Studies in the replication of viral RNA

Abstract

Experiments will be described which demonstrate that the product of purified "Q@replicase" is fully competent to serve both as a template and as a program for the synthesis of complete virus particles. The use of mutant RNA has permitted the demonstration that nucleic acid is the instructive agent in the replication process and hence satisfies the definition of a self-duplicating entity. Methods have been devised to examine the product both physically and biologically with a minimum of manipulation and with complete recovery of product and input template. A detailed analysis of every interval of synthesis has thus become possible. These technical advances have permitted us to demonstrate the existence of a latent period prior to the appearance of the first new complete infectious RNA molecules. Further, this latent period is accompanied by an eclipse of the input templates as infectious agents. The use of electrophoretic separation on acrylamide gels has yielded a detailed account of both templates and early product during the latent period, with the consequent identification of the intermediate stages. The resulting data and their implications for the mechanism of RNA replication will be discussed.RNA viruses must carry out a major part of their life cycle in cells which use DNA as their genetic material and RNA as genetic messages. On entry, the viral RNA is faced with the problem of inserting itself into the cellular information flow pattern in order to communicate its own instructions to the synthesizing machinery. A possibility one might entertain centers on whether an RNA virus employs the DNA to RNA to protein pathway of information flow. This could occur either because the DNA of the host already contains a sequence homologous to the viral RNA (ie., the "escaped genetic message" hypothesis) or because DNA sequences are generated subsequent to infection by reversal of the DNA-dependent RNA-synthesizing reaction. Either mechanism would predict homology between viral RNA and some segment of DNA derived from infected cells.It is clear that a decision on the existence or nonexistence of homology between viral RNA and the host DNA is a necessary prelude to further experiments designed to delineate the molecular life history of an RNA genome. To answer questions of this nature, Doi and Spiegelman ('62) employed the specific hybridization test (Hall and Spiegelman, '61), combined with the subsequently developed use of RNase to J. CELL. PHYSIOL., 70: Sup. 1 35-64.eliminate "noise." The sensitivity required had already been achieved in earlier experiments which identified the DNA complements of ribosomal RNA (Yankofsky and Spiegelman, '62a, b; '63) and s-RNA (Giacomoni and Spiegelman, '62; Goodman and Rich, '62). Under conditions where complexes between 23s r-RNA and DNA were readily observed, no complexes were detected between the viral RNA and the infected host DNA.Since the negative outcome of the hybridization test implies that the DNA to RNA pathway is not employed, we must conclude that these RNA viru...

Evolution of Hepatitis Delta Virus RNA Genome following Long-Term Replication in Cell Culture

“…Two important examples of this are studies of the phage Q␤ minireplicon (27,33) and the short RNAs replicated by a phage RNA polymerase (19,20). In our studies, the HDV RNA genome was replicated in a cell line that had been transfected with RNA 1 year earlier.…”

Inhibition of Vesicular Stomatitis Virus Replication by Poly(2′-Fluoro-2′-Deoxyuridylic Acid)

“…and not affected by the presence of (G)n. They suggested that their data might indicate that polynucleotides might be highly selective inhibitors of the "recognition mechanism between a replicase and homologous template." Subsequent studies by Spiegelman and associates (17) demonstrated that. when selective pressures (i.e., short periods of incubation) were applied to an in vitro Q,3 RNA replicating system, variant small RNA molecules evolved that possessed a very high affinity for the replicase.…”

“…Recently, a more direct mechanism of antiviral activity has been attributed to this class of polymers by the demonstration that certain single-stranded polynucleotides can block the activity of virus-associated nucleic acid polymerases (8,16,17,22). The majority of these data was obtained in studies utilizing the ribonuleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase ofthe oncornaviruses and indicated that the inhibitory polynucleotides blocked the association of enzyme and active nucleic acid template (7).…”

Stability of host-parasite systems: you must differ to coevolve

“…In our previous theoretical analysis of the evolution of genetic parasites in simple replicator systems [26] which was, to a large extent, inspired by the seminal early experiments of Spiegelman and colleagues on reductive evolution of bacteriophages genomes in vitro [27][28][29][30], we present a semi-formal argument that the parasite-free state of a replicator is inherently evolutionarily unstable. Here, we explore these and derivative models analytically and show that, in order for a replicator and a parasite to stably coevolve, the parasite must substantially differ from the host in its reproduction strategy.…”