Cellular RNA synthesis

RNA-synthesis is a basic function of living cells, and its rate can be influenced by various factors. Measurement of the cellular RNA-synthesis rate after exposure to the test agent by following the incorporation of radio... 

Incorporation of precursors into DNA, proteins, and lipids is affected less rapidly or to a lesser extent. Respiration is not inhibited. Since the phenylamides at low concentrations do not affect the uptake of precursors or the conversion of nucleosides into nucleotides, inhibition of RNA synthesis must be responsible for the observed effects on uridine incorporation. However, at phenylamide concentrations that are fully inhibitory to growth, a complete inhibition of uridine Incorporation does not occur. Depending on the fungal species used incorporation is reduced to 20-60 X of the control value (12 ). This indicates that only part of the cellular RNA synthesis is sensitive to phenylamides. 

Huang AS, Wagner RR (1965) Inhibition of cellular RNA synthesis by non-rephcating vesicular stomatitis virus. Proc Natl Acad Sci USA 54 1579 Hully JR, Chang L, Schwall RH, Widmer HR, Terrell TG, Gillett NA (1994) Induction of apoptosis in the murine liver with recombinant human activin A. Hepatology 20 854-861... 

Rhodanine (XXXIV) has been found to inhibit the multiplication of ECHO 12 virus, one of the enteric RNA viruses. Other strains of ECHO and various other RNA and DNA viruses were insensitive. It produced 95 per cent inhibition of virus growth in monkey kidney cells at 17 pg/ml. Cellular RNA synthesis and morphological appearance were unaffected by 150 pg/ml. This work indicated that rhodanine prevented synthesis of the viral protein coat. All the substituted rhodanines examined were inactive or only slightly active and generally more toxic than the unsubstituted compound [177]. 

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) ... 

It has long been known that cells vary greatly in their susceptibility to viral infection, even when no differences can be demonstrated in their surface receptors for virus adsorption. Moreover, the same virus clone can inhibit macromolecular synthesis in one cell type to a greater extent than another, even though virus yields may not differ significantly. For example, Baxt and Bablanian (1976 ) showed that VSV inhibits nucleic acid synthesis in BHK-21 celts more readily than it does in LLC-MK2 cells. Week and Wagner (1978) also reported that MFC-11 mouse myeloma celts were more susceptible to VSV shut-off of cellular RNA synthesis than were BHK-21 or mouse L cells. 

Use of the general protein synthesis inhibitor, cycloheximide, led to the hypothesis that de novo VSV protein synthesis is required for inhibiting cellular protein synthesis because, when cycloheximide was removed from infected cells, viral protein synthesis preceded suppression of cellular protein synthesis inhibition (Wertz and Young-ner, 1972). In our experience, experiments such as these are difficult to interpret because cycloheximide has many side effects, such as inhibition of cellular RNA synthesis (Week and Wagner, unpublished data). 

The importance of cell type in susceptibility to VSV inhibition of cellular RNA synthesis was also demonstrated much later by Rob-... 

In the previous section, we dealt with the question whether VSV inhibition of cellular protein synthesis was a primary effect of the virus or was secondary to its effect on cellular RNA synthesis. We came to the conclusion, based on kinetics of the reactions and UV sensitivity, that inhibition of cellular protein synthesis was largely, if not entirely, due to a primary effect of the virus and not secondary to inhibition of cellular RNA synthesis. 

This statement still applies despite the recent elegant study of Dunigan and Lucas-Lenard (1983) which provides evidence for two sites of UV inactivation of protein synthesis inhibition, one of which may be identical to the leader sequence presumably responsible for inhibiting cellular RNA synthesis (Week et al., 1979 McGowan et al., 1982). In any case, the kinetic data and UV inactivation data seem to implicate the same or closely-related viral functions for inhibiting both cellular RNA and DNA synthesis and not inhibition of cellular protein synthesis, at least not entirely. 

As discussed in the previous section on VSV inhibition of cellular protein synthesis, inhibition of cellular nucleic acid synthesis could be caused by toxic effects of input virion components or by newly synthesized viral products. As stated above, there appears to be a multiplicity-dependent effect of VSV for inhibition of cellular RNA synthesis in different cells (Wertz and Youngner, 1970). Marked and rapid inhibition of RNA synthesis in Krebs-2 cells infected with nonreplicating DI particles (Huang et al., 1966) or UV-inactivated standard (B) VSV (Huang and Wagner, 1965) led to the hypothesis that structural components of input (parental) VS virions were responsible for inhibition of cellular RNA synthesis. An alternative explanation, in retrospect, is that our DI particles were heavily contaminated with infectious B virions, the transcription function of which is not compromised by DI particles (Schnitzlein et al., 1983) and, as will be discussed presently, UV irradiation does not readily inactivate the capacity of standard B virions to inhibit cellular RNA synthesis (Week et al., 1979 McGowan and Wagner, 1981). 

The purpose of these experiments was to determine whether primary transcription from the 3 end of the VSV genome was essential to express its capacity to inhibit cellular RNA synthesis. Quite obviously, such studies could not distinguish between viral RNAs and proteins as the potential inhibitors. The use of protein synthesis inhibitors, such as cycloheximide, puromycin, and amino acid analogues, always resulted in inhibition of cellular RNA synthesis as well (Week and Wagner, unpublished data), thus, precluding this approach to the problem of identifying the viral inhibitor. 

Use of UV Irradiation to Identify VSV Genetic Information Responsible for Shutting Off Cellular RNA Synthesis... 

Among the potential targets for VSV inhibition of cellular RNA synthesis are transport of nucleoside triphosphates across the cell membrane, enzymatic conversion to nucleotides, initiation/chain elongation/termination of nuclear chromatin transcription, polyaden-ylation, processing, transport to the cytoplasm of completed RNA transcripts, and stability. Quite obviously, RNA synthesis inhibition could be at the level of ribosomal, messenger, and/or transfer RNA catalyzed by polymerases I, II, and III, respectively. 

Although the case for inhibition of DNA-dependent RNA synthesis by wt VSV leader RNA sequences is building, all that can really be said at this time is that wt VSV leader RNA contains nucleotide sequences potentially capable of interacting with promoters or with host cell protein cofactors that interact with nucleotide sequences essential for accurate transcription. Perhaps the most intriguing possibility is that VSV wt leader RNA can serve as a surrogate for other small RNA species found inside the cytoplasm and nucleus of cells (Lerner and Steitz, 1981 Lerner et ah, 1981 Zieve, 1981). Similar sequences do not appear to be present in leader RNAs transcribed from 5 -DI particles (Fig. 2), possibly explaining why DI particles do not possess the capacity to inhibit cellular RNA synthesis (Week and Wagner, 1978). 

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