RNA Genetics

Volume III: Variability of RNA Genomes

Esteban Domingo

• Publisher: CRC Press
• Year: 2018
• Print ISBN: 9780367657451
• eBook ISBN: 9781351093347
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

This book reviews the early evidence of genetic variability of rhabdoviruses. It describes data on the variability in the genomes of closely related virus strains and the variability that can be observed within a given virus strain.

Genetic Heterogeneity of RNA Genomes

Chapter 1

HIGH ERROR RATES, POPULATION EQUILIBRIUM, AND EVOLUTION OF RNA REPLICATION SYSTEMS

Esteban Domingo and John J. Holland

TABLE OF CONTENTS

I.	Introduction: Early Evidence of Genetic Variability of RNA Viruses
II.	RNA Genome Populations and Methods for their Analysis
	A.	Direct Methods
		1.	Oligonucleotide Fingerprinting
		2.	RNA Sequencing
		3.	cDNA Synthesis, Molecular Cloning, and DNA Sequencing
	B.	Indirect Methods
III.	Genetic Heterogeneity of Natural Populations of RNA Viruses
	A.	Negative Strand RNA Viruses
	B.	Positive Strand RNA Viruses
IV.	Antigenic Heterogeneity of Natural Populations of RNA Viruses
V.	Genetic and Antigenic Heterogeneity of Clonal Populations of RNA Viruses
VI.	Mutation Frequencies, Mutation Rates, and Variability of RNA Genomes
	A.	Reversion Rate of an Extracistronic Mutant Phage of Qβ
	B.	Direct Measurement of Mutation Frequencies
	C.	Other Measurements of Mutation Frequencies
	D.	Mutation Rates of Retroviruses
	E.	RNA Recombination and Reassortment in the Generation of Variability
	F.	RNA and DNA Mutation Frequencies
VII.	The Population Equilibrium Model for RNA Genomes: Formulation and Biological Implications
VIII.	Conclusions and Prospects
Acknowledgments
References

I. INTRODUCTION: EARLY EVIDENCE OF GENETIC VARIABILITY OF RNA VIRUSES

It was often observed in the initial studies on mutagenesis of RNA viruses that wild-type revertants arose and contaminated clonal mutant preparations. In addition, it was noted that the so called “wild-type” virus included significant proportions of mutant genomes. Granoff^1,2 identified several spontaneous plaque-type (small, turbid) mutants of Newcastle disease virus (NDV) which occurred at variable frequencies that occasionally reached 1.5 × 10⁻¹. Pringle³ characterized many ts mutants of vesicular stomatitis virus (VSV) Indiana serotype that had been induced by various mutagenic agents. The yield of revertant phenotypes in such preparations was quite high and variable. For one mutant it reached 0.19 (ratio of plaques of mutant and wild type plated at the restrictive temperature). For others, it was <0.001, either because they were affected in more stable genomic positions (Section VII) or because their ts phenotype was the result of multiple point substitutions or of deletions. When analyzed for its ability to synthesize RNA at the restrictive temperature, one mutant was shown to contain 10⁻³ revertants. One “subclone” derived from this mutant did not synthesize viral RNA in the infected cells, while another “subclone” of the same preparation did so normally.⁴ Genetic instability was found with other VSV mutants.^5,6 In those studies the frequency of spontaneous ts phenotype in VSV was 1% and 2% with nonpermissive temperatures of 39.0 and 39.8°C, respectively. The early evidence of genetic variability of rhabdoviruses has been reviewed.⁷, ⁸, ⁹

From the onset, influenza viruses became the prototype of “variable” virus, although it is now clear that the genetic variation of most RNA viruses is generally comparable to that of orthomyxoviruses (Sections III to VI). During the early 1940s, F.M. Burnet studied the rapid phenotypic variations represented by the O (original) to D (derivative) transition in agglutinating ability of influenza virus.¹⁰ Burnet stated that passage of a natural isolate of a virus in some laboratory host until it becomes less pathogenic is a process that “must necessarily be something of an exercise in population genetics”.¹¹ He recognized the need to define the distribution of genotypes in the initial and final virus populations, the frequency of mutation, and the selective effect of the environment, as well as the difficulty in obtaining genetically homogeneous populations of viruses.

For reovirus, the frequency of spontaneous ts mutants was estimated to be 0.3%.¹² Among picornaviruses, variation in the behavior of individual mutants made very difficult the early genetic studies on RNA recombination. To keep mutants free of revertants, passages of viral stocks were kept to a minimum; even with such precautions, some mutants could not be used because of their genetic instability (review in Cooper,¹³ and references therein).

Lesion-type mutants of plant viruses were recognized early and Kunkel showed that a porportion as high as one out of 200 lesions were induced by variant tobacco mosaic virus (TMV).¹⁴ Further evidence of high mutability of TMV was provided by Mundry and Gierer.¹⁵ As documented in other chapters and as reviewed by van Vloten-Doting,¹⁶ plant viruses show extreme adaptability to new environments, and adaptation has often been shown to be concomitant with selection of mutant genomes.

Extreme variability was also clearly documented in initial studies with the RNA bacteriophages. Valentine et al.¹⁷ found that a stock of bacteriophage Qβ contained 8% ts mutants. Furthermore, wild-type revertants frequently arose on the plaques formed by ts mutants. They concluded that replication errors constantly “replenished the mutant pool” and that roughly “one base in 3 × 10⁴ was misread.” Plaque morphology mutants, antiserum-resistant, amber, ts, and azure mutants of the RNA phages were described, and they included revertants at frequencies of 10⁻³, except for several ts mutants that did not revert and were assumed to contain either multiple point substitutions or deletions (reviewed by Horiuchi¹⁸).

A fruitful line of research was opened by Spiegelman and colleagues who developed an in vitro system for the replication of purified Qβ RNA. They amplified RNA molecules in a “serial transfer experiment”: a series of tubes contained the standard reaction mixture with Qβ replicase, but no template; the first tube was seeded with Qβ RNA and incubated to allow synthesis of progeny RNA; then an aliquot was transferred to the second tube and the process of synthesis and amplification of Qβ RNA could continue for extended periods of time.¹⁹ This experiment in its exact format, or in modified versions, has been useful in the study of the evolution of RNA replicated in vitro and of viruses passaged in cell culture. The results of Spiegelman’s group represented the first amplification of an infectious genome in the test tube and paved the way for numerous interesting experiments on evolution of self-replicating RNA molecules in vitro.²⁰ Phage RNA replication is reviewed by Biebricher, C. and Eigen, M. in Chapter 1 of Volume I.

The first evidence of a nucleotide sequence variation within “one virus” was obtained by Wachter and Fiers,²¹ who showed two different 5′ terminal sequences in RNA from cloned phage Qβ. Weissman et al.²² considered that a possible complication in nucleotide sequence determination of phage RNA was that “an apparently phenotypically homogeneous phage stock might contain multiple variants at various sites on the RNA” (see Section V). Phage RNA sequencing, however, progressed quickly and by the mid 1970s, Fiers and colleagues had elucidated the complete nucleotide sequence of phage MS2,²³, ²⁴, ²⁵ a historic achievement in biology. Work originated in Spiegelman’s, Fiers’, and Weissmann’s groups has had many important consequences for our understanding of RNA genome organization, replication, and variability, as documented in several other chapters of these volumes.

Most of the nucleotide sequences of viral RNAs during the 1960s and eary 1970s were elucidated by time-consuming enzymatic methods, since molecular cloning of cDNA and rapid nucleotide sequencing techniques were not yet available. This precluded the analysis of even a few of the genomes that constitute viral populations and, thus, the early suggestions of considerable variability of RNA viruses relied mostly on indirect genetic evidence. More recently, as methods of sampling of viral nucleic acid sequences have become avail...

Table of contents

• Cover
• Title Page
• Copyright Page
• Table of Contents
• GENETIC HETEROGENEITY OF RNA GENOMES
• GENE REASSORTMENT AND EVOLUTION IN SEGMENTED RNA VIRUSES
• ROLE OF GENOME VARIATION IN DISEASE
• ROLE OF GENOME VARIATION IN VIRUS EVOLUTION
• INDEX