2024 年 100 巻 4 号 p. 253-263
I. Watanabe et al. isolated approximately 30 strains of RNA phages from various parts of Japan. To isolate RNA phages, they assessed the infection specificity of male Escherichia coli and RNase sensitivity. They found that the isolated strains of RNA phages could be serologically separated into three groups. Furthermore, most of them were serologically related, and the antiphage rabbit serum prepared by one of these phages neutralized most of the other phages. The only serologically unrelated phage was the RNA phage Qβ, which was isolated at the Institute for Virus Research, Kyoto University, in 1961.
The discovery of the virus was attributed to Dmitri Ivanovsky (1892), who showed that filtrates that pass through a filter with small pores that do not pass bacteria still retain infectivity. M. Beijerinck first described the concept of a virus that multiplies only in infected cells.
F.W. Twort discovered the bacteriophage or phage in 1915,1) and F. d’Hérelle independently discovered it in 1917.2) Interested readers may refer to “Who discovered bacteriophage?” by D.H. Duckworth.3)
Bacteriophages were the most important model systems in the development of molecular biology in the 1950s and 1960s, and important discoveries were made through bacteriophage studies. For example, the proof that DNA carries genetic information,4) the discovery of the double-helix DNA structure,5) the discovery of mRNA,6) and elucidation of the genetic code7) were discovered in bacteriophage studies.
In contrast, T. Loeb and N.D. Zinder first reported RNA phages in 19618) and described a coliphage containing RNA as its genetic material. The RNA phages specifically infect F+ Escherichia coli. They were spherical and similar to ΦX174, as observed by electron microscopy with negative staining.
In 1967, I. Watanabe et al. isolated RNA phages from various parts of Japan and serologically classified them into three groups9): Groups I (MS2 type), Group II (GA type), and Group III (Qβ type). The previously isolated phages MS2,10) f2,8) and R1711) were classified into Group I. The RNA phage Qβ, which was first isolated by Watanabe et al. in 1961, was distinguished from the known RNA phages and was classified into a different group, Group III. Groups I and II had immunologically slight cross-reactions, but there was no cross-reaction between Group III and the other groups. Haruna et al.12) also revealed that RNA replicase from Qβ and KV can use RNA from Group III, but not RNA from Group I or II.
The genetic map of the Qβ phage is presented in Fig. 1.13) There are three cistrons, the first coding for maturation protein A2, the second coding for coat protein, and the third coding for the b subunit of viral RNA polymerase. The A2 gene possesses the lysis function and is essential for infection, where the gene product is required for phages to attach to host pili. In the case of Qβ RNA, ribosomes cannot bind to the A2 gene because of secondary and tertiary structures, but they can be translated only in the early stage of infection. By the time of publication, approximately 20% of the sequence had been determined. The life cycle of the RNA phage is depicted in Fig. 2.14)
Map of the Qβ genome.13) Nontranslated regions are dark; arrows mark the locations of the cistrons. Narrow bars under the map indicate the areas of known nucleotide sequence. At the time of publication, 20% of the genome had been sequenced.
The life cycle of bacteriophage Qβ14) and related RNA phages. The phage attaches to the host F-pilus through interaction with A2 protein and enters the cytoplasm along with RNA. A2 protein is then cleaved. The host ribosomes attach to the viral RNA and produces viral-specific protein synthesis. The synthesized coat protein assembles and encapsidates viral RNA, and lysis of the host cell will be caused by the synthesized A2 protein to release the infectious virus particles.
In their serological classification of viruses, the authors used antisera from several RNA phages. The binding specificity to host bacteria was based on the interaction between the maturation protein A2 or A protein of the phage and the surface structure of the pili, indicating that there were at least three different surface structures or antigens on the pili. Therefore, it is not surprising that other sequence differences were observed among the phages in the same serological group.
Gene expression is normally regulated at the transcription level; however, in the case of RNA phages, it must be regulated otherwise. The gene expression of Qβ depends on the competition between the binding of ribosomes and replicase to the genome.
Two important RNA polymerases were identified through studies of RNA viruses: RNA-dependent RNA polymerase, RdRp, and reverse transcriptase. RdRp was discovered in the early 1960s through studies of mengo virus and poliovirus.15) The fact that these viruses were insensitive to actinomycin D indicated that they had their own RNA polymerase and required RNA as a template, not DNA. As will be discussed, RNA phages including MS2 and Qβ possess RdRp. In fact, RdRp is an essential enzyme for all RNA viruses.
In 1970, D. Baltimore16) and H.M. Temin17) independently discovered reverse transcriptase. The discovery expands the central dogma in that RNA not only transfers genetic information from DNA to proteins but also reverses this information from RNA to DNA. Reverse transcriptase is essential for the retrovirus growth. Thus, the synthesized double-stranded DNA is incorporated into the host genome as provirus. The discovery of retrovirus and reverse transcriptase has played an important role in the development of molecular biology. Different from animal cells, in which viral RNA is reverse-transcribed to DNA, RNA phages replicate only to and from RNA, and there is no DNA stage during RNA phage replication.
In 1971, Baltimore published a brief note on the classification of viruses.18) The title of the report implied animal viruses, but in fact, this was true for all viruses, including phages. He classified viruses into six classes, i–vi. The RNA viruses were classified into Groups iii–vi. Group iii included double-stranded RNA viruses such as phage ⲫ6.19) When infected, the capsid-attached RdRp is used to produce a complementary strand that functions as a pregenome for replication and as mRNA for viral protein synthesis. The newly assembled capsid protein and mRNA associate and replicate the complementary strand by RdRp to generate double-stranded RNA. Group iv is a positive single-stranded RNA virus in which genomic RNA directly attaches to host ribosomes to produce phage proteins. Group iv included RNA phages MS2 and Qβ. Group v or negative RNA viruses replicate their RNA via capsid-bound RdRp to produce complementary RNA for translation. Negative single-stranded RNA phage has not been found. Group vi included RNA viruses that have reverse transcriptase and are called retroviruses; hepatitis B virus is an example.20)
I. Haruna and S. Spiegelman15) (1965) and L.R. Overby et al.21) (1966) studied the template specificity of the phage-encoded RdRp from MS2 and Qβ phages and found that they only replicate their own RNA, ensuring replication of the infecting virus RNA. It is also important that double-stranded RNA viruses and negative single-stranded viruses replicate themselves without going through the DNA stage. Because RdRp is not produced by the host cell and is attached to the viral capsid, RNA phages must enter the host cell together with their capsid despite the general idea that phages inject nucleic acids into the host cell, leaving the capsid outside the cell.
The development of nucleotide sequencing was slower than that of amino acid sequencing. F. Sanger developed an amino acid sequence determination method and applied it to determine insulin amino acid sequences in 1955.22) Today, RNA is sequenced after being reverse-transcribed to DNA. The DNA sequencing method was not available in the early 1970s, and at that time, RNA was directly sequenced. The first total RNA sequence was that of phage MS2, which was reported in 1976,23) before ΦX174 DNA was sequenced in 1977.24),25)
RNA was sequenced just as in the case of amino acid sequence determination, except that the number of different nucleotides was only 4 compared with the 20 amino acids in proteins. A protein whose sequence is to be determined will be digested by protease into several peptides, and each peptide may be sequenced by Edman degradation. At least two proteases with strict substrate specificity are necessary to determine which peptide is connected to which peptide. In the case of RNA sequence determination, the two commonly used types of nucleases were bovine pancreatic RNase A and RNase T1, which were isolated by K. Sato and F. Egami.26) As the complete digestion of RNA produces too many small oligo nucleotides, RNase digestion was conducted at low temperatures, retaining the quaternary structure, and reasonably long oligo nucleotides were obtained for sequence determination by column chromatography and electrophoresis.23)
In general, transcription levels control gene expression. The gene expression of RNA viruses should also be controlled, but a strategy other than transcription is required. The lysis gene should not be expressed before the replicase gene or coat protein gene. The upstream of A and replicase genes are assumed secondary structures, and only the ribosome binding site upstream of the coat gene is used for translation. As the coat gene is translated, secondary structures upstream of the replicase gene are released, and the ribosome binds upstream of the replicase gene. As the replicase gene is synthesized, it will synthesize the minus-RNA. The phage replicase then synthesizes the plus-strand RNA using the synthesized minus-RNA strand as the template. The ribosome binds upstream of the A gene as the plus strand RNA begins to be transcribed. The A gene product of MS2 or Qβ is essential for binding to the host pili. Translation of the lysis gene is believed to start with the ribosome, which changes the reading frame following translation of the coat gene.
The Nobel Prize in Physiology or Medicine 1969 was awarded to M. Delbrück, A.D. Hershey, and S.E. Luria for their “discoveries concerning the replication and genetic structure of viruses”. Delbrück began as a theoretical physicist, later changing his field to experimental physics and then turning into virology.
Similar to Delbrück, I. Watanabe was originally a physicochemist and interested in the mechanism of virus growth when he was thinking about his new research field after World War II. He tried introducing an ultracentrifuge because he thought that an analytical and preparative ultracentrifuge would be useful for virus research. In fact, based on I. Watanabe’s proposal, Prof. S. Mizushima, who was the mentor of I. Watanabe, made efforts to collaborate with the University of Tokyo and Tokyo Institute of Technology to design and construct an analytical and preparative ultracentrifuge based on a paper by J.W. Beams and E.G. Pickels27) and another paper by E.G. Pickels and J.W. Beams.28) The completed analytical ultracentrifuge was brought to the University of Tokyo, but at almost the same time, Beckman company began to commercialize Model E. Model E was eventually introduced in Japan. Prof. Mizushima’s laboratory produced a number of well-known scientists in Japan such as Drs. T. Shimanouchi, S. Nagakura, H. Noda, K. Imahori, I. Tanaka, Y. Kyogoku and others.
“Isolation and grouping of RNA phages” by I. Watanabe et al. (1967) was introduced, the effect of the paper on the development of virology and molecular biology was described, and the historical background of phage research was reviewed. RdRp was discovered in the early 1960s from the study of poliovirus as an actinomycin D-insensitive polymerase. Reverse transcriptase was discovered independently by D. Baltimore and H.M. Temin in the 1970s. In 1967, the genetic code was fully elucidated. At that time, an RNA sequencing method was not yet available. In 1976, the first total RNA sequence of phage MS2 was reported by W. Fiers et al.23) In 1977, the first total DNA sequence of ΦX174 was reported by F. Sanger et al.24),25) Both RdRp and reverse transcriptase introduced a new concept in molecular biology and expanded the central dogma in which genetic information transfers not only from DNA to RNA to protein but also RNA to RNA and RNA to DNA. However, the control mechanism of gene expression in RNA viruses was clarified after determining the total nucleotide sequence of the phage.
Edited by Shigekazu NAGATA, M.J.A.
Correspondence should be addressed to: F. Arisaka, e-mail: fumio.arisaka@gmail.com.
This paper commemorates the 100th anniversary of this journal and introduces the following paper previously published in this journal. Watanabe, I., Miyake, T., Sakurai, T., Shiba, T. and Ohno, T. (1967) Isolation and grouping of RNA phages. Proc. Jpn. Acad. 43 (3), 204–209 (https://doi.org/10.2183/pjab1945.43.204).
[From Proc. Jpn. Acad., Vol. 43 No. 3, pp. 204–209 (1967)]
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Fumio Arisaka was born in Kanagawa Prefecture in 1948 and graduated from the University of Tokyo in 1972. After obtaining a master’s degree at the University of Tokyo, he entered the Graduate School of Biophysics and Biochemistry, Oregon State University, and obtained his PhD in 1977. He worked as a postdoctoral fellow at the Biocenter of the University of Basel between 1977 and 1980 and served as an Assistant Professor at the Department of Pharmaceutical Sciences, Hokkaido University, between 1980 and 1990 and as an Associate Professor at the Tokyo Institute of Technology and became Professor in 2010. He has been working on the structure and molecular assembly of Bacteriophage T4. He was a Vice-President of the Protein Science Society of Japan between 2012 and 2013 and a Vice-President of the Biophysical Society of Japan between 2013 and 2014. Since 2014, he has been a Professor Emeritus at the Tokyo Institute of Technology.
