Virus-specific polymerase

The experimental results described above leave one important question unanswered, namely What is the mechanism of RNA synthesis in RF-RNA-infected F. coli Does the RF-RNA serve as a template for an E. coli enzyme directing the synthesis of new virus-specific RNA — both virus plus- and minus-strand RNA — or is the adsorbed RF-RNA melted in E. coli, with the result that its viral plus-strand in turn serves as mRNA for the synthesis of a virus-specific polymerase responsible for the subsequent accumulation of new virus-specific RNA We attempted to answer this question by studying the fate of viral RNA in E. coli incubated in the absence and presence of inhibitors of protein synthesis, using the methods outlined above. 

Provided the synthesis of virus-specific RNA is dependent on the prior production of a virus-specific polymerase, RNA synthesis should be inhibited or abolished by the presence of inhibitors of protein synthesis. Protein synthesis in E, coli was more than 90% inhibited by either puromycin or chloramphenicol, and the fate of single-stranded RNA in E, coli investigated. The rate of conversion of viral RNA into double-stranded RNA was unaffected by chloramphenicol but was reduced to about half in the presence of puromycin. These results indicate that an RNA polymerase is already present in E, coli. 

Toniutto P, Fabris C, Bitetto D, Fornasiere E, Rapetti R, Pirisi M (2007) Valopicitabine dihydrochloride a specific polymerase inhibitor of hepatitis C virus. Curr Opin Investig Drags 8 150-158... 

Virus messenger RNA In order for the new virus-specific proteins to be made from the virus genome, it is necessary for new virus-specific RNA molecules to be made. Exactly how the virus brings about new mRNA synthesis depends upon the type of virus, and especially upon whether its genetic material is RNA or DNA, and whether it is single-stranded or double-stranded. Which copy is read into mRNA depends upon the location of the appropriate promoter, since the promoter points the direction that the RNA polymerase will follow. In cells (uninfected with virus) all mRNA is made on the DNA template, but with RNA viruses the situation is obviously different. 

A virus-specific RNA RNA polymerase is needed, since the cell RNA polymerase will generally not copy double-stranded RNA (and ribosomes are not able to translate double-stranded RNA either). A wide variety of modes of viral mRNA synthesis are outlined in Figure. By convention, the chemical sense of the mRNA is considered to be of the plus (+) configuration. The sense of the viral genome nucleic acid is then indicated by a plus if it is the same as the mRNA and a minus if it is of oppposite sense. If the virus has double-stranded DNA (ds DNA), then mRNA synthesis can proceed directly as in uninfected cells. However, if the virus has a singlestranded DNA (ss DNA), then it is first converted to ds DNA and the latter serves as the template for mRNA synthesis with the cell RNA polymerase. 

Pharmacology Foscarnet exerts its antiviral activity by a selective inhibition at the pyrophosphate binding site on virus-specific DNA polymerases and reverse transcriptases at concentrations that do not affect cellular DNA polymerases. CMV strains resistant to ganciclovir may be sensitive to foscarnet. Acyclovir- or ganciclovir-resistant mutants may be resistant to foscarnet. 

Mechanism of Action An antiviral that selectively inhibits binding sites on virus-specific DNA polymerase and reverse transcriptase. Therapeutic Effect Inhibits replication of herpesvirus. 

Selective inhibition at the pyrophosphate binding site on virus-specific DNA polymerases and reverse transcriptases... 

Note Herpes simplex virus encodes a virus-specific thymidine. kinase, which phosphorylates the nucleoside analog acyclovir (acycloguanosine) to form acycloguanosine monophosphate. After further phosphorylation, the resulting acycloguanosine triphosphate is incorporated by the viral DNA polymerase into viral DNA, causing chain termination in virus-infected cells.]... 

A third type of virus, exemplified by bacteriophage N4, carries a virus specific RNA polymerase in its virion. This polymerase enters the cell together with the viral DNA and transcribes some early viral genes. Some of these genes code for specificity factors that direct the host RNA polymerase to transcribe late genes. Vaccinia virus is another example of a virus that contains a virion-encapsulated RNA polymerase. 

Figure 4. The technique of serial transfer. An RNA sample which is capable of replication in the assay is transferred into a test-tube containing stock solution. This medium contains the four nucleoside triphosphates (ATP, UTP, GTP and CTPJand a virus specific RNA polymerase, commonly QP-replicase because of the stability of this protein, in a suitable buffer solution. RNA replication starts instantaneously. After a given period of time a small sample is transferred to the next test-tube and this procedure is repeated about one hundred times. The transfer has two consequences (i) the material consumed in the replication is replaced, and (ii) the distribution of RNA variants is subjected to a constraint selecting for the fastest replicating species. Indeed, the rate of replication is increased by several orders of magnitude in serial transfer experiments starting out from natural QB RNA and leading to variants that are exclusively suited for fast replication and hence are unable to infect their natural hosts, Escherichia coli.

Figure 49-1 provides a schematic diagram of the replicative cycle of a DNA virus (A) and an RNA virus B). DNA viruses include poxviruses (smallpox), herpesviruses (chickenpox, shingles, oral and genital herpes), adenoviruses (conjunctivitis, sore throat), hepadnaviruses (hepatitis B virus [HBV]), and papiUomaviruses (warts). Typically, DNA viruses enter the host cell nucleus, where the viral DNA is transcribed into messenger RNA (mRNA) by host cell polymerase and mRNA is translated into virus-specific proteins. 

Mechanisms Ganciclovir, a guanine derivative, is triphosphorylated to form a nucleotide that inhibits DNA polymerases of CMV and HSV but does not cause chain termination. The first phosphorylation step is catalyzed by virus-specific enzymes in both CMV-infected and HSV-infected cells. CMV resistance mechanisms include changes in DNA polymerase and mutations in the gene that codes for the activating viral phosphotransferase. Thymidine kinase-deficient HSV strains are resistant to ganciclovir. 

Enzymes Animal viral particles often contain enzymes (Table 1). These enzymes are virus-specific. In addition to the enzymes summarized in Table 1, viruses often contain other enzymes. Among them are the enzymes that modify both ends of m-RNA molecules synthesized by their capping enzymes and poly(A) polymerases. Protein kinases, deoxyribonucleases, DNA-dependent phosphohydrolases and topoisomerases are also often present in viruses [7]. 

Recently, it has been shown that some virus-infected cells induce virus-specific enzymes such as thymidine kinase or DNA polymerase. These virus-induced enzymes have been proven to differ from their cellular enzymes in terms of such properties as substrate specificity. 

Although a stable preparation of a picornavirus replicase has not yet been obtained, it may be instructive for fut ire work to summarize some of the experience gained so far in attempts to purify and study the properties of the replicase of encephalomyocarditis (EMC) virus. We will therefore describe in some detail our experimental approach to the isolation of an RNA-dependent polymerase from baby hamster kidney cells (BHK 21) infected with EMC virus, and also results of experiments aimed to identify EMC virus-specific proteins in the piirified enzyme (15) ... 

Most of the 29 early RNA species are synthesized in vivo in cells where virus-specific protein synthesis does not occur because of the presence of chloramphenicol. This indicates that early RNA is synthesized by the bacterial RNA polymerase. In vitro, B. subtilis RNA polymerase binds to 29 DNA in four sites, which can be visualized by electron microscopy. Three of these sites correspond or are close to the three initiation sites of transcription of the early genes. 

The inhibition of cellular protein synthesis is inevitably followed by the decline of the functions of unstable proteins with possible widespread effects on the cell. It has been observed that the activity of enzymes controlling polyamine synthesis (McCormick and Newton, 1975) and of RNA polymerases I and II, measured in isolated nuclei, declined after infection (Preston and Newton, 1976). However, the rate of decline was faster than occurred after adding cycloheximide to uninfected cells, suggesting that the polymerases may have been inactivated by interaction with some virus-specific protein. Lowe (1978) could detect no changes in the activities of purified RNA polymerases in infected cells but extraction of the enzymes may possibly restore their activity by dissociation of an inhibitor. It would be interesting to have a direct comparison between the polymerase activities measured in intact nuclei and after purification. 

In this case RNA is synthesized only in the presence of all four ribonucleoside triphosphates, magnesium and manganese ions, and the enzyme RNA polymerase (sometimes called DNA-dependent RNA polymerase, to distinguish it from certain forms of virus-specific RNA polymerase responsible for RNA synthesis in the presence of primer RNA). 

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