Replicative intermediate

Radioactive RF has been used to infect cells and search for the modifications of the parental molecules in each sub-cellular compartment. Such studies revealed that soon after infection with RF, the input d-s molecules are found in the cytoplasm as part of a Replicative-Intermediate-like structure (18). Treatment of the host-cell with interferon abolishes the infectivity of RF, but does not prevent the intracellular processing of RF into RI. In contrast, exposure of the cells to Actinomycin D inhibits the infectivity of RF and the intracellular transition to RI as well (36). As it was found that a cellular RNA polymerase specifically binds to RF (37) it was postulated that RF served as abnormal template for a cellular RNA polymerase to transcribe the first viral messenger (18, 57). 

A third RNA structure can be isolated from picornavirus-infected cells the complex Replicative Intermediate (RI), partially single- and partially double-stranded. 

First found as an intermediate in the replication of the small RNA-containing phages (38, 39) the RI was soon identified as the actual site of synthesis of the viral RNA. 

It consists of a RNas e-resist ant, d-s core plus several s-s nascent chains of different length, attached to the template by a hydrogen-bonded region (40-42). 

Intact RI sediments in sucrose gradients as a polydisperse band between I8-7O S. However its sedimentation behaviour depends to a considerable extent on the extraction procedure phenolization at 60 G causes the release of nascent chains and, accordingly, the RI so obtained sediments between IO-4O S (42). 

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Rothmund-Thomson (Recql 4) proteins. The genes that encode these proteins are altered in human syndromes of the same name. Patients with these diseases are predisposed to different forms of cancer. Cell lines derived from patients with these diseases exhibit chromosome instability and accumulate abnormal replication intermediates. When these helicase proteins are studied in vitro, they can unwind Holliday junction-like structures. A characteristic of many proteins involved in homologous recombination is that they can be visualized as forming subnuclear structures in cells in response to DNA damage. This can be directly visualized using immunofluorescence and the structures that are formed are called foci (see Section 23.8.2). 

Sells, M. A., Zelent, A. Z., Shvartsman, M., and Acs, G. (1988) Replication intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J. Virol. 62, 2836-2844. 

In cultured primary duck hepatocytes (PDH) congenitally infected with duck hepatitis B virus (DHBV), all major viral replicative intermediates are generated, and CCC DNA, present initially at a low copy number, is amplified in the PDH after a few days in culture (4,5), The amplification of CCC DNA in vitro provides an alternative to liver analysis for studying the structure of this molecule and its sensitivity to potential antiviral agents. 

In this chapter, procedures are described for the enrichment and isolation of CCC DNA and total DNA from both cultured PDH and fresh and frozen liver (or other organ) specimens. The technique for the former is essentially a modified Hirt (6) high-salt extraction (5), Detection of the replicative intermediates is by Southern blot hybridization analysis, which can be performed using... 

To distinguish DHBV CCC DNA from other viral replicative intermediates, a modified salt extraction of the extracts must be performed, which enriches for viral DNAs noncovalently bound to protein (5). 

Endogenous HBV polymerase assays measure the ability of the viral polymerase to incorporate dNTPs into viral replicative intermediates present within viral core particles. If the cores are derived from secreted virions, the assay measures predominantly DDDP activity, whereas if the particles are isolated from cytosol of infected cells, the assay measures predominantly RDDP activity. In these assays, one dNTP is usually radiolabeled, and polymerase activity is detected by scintillation counting of radioactivity incorporated into acid-precipitable material, presumed to be HBV DNA. In most published assay methods, a large excess of unlabeled dNTPs is present (see Note 1). In addition to dNTP substrates, reaction mixtures used for polymerase assays typically contain a buffer (usually Tris-HCl, pH 7.5-8.0), a monovalent salt (usually KC1), essential divalent metal ions (Mg2+ or Mn2+), a reducing agent (dithiothreitol [DTT] or 2-mercaptoethanol), and a nonionic detergent (usually Nonidet-P40 or Triton X-100). Concentrations of individual components of the reaction mixture in different published assay methods vary considerably. 

DNA replication intermediates contain "forked" structures at the site of replication (Figure 24.1). 

Thymosins. Thymosins are hormone-like polypeptides produced by thymus epithelial cells. They regulate the maturation of T-cells moreover, they modulate interferon production and stimulate the expression of interleukin-2 and its receptor. A trial of thymosin fraction-5 in four homosexual patients with chronic hepatitis B demonstrated no effect after six months of treatment (202), while treatment of WHV-infected woodchucks with thymosin alpha-1 resulted in clearance of circulating WHV-DNA and a 50-300-fold decline in the levels of replicative intermediates in liver tissue (203). Subsequently, thymosin alpha-1 and thymosin fraction-5 were used to treat chronic hepatitis B in a human pilot study (204). Results were encouraging, and this was followed by several randomized, controlled multicenter trials of thymosin alpha-1 in the U.S. The primary action of thymosin is unknown, but it is probably related to its immune-enhancing effects. 

Chloroplasts of land plants contain multiple identical circular double-stranded DNA molecules, whose size, according to different data, varies from 120 to 160 or to 220 kb. In the population of cpDNA molecules obtained by chloropiast lysis, monomeric circles prevail (-60% of all circular DNA molecules in tobacco). There also ate oligomeric forms, which are concatemers or head-to-tail associates that most probably arose during DNA recombination and/or replication, as well as atypical molecules, presumably replicative intermediates. Accordingly, the chloropiast genome is not uniform and exists in the cell as a DNA population heterogeneous in molecule size and conformation. The first complete nucleotide sequences of cpDNA were determined in 1986 for tobacco ... 

Birkenmeyer L, Ray DS. Replication of kinetoplast DNA in isolated kinetoplasts from Crithidia fasciculata. Identifrcation of minicircle DNA replication intermediates. J Biol Chem 1986 261(5) 2362-8. 

Hines JC, Engel ML, Zhao H et al. RNA primer removal and gap filling on a model minidrcle replication intermediate. Mol Biochem Parasitol 2001 115(l) 63-7. 

Drew ME, Englund PT. Intramitochondrial location and dynamics of Crithidia fiisciculata kinetoplast minicitcle replication intermediates. J Cell Biol 2001 153(4) 735-44. 

It was reported that VPg is linked to each nascent strand present within the poliovirus replicative intermediate form (20, 21). Flanegan et (2l) find that the amount of VPg-pUp/unit weight arising from nuclease digestion of the replicative intermediate ERA is the same as that obtained from virion ERA. The intermediate has a mass about four times that of the virion ERA (consisting of a (-)-strand ERA and 6 nascent chains, on average half-completed (8). If one molecule of VPg is attached to the (-)-strand template (see below), then the remainder must be attached to the nascent (+)-strands. 

YOGO, Y. and WIMMER, E. Sequence studies of poliovirus ERA. III. Polyuridylic acid and polyadenylic acid as components of the purified poliovirus replicative intermediate. J. Mol. Biol. (1975), 2i, 467-477. 

Protein synthesis in extracts of cells infected with other Ml viruses is not inhibited to the same extent as in the case of picornaviiTuses. This has been clearly shown for VSV extracts prepared from HeLa cells at different times after infection with VSV are very active in cell-free protein synthesis and synthesize only viral proteins (our unpublished observations). The replicative intermediate of VSV does not seem capable of activating the protein kinase of HeLa cells and of inhibiting protein synthesis. 

Separation of RNase T2 digests of 52p-labeled polio virion RNA (a) mRNA (b) and replicative intermediate ENA (c) on DEAE-cellulose in the presence of marker nucleotides. Columns were in 5 i al plastic pipettes (Falcon, Serological) elution with triethylammonium acetate, pH 5, was as described (5, 111 12). 

Our strategy to identify the function of the genome-linked protein has been then, to analyze the step in viral replication at which VPg becomes attached to the viral RHA. For example, if VPg were to be found in replicative structures of polio RHA, its attachment to RHA (and consequently its function) might be related to replication of RNA. As polio replicative intermediate (Rl)... 

PLAUEGAB, J.B., PETTERSSON, R.F., AMBROS, V., HEWLETT, M. and BALTIMORE, B. Covalent linkage of a protein to a defined nucleotide sequence at the 5 terminus of virion and replicative intermediate RNA of poliovirus. Proc. Nat. Acad. Sci. U.S.A. 

The rate of synthesis of each molecular species varies considerably during the infectious cycle viral s-s RNA synthesis is maximal during the linear phase. There is a constant production of d-s replicative form (RP) which, therefore, acoiMulates towards the end of the cycle. By contrast, the replicative intermediate Rl) is the major molecular species only during the early (exponential) phase of RRA synthesis (50). 

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