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The content and size of the poly(A) on Mengovirus RNA grown in both mouse L cells and HeLa cells have been examined. Virion RNA from either cell line could bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. The size of the poly(A) on the Mengovirus RNA was independent of the host cell and averaged from 50 to 70 nucleotides.
The effect of zinc ions on the post-translational cleavage of mengovirus polypeptides has been examined. The cleavage of the "A" precursor, which gives rise to the capsid proteins, was the most sensitive at concentrations of zinc chloride from 0.1 to 1.0 mM. Beta-mercaptoethanol and dithiothreitol antagonized the zinc-promoted inhibtion of clevage. Our results indicate that zinc ions interfere with the proper folding of the nascent polypeptide precursor rather than inhibit the proteases responsible for the cleavages. Thus, proper folding of mengovirus polypeptide "A" appears to be necessary for subsequent processing by proteases.
Mengovirus infection of Ehrlich ascites tumor cells caused a change of the intracellular ATP concentration. It increased by 35% within the first 3 h postinfection and then declined to zero within the next 5 h. The decrease in the ATP concentration was due, at least in part, to leakage of ATP into the medium, where it could be demonstrated by the luciferin-luciferase assay. Gross leakage of ATP was observed at 4.5 h postinfection, concomitant with the production of the first intracellular, infectious virus particles. A similar concentration decrease was detected for Mg(2+), the polyamines, and K(+), whereas an increase in the Na(+) concentration was observed. The intracellular Mg(2+) concentration varied synchronously with the ATP level, rising by 16% during the first 3 h postinfection and then progressively falling to lower values in the late period of the infectious cycle. After an initial slight enhancement, the putrescine, spermidine, and spermine concentrations declined at about 1.5 h postinfection. Wherease the intracellular K(+) concentration increased by 17% during the first hour postinfection, the Na(+) concentration diminished by the same value within the same time period, leaving the internal ionic strength unchanged early in infection. Three hours after the beginning of virus infection, there was a rapid decline of K(+) and enhancement of Na(+) within the cell. These alterations of the intracellular energetic and ionic conditions seem to be, at least in part, responsible for the cessation of virus-specific protein synthesis in mengovirus-infected Ehrlich ascites tumor cells commencing 3 to 3.5 h postinfection.
Employing high-resolution polyacryl-amide gradient slab-gel electrophoresis in 6M urea and 6% acetic acid, a distinct change in the microheterogeneity of histone H1 was detected after mengovirus infection of Ehrlich ascites tumor cells. Whereas in uninfected cells the band multiplicity was found to be 6, it was reduced to 4 in the course of the infectious cycle (8 to 9 h). It could be deomonstrated that, while the electrophoretically slowly moving subspecies H1e and H1f disappeared from the band profile of histone H1, the faster migrating bands H1a and H1b increased in relative intensity. The relative intensity of band H1d was also drastically reduced, that of band H1c stayed practically constant throughout infection. When Ehrlich ascites tumor cells were labeled with a mixture of [14C]amino acids prior to mengovirus infection, the radioactivity incorporated into histone H1 subspecies of low electrophoretic mobility was chased into histone H1 components of high mobility in the course of infection, suggesting a virus-induced, unidirectional interconversion of the multiple histone H1 subunits. With the exception of a histone H2B subspecies, which also decreased in amount, the relative quantities of the other histones stayed constant throughout infection.
Treatment of L cell monolayers with actinomycin D resulted in an increase in both the size and number of mengovirus plaques when compared to untreated cells. Addition of the drug to the agar overlay was as effective as pretreatment provided the addition was immediately after virus adsorption. The increased plaque number observed was apparently not due to rescue of defective particles or inhibition of interferon production. The data indicate that more mengovirus particles can successfully complete the infectious cycle in L cells in the presence of actinomycin. The data also suggest that indigenous C-type particles in L cells may interfere with picornavirus replication.
Virus-specific RNA polymerase activity of mengovirus-infected Ehrlich ascites carcinoma (EAC) cells was maximal 7 hours after infection in a one-step growth cycle. In a cell-free system with the mitochondrial-microsomal fraction (MMF) derived from mengovirus-infected EAC cells in a nucleoside-triphosphate medium, polymerase activity increased up to 60 min (when MMF was obtained by a homogenizer) or 120 min (when MMF was prepared by sodium deoxycholate disruption). Subsequently RNA polymerase activity decreased between 150-180 min of incubation at 37 degrees C and then formed a plateau up to 300 min. The cell-free system used is able to give valuable information as regards testing of RNA polymerase inhibitors in an antiviral screening programme.
Eleven pyrimido-pyrimidine derivatives, seven with significant antiviral activity against Mengovirus, five against Coxsackie B1 virus and four antiviral negative compounds were tested for their photosensitizing ability. All seven compounds with antiviral activity in vitro showed an enhanced antiviral action against Mengovirus under irradiation with visible light, a fact that may be caused by photodynamic processes. It was tried to correlate the oxidation potentials of sensitizers with their photodynamic activity. By means of mass-spectrometric investigations, molecular fragmentation was examined following thin layer chromatography (TCL) before and after irradiation. Furthermore, binding affinity to biopolymers (BSA and RNA) was investigated to reveal conformity in differences of antiviral activity. The main results are the following: 1. Generally, strong antiviral activity can be correlated with strong binding affinity. 2. No significant correlation could be detected between oxidation potentials of antiviral compounds and their enhanced antiviral activity under irradiation conditions, although in some cases sensitizer with higher oxidation potentials are more effective than those with lower ones. 3. The lower the photostability of the compounds the higher was the light-induced antiviral activity. 4. No alteration of the molecular ion peak and fragmentation pattern before and after irradiation was indicated by means of mass-spectrometry and TLC using fairly comparable conditions.
Mengovirus particles adsorbed to Ehrlich ascites tumour cells are redistributed into patches and caps without the action of antibodies. Thus capping may be an early signal in picornavirus infection.
Substituted amino pyrazoles were found to exhibit plaque reduction and inhibition of the cytopathic effect of mengovirus on FL cells. Their antiviral activity was not caused by a virucidal effect or by inhibition of viral adsoprtion or penetration but by suppression of the virus multiplication. During a one-step growth cycle maximum suppression of virus yield occurred after compound addition from 0 to 2 h after infection. Progressively less viral inhibition appeared when compound was applied during the later part of virus replication. The antiviral effect was reversible by removal of the compound, and no inhibitor-resistant period occurred.
The polyadenylic acid contained in 35S mengovirus RNA produced in infected BHK-21 cells contained approximately 94% AMP and was estimated to contain an average of 50 to 55 nucleotides. The polyadenylic acid is placed at the 3'-end of the genomic RNA based on the presence of significant levels of [3H]adenosine in complete alkali or RNase T2 digests of polyadenylic acid from [3H]adenosine-labeled 35S viral RNA.
Dipyridamole showed an antiviral activity aganinst mengovirus in FL cells using the agar diffusion plaque inhibition test, plaque reduction test, tube titration test, and virus yield test after one replication cycle. With the last two tests mentioned above the inhibitory action was also confirmed in L cells. In consequence of the known transport inhibition of uridine into the cell in presence of dipyridamole only a very small incorporation of 3H-uridine into acid-insoluble material could be demonstrated. Applying the method of prelabelling of FL cells at 16 degrees C for 1 h with subsequent addition of dipyridamole the drug failed to show an effect on cellular RNA synthesis per se in uninfected cells whereas the viral RNA synthesis in mengo-virus-infected L cells was completely depressed.
Cellular RNA synthesis was studied in mouse L-929 cells and in these cells infected with mengovirus. RNA polymerases I, II, and III were partially purified and their chromatographic properties were analyzed by DEAE-Sephadex A-25 chromatography. RNA polymerase II was purified from mouse liver and its subunit structure was compared to that of normal and virus-infected L-929 cells by two-dimensional gel electrophoresis. By these criteria, the enzymes from all three sources were identical. The RNA synthetic activities and capacities of chromatins from normal and virus-infected cells were compared under a variety of conditions. The endogenous activity in chromatin from infected cells was inhibited relative to controls but the residual activity responded normally to stimulation by ammonium sulfate, heparin, and Sarkosyl. The template capacity of the chromatins was compared with added RNA polymerase II and by a rifampicin challenge assay utilizing Escherichia coli RNA polymerase. Identical results were obtained in each case. The number of growing RNA chains and the rates of their elongations were determined. The results showed that nuclei and chromatin from infected cells have a smaller number of RNA polymerase II molecules engaged in RNA synthesis than normal cells do but that the active molecules elongate RNA chains at the same rate.
One host polypeptide (40,000 daltons) synthesized prior to infection is associated with the 250S RNA polymerase structure partially purified by a combination of velocity sedimentation and isopycnic separation. A series of pulse-chase experiments have shown that a 56,000 dalton polypeptide made during the eclipse phase of infection is inserted into the 250S viral RNA polymerase structure. This 56,000 dalton polypeptide is bound in a stable manner since labeled 56,000 dalton polypeptide is not removed from the 250S polymerase structure by a 2-hour chase (3 to 5 hours after infection) and it is the major labeled polypeptide species remaining. However, the 56,000 dalton polypeptide (viral-specific polypeptide E) made at 4 hours after infection is not present in the 250S polymerase structure following a 50 minue chase. Levels of cycloheximide which inhibit protein synthesis 95 per cent in the infected cell have no effect on the amount of viral-specific RNA polymerase activity (in vitro) when the inhibitor is added for 30 minutes at the time of maximum rate of viral RNA synthesis in whole cells. These inhibitor studies support the hypothesis that the viral-specific RNA polymerase polypeptide may be a stable polypeptide that is not rapidly turning over in the infected cell. In view of these results the stable 56,000 dalton polypeptide (polypeptide E) made early in infection may be a candidate for the viral-specific polymerase polypeptide.
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