virus yield after one-step growth. For the latter test, the cultures were washed twice with balanced salt solution, challenged by SbV with an input multiplicity of infection of 1-10 PFU (plaque-forming unit) per cell, washed four times after an adsorption period of 1 hr, incubated at 37 C for 8-12 hr, and then freeze-thawed.…”