RNA replication of bacteriophage

In addition, they assist in replication of RNA viruses, another process that requires RNA synthesis. Viruses sometimes make use of host RNA polymerases but often synthesize their own catalytic subunits. Bacteriophage T4 uses the E. coli RNA polymerase and o factors but modifies their action through the binding of several phage-encoded proteins.248 In contrast, phage T7 encodes its own relatively simple RNAP whose initiation complex (Section A,2)29 and elongation complexes have been studied 249-249b... 

Yarosh, E., Levinthal, C. Exclusion of RNA bacteriophages and interference with their RNA replication by bacteriophage T4. J. molec. Biol. 30, 329-348 (1967). 

The T-odd bacteriophages Tl, T3, T5, and T7 are medium-sized phage with linear duplex DNA genomes. Replication of linear DNA in these and in many other genomes presents a problem. Even if the RNA primer segment is made at the very 3 end of the template strand, there will be a gap in the final replicated strand when the primer is digested out. Since there is no known enzyme that will add to the 3 end of a chain, this gap will remain unfilled. The problem is solved by terminal redundancy, the presence of a common 260-nucleotide... 

The first successful attempts to study RNA evolution in vitro were carried out in the late sixties by Sol Spiegelman9 and his group at Columbia University (Spiegel-man, 1971). They made use of an RNA replicase isolated from Escherichia coli cells infected by the RNA bacteriophage QP and prepared a medium for replication by adding the four ribonucleoside triphosphates (GTP, ATP, CTP, and UTP) in a suitable buffer solution. QP RNA, when transferred into this medium, instantaneously started to replicate. Evolutionary experiments were carried out by means of the serial transfer technique (Figure 4). Materials consumed in RNA replication... 

Figure 6. Amplification of RNA molecules by assays that are sequence- insensitive. The first assay (upper part) combines the polymerase chain reaction (PCR) of DNA templates with reverse transcription and transcription. Commonly used enzymes are TAQ-polym-erase, HIV reverse transcriptase and bacteriophage T7 RNA polymerase. The assay requires a temperature program applying higher temperatures for double strand dissociation. The second assay (lower part) shows the self-sustained sequence replication reaction (3SR) which can be carried out isothermally because double strand dissociation is replaced by enzymatic digestion of the RNA strand in the RNA-DNA duplex. The enzymes used are HIV reverse transcriptase, RNase H and T7 RNA polymerase.

In addition, three enzymes involved in DNA replication, including DNA primases, prokaryotic DNA topoisomerase I and some hexameric DNA helicases, are also classic zinc-ribbon proteins. In bacteriophage DNA primases, mutations of the zinc-binding residues abrogate the synthesis of RNA primers for lagging strand DNA synthesis. Strikingly, each subunit of the mini-chromosomal maintenance (MCM) protein, a heterohexameric helicase that initiates DNA replication in S. cerevisiae, contains an independently folded zinc-ribbon domain that appears to stabilize the dodecameric structure (a dimer of hexamers) of this replication complex. ... 

A search for a specific inhibitor of the action of RNA replicase (RNA dependent RNA polymerase) another point at which viral replication could be inhibited independently of host cell metabolism, led to the screening of KXX) compounds against bacteriophage Q/S RNA replicase with E. coli as host cell. Thirty active compounds were obtained of which (LXIV) was the most active. This inhibited phage multiplication in E. coli by 99 per cent compared with control at 10 pg/ml whereas host cell growth was only inhibited by 50 per cent [243]. These authors hopefully conclude thus, further studies may produce a universal anti-RNA virus drug without side effects . 

The RNA replication complex of a picornavims is bound to smooth cytoplasmic membranes (10, 16). In order to obtain a soluble RNA dependent activity it is necessary to dissociate the enzyme from the membranes by means which adversely affect the activity of the enzyme. It is also more laborious and costly to grow and infect large quantities of animal cells than to carry out a large scale infection of E. coli with an RNA bacteriophage. So far, because of these limitations, the isolation of a picornavims replicase was carried out with very dilute solutions of enzyme-protein, a... 

The basic properties of viral RNA which are used in the life cycle of the bacteriophage are the capabilities to act as template for replication as well as to be recognized as messenger RNA by the host translation system. In this contribution we shall be concerned mainly with replication because this process can be regarded as a simple version of Darwinian evolution. 

The mean single digit accuracy of replication has been determined experimentally for three cases enzyme-free template induced replication (Lohrmann et al., 1980), enzyme-catalysed RNA replication in simple bacteriophages (Domingo et al., 1976) and DNA replication in procaryotes (Kunkel and Loeb, 1979 Kunkel et al.,... 

Let us now consider the physics of catalytic interaction between individual polynucleotides. The most simple example for higher order catalytic action can be observed with primitive RNA bacteriophages. We consider the replication of Q0 in the host cell (Fig. 15). Plus and minus strands act as templates in replication. 

Some E. coli bacteriophages, including f2, MS2, R17, and Qj8, as well as some eukaryotic viruses (including influenza and Sindbis viruses, the latter associated with a form of encephalitis) have RNA genomes. The single-stranded RNA chromosomes of these viruses, which also function as mRNAs for the synthesis of viral proteins, are replicated in the host cell by an RNA-dependent RNA polymerase (RNA replicase). All RNA viruses—with the exception of retroviruses—must encode a protein with RNA-dependent RNA polymerase activity because the host cells do not possess this enzyme. 

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