Viral RNA synthesis

The structure/activity relationships for the methisazone, 3a, derivatives against adenoviruses and poxviruses have been shown to be similar [78]. Pearson and Zimmerman [79] demonstrated that all three types of polioviruses are inhibited by 2-acetylpyridine JV-dibutylthiosemicarbazone, which is similar to 3a, by blocking viral RNA synthesis. A 3-substituted triazinoindole derivative of isatin was effective against several strains of rhinovirus in tissue culture the mechanism of action is unknown [80]. 

The antiviral nucleosides aciclovir and ganciclovir are also converted to their respective nucleoside triphosphates in the cytoplasm of infected cells. They proceed to inhibit viral DNA replication either by inhibition of the DNA polymerase or by incorporation into DNA with subsequent termination of chain extension. Finally the anti-HIV drug AZT acts in an analogous manner, being converted to the corresponding triphosphate and inhibiting viral RNA synthesis by the HIV reverse transcriptase. 

Wu, T.-T., Coates, L., Aldrich, C., Summers, J., and Mason, W. S. (1990) In hepatocytes infected with duck hepatitis B vims, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 175, 255-261. 

The early studies on the antiviral effects of benzimidazoles by Eggers and Tamm are now regarded as classical . Since then interest has concentrated on benzimidazole derivatives which have a more selective toxicity towards the virus rather than the overall toxicity shown particularly by 5,6-dichloro-l-( -D-ribofuranosyl)-benzimidazole (DRB) and its trichloro analogue (TRB) [157]. a-Hydroxybenzylbenzimidazole (HBB, XXVII) and its derivatives have been most thoroughly studied since HBB was found to inhibit selectively the replication of several picomaviruses (for example, poho and ECHO) by interfering with viral RNA synthesis [158]. 

Y-Methylisatin-/ -4,4-dibutylthiosemicarbazone (Busatin) inhibited the growth of all three strains of poliovirus in vitro by 10-40 per cent at 3 pM. Maximum inhibition of >99 per cent was produced at 10 pM. However, at 20 pM the compound inhibited cellular growth and DNA synthesis completely after 1 hour contact, but RNA synthesis was only slightly affected. Busatin was thought to block a stage in the viral RNA synthesis. All the above effects were reversible on removal of the compound from the medium [166]. 

Survival was reduced in adult mice infected with various viruses - including Semliki Forest vims, various strains of yellow fever vims, and West Nile vims - after intraperitoneal injection of gold sodium thiomalate. Adverse effects of therapeutic monovalent gold compounds may be linked to their ability to induce membrane proliferation. For example, the virulent strain of Semliki Forest virus in adult mice is characterized by the development of numerous membrane vesicles in brain with mature vims budding from these stmctures. In contrast, infection of the avirulent strain of Semliki Forest vims results in the formation of very few membrane vesicles and no mature vims particles in adult mouse brain. Proliferation of smooth membrane vesicles from whole mouse brain was induced in mice treated with gold sodium thiomalate. In certain vims infections, smooth membranes are a prerequisite for vims RNA synthesis and maturation. The ability of a virus to stimulate the smooth membranes may be the limiting factor in determining both the extent of viral RNA synthesis and maturation. This mechanism could... 

Inhibition of protein synthesis in intact HeLa cells infected with EMC virus was observed at the same time that viral RNA synthesis reached its peak (Figure l). The shut-off of host protein synthesis, however, preceded this general inhibition of protein synthesis (see below). When cell extracts were prepared at different times after infection and tested for endogenous protein synthesis, a progressive loss of activity with time of infection was observed. A similar result was obtained in L-cells infected with EMC (Figure 2). 

The shape of this curve, however, depends to some extent on the metabolic state of the cells in synchronized cultures, viral RNA synthesis was shown to proceed at the highest rate when infection started at the end of the S-phase, that is, at a time when the rate of cellular RNA synthesis was maximal (85) ... 

Although in the past there was some controversy on this point, there is now little doubt that these RNA species are involved in the viral RNA synthesis, and that they do represent true intermediates in the replication process (see below). 

Additional support for this viewpoint comes from the substantially different kinetics of formation of RF and RI in the presence of guanidine, an inhibitor of viral RNA synthesis (see section Vl), or cycloheximide. This finding led Noble and Levintow (94) to suggest that "the two extracted forms represent distinct structural and functional entities". 

There have been conflicting reports regarding the nature of the RNA which serves as template in the RC Based on electron microscopic examination, it was concluded that the template for viral RNA synthesis was mainly s-s RNA (60). On biochemical grounds, Sberg and Phillipson suggested that the nascent chains are hydrogen bonded to the template over a very short area, perhaps held in place just by the polymerase (48) ... 

GLVII, a mouse encephalomyocarditis virus, is restricted in HeLa cells. Both viral yield and viral RNA synthesis are reduced in infected restricted cells. However, the restriction in viral yield is more than ten-fold greater than the restriction of viral RNA synthesis. This observation that the viral yield is more severely depressed than the yield of viral RNA was also seen in the mengoviirus-MDBK system (20). A non-restrictive variant of GDVII was obtained following alternate viral passage on restrictive and permissive hosts. The authors were unable to identify a mechanism of restriction (19) ... 

That viral penetration and uncoating are normal in the restricted system is indicated by 1) The irreversible eclipse of the virus, 2) the cessation of host protein sjmthesis, and 5) the simultaneous initiation of viral RNA synthesis in both permissive and restrictive hosts (24f 25). Indeed, no major differences occur (are detectable ) in the infected restrictive cell compared to the permissive host until four hours post-infection, at which time there appears to be a cessation of viral RNA accumulation. Initial data (25) supported the hypothesis that this was due to RNA degradation superimposed upon ongoing RNA synthesis. However, others in this laboratory have been unable to confirm this. 

Some viral protein synthesis (translation) must be occurring in the restrictive cells early in infection, since the viral replicase is required to initiate viral RNA replication and we can detect some replicase activity. Since some viral RNA synthesis does occur, cleavage of the viral polypeptide must have occurred... 

As indicated above, these data do not tell us whether a function" is lacking in MDBK which is essential for viral protein or RNA synthesis, or whether a macromolecule is synthesized which prevents the translation of viral proteins or viral RNA synthesis. Since MDBK can be infected productively by other viruses (Bovine enterovirus, influenza), it must be a molecule specific for the mengo-MDBK cell interaction. The greatest enigma in our data is the origin of the early ENA replicase, since very little (or no) protein synthesis occurs. 

Crucial to the understanding of viral restriction is the realization that mengovirus protein and RNA synthesis are interconnected processes. Viral protein synthesis provides the enzymes (the polymerase molecules) needed for viral transcription, while viral RNA synthesis supplies the templates on which viral protein synthesis proceeds. A third activity, viral protein processing, cleaves the product of viral translation to generate the structural (capsid) and functional (polymerase) proteins needed for viral replication. The intimate relationship between viral protein and viral RNA synthesis demands that an early inhibition of any one of these essential fxmctions - translation, transcription, or viral protein processing - may affect both viral RNA and protein synthesis. 

Bossart and Bienz (1979) showed that poliovirus-infected enucleated HEp-2 cells exhibited the same cytopathic effect as poliovirus-infected nucleated cells. However, enucleated cells did not show the same redistribution of lysosomal enzymes (p-glucuronidase and p-glucosaminidase) into the cytoplasm as do infected nucleate cells however, it should be noted that enucleated cells do not support the replication of poliovirus as well as do nucleated cells. A temporal analysis of the events occuring in poliovirus-infected cells, as well as their cellular location, was made by Bienz et al. (1980). By kinetic analysis, viral protein synthesis was found to reach a maximum at 2.5 hr before cell alterations can even be detected. Poliovirus RNA synthesis reached a peak later (at 3.0-3.5 hr postinfection) when new vacuoles can be seen, although viral RNA synthesis continues as vacuoles coalesce to form the typical poliovirus cytopathic effect. Therefore, these authors consider that viral RNA synthesis and not protein synthesis is more closely related to structural changes possibly this could also include lysosomal structural changes. 

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