The Replicative Form

The Replicative Form (RF) of picornaviruses is a very stable double-stranded molecule, formed by an intact viral ERA chain hydrogen-bonded to a full-length complementary strand. 

The crucial discovery of this structure by Montaigner and Sanders (19) P t an end to speculations on the possible involvement of mechanism(s) of RNA replication by-passing the use of complementarity. Their finding was soon extended to RRA-containing phages (20), and other picorna- and plant-viruses as well (21-23). 

From the beginning of the infectious cycle a small but defined fraction of the virus-induced RNAs is in a d-s form. At early times the ERas e-resist ant material accounts for not more than 0.5% of the virus-induced RRAs, but at the end of the cycle (6-8 hours post infection), the d-s molecules may represent up to 10% of the total ENA synthesized at that time. 

With a molecular weight of about 5x10, the RF of picornaviruses moves as a sharp peak at 18-20 S in sucrose gradients, and the ionic environment has little influence on its sedimentation behaviour. 

The buoyant density of RF in Cs salt gradients (about 1.62 g/ml for mengovims RF, ref.21, 24) is less than that of s-s viral ERA. 

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Note Data on size of DNA are for the replicative form (double-stranded). The contour length is calculated assuming that each base pair occupies a length of 3.4 A (see Fig. 8-15). 

Canine parvovirus, first identified in 1978, is now endemic.455,456 Childhood fifth disease is also caused by a parvovirus.456,457 When these single-stranded DNA viruses infect cells a double-stranded replicative form of DNA arises by synthesis of the complementary negative strand alongside the original positive DNA strand. Many copies of the replicative form are then synthesized. The negative strands of the replicative forms serve as templates for synthesis of numerous new positive strands that are incorporated into the progeny viruses. The whole process may take only 20 minutes. Some parvoviruses are unable to reproduce unless the cell is also infected by a larger adenovirus. 

When the "replicative form" of DNA of the virus (()X174 (Chapter 27), a small circular molecule containing 5000 bp, was treated with proflavine, the binding of 0.06 mol of proflavine per mole of nucleotides reduced Wr to zero. From this it was estimated that o = 0.055, corresponding to -27 superhelical turns at 25°, pH 6.8, ionic strength 0.2. 

The single-strand DNA penetrates the cell, where it has converted by enzymes to a duplex replicative form through Watson-Crick base pairing. The replicative form is then reproduced by a mechanism similar to that used for the semiconservative replication of the duplex chromosome of the host cell. Finally, after this stage of replication, the mechanism shifts to one in which the replicative form serves as a template to produce copies of the single-strand DNA found in the mature virus. 

Hilleman and his co-workers have demonstrated interferon production following the administration of reovirus RNA or the replicative form of RNA isolated from E. coli infected with MS2 coliphage. These BNAs are effective in inducing resistance to virus infection m vitro and vivo. The induction of interferon and the broad-spectrum protection against viral infection conferred by double-stranded RNA suggested the use of synthetic polynucleotides, including polyriboinosinic-polyribocytidilic acid, which possess a double-stranded conformation. Chemically modified RNA may also induce the production of interferon. ... 

Chandler, B., Hayashi, M., Hayashi, M.N., and Spiegeltrtan, S. (1964) Circularity of the replicating form of a single-stranded DNA vims. Science, 143,47 9. 

DNA-dependent RNA synthesis in E.coli cells was inhibited under experimental conditions by actinomycin D. After 50-90 sec, almost 70% of the of the parent RNA was found in one fraction of RNA with a sedimentation coefficient of about 20S, which these workers called the replicative form of RNA, because it was resistant to ribonuclease (double-helical form). The quantity of P of ribonuclease-resistant RNA then fell sharply, and a new peak of this RNA appeared after 3.5 min (Fig. 12). In one case this wave of change of the parent RNA into the double-helical RNAase-resistant form was observed on four successive times. In addition, slight but definite incorporation of parent P material was observed into a special low-molecular weight 6S fraction (mol.wt. from 1 to 30 X10 ) was observed, which, however, contained much newly synthesized RNA (in this fraction the ratio of parent RNA/new RNA was much lower than in the 2OS fraction of RNA). 

After penetrating into bacterial cells the DNA of this phage undergoes the typical fate which we saw for RNA in cases of virus reproduction considered above it is replicated by complementary synthesis with the preliminary formation of double-stranded DNA. This process was discovered in later experiments (Sinsheimer, 1961 Sinsheimer et al., 1962) by fractionation of nitrogen- and phosphorus-labeled DNA of infected cells in a density gradient of cesium chloride at various times after infection. This double-stranded form of phage DNA was called the replicative form (RF). 

The formation of the replicative double-stranded form of DNA can therefore be regarded as the first act in molecular morphogenesis of this virus. The replicative form also has infective properties, i.e., it preserves the complete program of phage development. 

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