key: cord-0924164-7w1t5v18
authors: Mukherjee, Pranab K.; Simpson, Robert W.
title: Inhibitory effect of papaverine on RNA and protein synthesis of vesicular stomatitis virus
date: 1985-07-31
journal: Virus Research
DOI: 10.1016/0168-1702(85)90041-3
sha: 30902c33683438d343f4cb8c1858819bb16de596
doc_id: 924164
cord_uid: 7w1t5v18

Abstract Papaverine, an inhibitor of cAMP phosphodiesterase, reduced yields of infectious vesicular stomatitis virus in HEp-2 cells approximately 100-fold if added to cultures at a concentration of 30 μM before and after virus infection. The extent of papaverine-induced suppression of viral growth was dependent on drug dose and treatment regimen. Cells progressively recovered their viral permissive state after removal of drug. The cyclic nucleotide, cGMP, nullified the inhibitory effect of papaverine if added to cells during drug treatment. Pulse labeling experiments with [35S]methionine showed that papaverine compromises production of all virus-specific proteins in infected cells without adversely affecting host cell protein synthesis. Treatment of cells with papaverine strongly inhibited the production of viral RNA and both cellular RNA and DNA. It was found that VSV causes an immediate but transient stimulation of DNA synthesis in HEp-2 cells which is prevented by papaverine treatment. This drug also selectively blocked primary transcription of VSV in vivo and to a lesser extent in vitro RNA polymerase activity of the virion-bound transcriptase. The finding that papaverine has a strong inhibitory effect on viral biosynthesis including early transcription suggests that VSV replication may depend on host factors that regulate intracellular levels of cyclic nucleotides such as cAMP.

Virus Research, 3 (1985) methionine showed that papaverine compromises production of all virus-specific proteins in infected cells without adversely affecting host cell protein synthesis. Treatment of cells with papaverine strongly inhibited the production of viral RNA and both cellular RNA and DNA. It was found that VSV causes an immediate but transient stimulation of DNA synthesis in HEp-2 cells which is prevented by papaverine treatment. This drug also selectively blocked primary transcription of VSV in vivo and to a lesser extent in vitro RNA polymerase activity of the virion-bound transcriptase. The finding that papaverine has a strong inhibitory effect on viral biosynthesis including early transcription suggests that VSV replication may depend on host factors that regulate intracellular levels of cyclic nucleotides such as CAMP.

vesicular stomatitis virus, papaverine, cyclic AMP, phosphodiesterase inhibitor

The rhabdovirus, vesicular stomatitis virus (VSV), has an unusually broad host range that may reflect, in part, an ability to utilize host regulatory factors which are 58 common to most eurkaryotic cells of higher animals. For example, we have recently shown that inhibitors of cellular prostaglandin biosynthesis prevent VSV replication at the level of viral RNA and protein synthesis (Mukherjee and Simpson, 1984 Simpson, , 1985 . These studies also demonstrated that cells can recover their permissive state for productive viral infections after removal of such inhibitors.

The involvement of cyclic nucleotides and their modulators in the replication of animal viruses has been reported earlier. Exogenous addition of CAMP to cell cultures has been shown to inhibit replication of measles virus (Robbins and Rapp, 1980) . We have recently demonstrated that CAMP can partially reverse the inhibition of VSV replication by indomethacin, a drug which targets various enzyme systems including phosphodiesterase (Mukherjee and Simpson, 1984) . Miller and Carrigan (1982) showed that treatment of neural cells with papaverine. a potent agonist of phosphodiesterase which elevates intracellular CAMP levels, caused a marked but reversible suppression of measles virus growth. Others have found that RNA but not protein synthesis of measles virus can be inhibited by papaverine in neural and non-neural cells (Yoshikawa and Yamanouchi, 1984) . In this communication, it is shown that papaverine prevents both viral RNA and protein synthesis in HEp-2 cells infected with VSV and acts at an early stage of the viral growth cycle.

The Indiana strain of VSV was grown in cultures of BHK-21 cells for preparation of working stocks (Mukherjee and Simpson, 1984) . Cultures of BHK-21 or HEp-2 cells used in this study were maintained in reinforced MEM (RMEM) supplemented with 10% newborn calf serum (NCS). Infectivity titrations were performed by plaque assay in BHK-21 monolayer cultures as described earlier (Simpson et al., 1979) .

Virus used for primary transcription assays was labeled in its RNA by growth for 36 h in BHK-21 cells with f3H]uridine (specific activity 27.9 Ci/mmol) at 20 pCi/ml and purified by established methods (Bean and Simpson, 1973) .

The method of Baltimore et al. (1970) For determining primary transcription in vivo, the method of Bean and Simpson (1973) was used with only minor changes and HEp-2 cell monolayers (2 x lo6 cells) were infected with purified 3H-labeled VSV at an input of approximately 5 X 10' cpm (3 X lo6 pfu) per 60 mm Petri dish culture.

For determination of viral protein synthesis, HEp-2 cells infected with VSV were pulse-labeled with [35S]methionine under different regimens of drug treatment as described in the figure legends. Washed cells, lysed in Laemmli buffer (0.0625 M Tris-HCl, pH 6.8/2% SDS/lo% glycerol), were sonicated for 5 min at 4°C and combined with P-mercaptoethanol and 0.025% bromphenol blue before boiling for 5 min. The dissociated proteins were analyzed by standard discontinuous SDS-PAGE in 10% acrylamide resolving gels (Laemmli, 1970) . The 35S-labeled proteins were detected by autoradiography using Kodak XAR-5 X-ray film exposed with a Cronex lightning-plus intensifying screen for 7-10 days at -80°C. For analysis of viral RNAs, infected HEp-2 cells were pulse-labeled with inorganic [ 3'P]phosphate in phosphate-free RMEM at different times during infection and according to the drug regimens described under Results. Total RNA was extracted from washed monolayers solubilized with STE buffer containing 0.5% SDS. After the lysate was aspirated three times through a 22 gauge needle, the RNA was extracted with STE-saturated phenol and precipitated with 95% ethanol at -20°C (Bean and Simpson, 1973) . RNA preparations dissolved in STE buffer with 8 M urea and 20% glycerol were stored at -20°C until analyzed in 1% agarose acid-urea slab gels by the method of Lynch et al. (1979) . For autoradiography, gels fixed in 50% methanol were exposed to Kodak XAR-5 film with a Cronex lightning-plus intensifying screen at -80°C for 7 days. 

Preliminary experiments were carried out to establish conditions for maximum inhibition of VSV growth by papaverine in HEp-2 cells. Although papaverine was recently shown to inhibit measles virus replication by more than 99% in neural cell cultures treated at a dose level of 7 FM for 6 days (Miller and Carrigan, 1982) we found that HEp-2 cells required exposure to 30 PM papaverine (before and after infection) to obtain a 98% reduction of virus yields ( Table 1) . Use of lower doses or omitting drug after infection was less effective in reducing virus yields. Prolonged drug treatment of HEp-2 cells for more than 2 days produced cytotoxic effects. In all subsequent experiments, HEp-2 cells were treated with drug at a concentration of 30 PM and for periods not exceeding 48 h.

The reversibility of the drug effect was investigated by pretreating cells with papaverine, infecting with virus, and further incubating cells for 24 h before removal of drug. Under these conditions, there was a rapid and progressive recovery of the viral permissive state with increasing virus yields seen as a function of time after drug removal ( Table 2 ). The kinetics of virus production during the cell recovery period was similar to that of a group of untreated cultures infected at the same time (24 h p.i.) that papaverine was removed from test cultures, indicating that no viral shown). We also tested the effect of known modulators of cyclic nucleotide metabolism for their ability to counteract the viral inhibitory effect of papaverine. The most significant effect was obtained when cells were exposed to cGMP during treatment with drug (Table 3) . Cyclic GMP totally negated the papaverine block of viral replication if it was present during the entire drug treatment period. Inhibition of measles virus growth in vitro by papaverine is similarly antagonized by exogenous addition of cGMP (Miller and Carrigan, 1982) . Although papaverine is known to affect intracellular levels of Ca2+ ions which in turn can regulate phosphodiesterase activity (Carpendo et al., 1975) , we found that altering the calcium concentration of the culture medium had no apparent effect on inhibition of VSV by this drug (data not shown).

To determine the effect of drug on viral protein synthesis, papaverine-treated cells were pulse-labeled with [ 3sS]methionine during infection and total radiolabeled proteins were analyzed in SDS-PAGE gels as illustrated in Fig. 1 . The production of viral proteins was totally blocked in cells infected immediately after drug treatment (Fig. 1, lane 5 ) even though cellular protein synthesis was not apparently reduced (cf. Fig. 1, lane 4) . Under the conditions employed in these experiments, papaverine reduced yields of infectious virus to 1% of infected control cultures (data not shown). Calcium had no sparing effect on the inhibition of viral protein synthesis by this drug (Fig. 1, lane 6) . production or processing of viral mRNAs might be impaired. When papaverinetreated HEp-2 cells were analyzed for RNA species produced during the early stages of infection, none of the expected VSV mRNAs or genome-length 42s RNA were detected (Fig. 2) . Synthesis of cellular ribosomal RNA was also prevented by this drug (Fig. 2, lane 4) .

The effect of papaverine on host DNA synthesis was also examined. Fig. 3 illustrates that ['Hlthymidine uptake in drug-treated cells was totally blocked whether or not they were infected. An interesting aside of these experiments was the finding that immediately after infection with VSV, there was an appreciable stimulation of [3H]thymidine incorporation by comparison with uninfected cells (Fig. 3) . Although the true significance of this phenomenon is unknown, it appears to be related to virus uptake since it was multiplicity dependent and did not occur when Fig. 2 . Gel electrophoresis of RNA species synthesized in HEp-2 ceils treated with papaverine and infected with VSV. Cells pretreated with papaverine (30 PM) for 24 h at 37°C were infected with VSV (10 pfu/cell), incubated with fresh medium containing drug, and labeled with 32P (25 pCi/ml) from 2 to 4 h p.i. Total RNA was extracted and analyzed in acid-urea agarose gels (Materials and Methods). RNA species from: untreated, virus infected cells (Lane 1); infected cells treated with papaverine (Lane 2); mock-treated uninfected cells (Lane 3); uninfected cells treated with papaverine (Lane 4). Arrows indicate the position of 28s and 18s ribosomal RNA, respectively. infected cells were incubated in the cold (data not shown). Collectively. the foregoing experiments established that papaverine blocks host nucleic acid synthesis but not protein synthesis under conditions where no VSV-coded polypeptides or RNA species are produced.

In the absence of viral mRNA synthesis, it was of interest to determine whether papaverine directly affects viral transcription.

We tested for primary transcription in vivo in drug-treated cells by measuring the association of radiolabeled input viral genomic RNA with RNase resistant complexes during the first 2 h of infection (see Materials and Methods). Fig. 4 shows that primary transcription of VSV in ceils w 0 10 Incuba~nTime ( his. )

Time of platen (Evans and Simpson, 1980) .

[ 3H]Thymidine uptake is shown as a function of time of pulse-labeling following virus adsorption for drug-treated infected cells (A). drug-treated uninfected cells (A). mock-treated infected cells (0) and mock-treated uninfected cells (0). Fig. 4 . Effect of papaverine on primary transcription of vesicular stomatitis virus in monolayer cultures of HEp-2 cells. Cultures were exposed to RMEM (2% NCS) with or without 30 pM papaverine for 24 h at 37°C. The drug treated cultures were temperature equilibrated at 4°C and inoculated with gradient-purified VSV radiolabeled in its RNA with 'H. The virus input was approximately 10 pfu per ceil or 4 x IO5 cpm. Cells were washed with cold BSS after virus adsorption and incubated at 37°C with pre-warmed (37OC) serum-free medium containing or lacking papaverine (30 PM). Individual cultures were extracted for total RNA at different time intervals and the percentage of ribonuclease resistant radioactivity after annealing was determined (Materials and Methods). Percent RNase-resistant 3H counts as a function of time of incubation for untreated infected cultures (0) and cultures treated before and after infection with drug (0). treated with papaverine before and after infection was largely prevented during the early stages of infection.

To determine whether the impairment of primary transcription seen in these experiments might involve a direct action of drug on viral transcriptive complexes, we tested the activity of the virion transcriptase in in vitro reaction mixtures containing different concentrations of papaverine. As shown in Table 4 , transcriptase activity was incompletely inhibited in reaction mixtures with drug concentrations of 30 PM or higher. Considering that drug levels greater than 30 PM are toxic for the HEp-2 cells used in this study, this partial inhibition of in vitro viral transcriptase activity obtained suggests that the stronger block of VSV transcription induced in vivo may not necessarily reflect a direct effect of papaverine on viral transcriptive complexes under the conditions used. 

The use of papaverine in this study as a reversible inhibitor of viral replication further implicates the participation of endogenous host factors in biosynthetic activities of vesicular stomatitis virus during infection of permissive cells. Pharmacologically, papaverine (6,7-dimethoxy-l-veratryl-isoquinoline)

is an alkaloid derivative of Papaver poppies which acts as a smooth muscle relaxant by inhibiting phosphodiesterase, causing an increase in intracellular levels of 3',5'-monophosphate (cyclic AMP) (Triner et al., 1970; Carpendo et al., 1975) . Recent studies on the possible role of cyclic nucleotides as modulators of acute and persistent infections with measles virus have established that elevation of CAMP levels either by exogenous addition of this nucleotide (Robbins and Rapp, 1980) or by treatment of cells with papaverine (Miller and Carrigan, 1982; Yoshikawa and Yamanouchi, 1984) renders cells reversibly nonpermissive for measles virus replication. Yoshikawa and Yamanouchi (1984) reported that papaverine blocks measles virus in vitro at the level of viral RNA synthesis although their ability to detect relatively low levels of measles P protein in neural and non-neural cells treated with this drug would indicate that inhibition of viral transcription in this system was not absolute. Other workers have shown that papaverine can selectively inhibit production of intracellular matrix (M) protein in measles infected neural cells without negatively affecting synthesis of other viral structural proteins detected by immunofluorescent staining (Miller and Carrigan, 1982) . it would thus appear that host cell differences influence the extent to which papaverine interferes with virus-specific macromolecular synthesis observed in these systems.

Ribonucleic acid synthesis of vesicular stomatitis virus. II. An RNA polymerase in the virion

Primary transcription of the influenza virus genome in permissive cells

Calcium and papaverine interaction with soluble cardiac phosphodiesterase

Cyclic AMP, a nonessential regulator of the cell cycle

The coronavirus avian infectious bronchitis virus requires the cell nucleus and host transcriptional factors

Effects of papaverine on smooth muscle and their mechanisms

Inhibition of prostaglandin biosynthesis

Rapid and transient localization of the leader RNA of vesicular stomatitis virus in the nuclei of infected cells

Cleavage of structural proteins during assembly of the head of bacteriophage T4

Separation and purification of the mRNA's of vesicular stomatitis virus NS and M proteins

The plus-strand leader RNA of VSV inhibits DNA-dependent transcription of adenovirus and SV40 genes in a soluble whole-cell extract

Reversible repression and activation of measles virus infection in neural cells

Reversible restriction of vesicular stomatitis virus in permissive cells treated with inhibitors of prostaglandin biosynthesis

Indomethacin inhibits viral RNA and protein synthesis in cells infected with vesicular stomatitis virus

Inhibition of measles virus replication by cyclic AMP

Conditional lethal mutants of vesicular stomatitis virus. III. Host range properties, interfering capacity, and complementation patterns of specific hr mutants

Cyclic phosphodiesterase activity and the action of papaverine

Use of U.V. irradiation to identify the genetic information of vesicular stomatitis virus responsible for shutting off cellular RNA synthesis

Effect of papaverine treatment on replication of measles virus in human neural and nonneural cells

The authors thank Lesley Anderson for her cheerful and competent technical 67 assistance rendered throughout this work. This investigation was supported by funds from Research Grant AI-17262 from the National Institutes of Health, U.S. Public Health Service, and the Charles and Johanna Busch Foundation of Rutgers University.

The results of the present investigation contrast with those cited above since we found that VSV biosynthesis, including early transcriptive events, is strongly compromised by papaverine treatment of cells. This profound inhibitory effect parallels that observed in our recent studies on the ability of indomethacin, an antagonist of fatty acid cyclooxygenase, to reversibly block VSV transcription, RNA synthesis and protein synthesis in HEp-2 cells (Mukherjee and Simpson, 1985) . We also recently reported (Mukherjee and Simpson, 1984) that CAMP and specific modulators of cyclic nucleotide metabolism such as theophylline, can partially negate the inhibitory effect of indomethacin which itself can block phosphodiesterase activity at higher concentrations (Flower and Vane, 1974) . It is possible, therefore, that the inhibition of viral transcription in VSV-infected cells treated with either papaverine or indomethacin involves common host factors required for viral mRNA synthesis in vivo. Of relevant interest is our recent discovery of a host membrane protein factor from normal cells that can significantly restore in vitro RNA synthesis of functionally defective viral nucleocapsids recovered from VSV-infected cells treated with indomethacin (Mukherjee and Simpson, in preparation) .Whether this viral transcription enhancing factor can counteract the inhibitory effect of papaverine on VSV remains to be determined.The reversibility of the inhibition of virus replication by papaverine seen in this and other investigations (Miller and Carrigan, 1982; Yoshikawa and Yamanouchi. 1984) indicates that compromised host factors required for productive infections can be restored upon removal of drug. Alternatively, the elevation of intracellular CAMP resulting from exposure of cells to this drug may arrest the cell cycle (Coffin0 et al., 1975) and require a return to normal levels of this nucleotide before cells recover their permissive state for viral replication. That CAMP plays an important role in the inhibitory effects seen in this study is strongly suggested by the ability of cGMP to block papaverine action (Table 3) . However, since papaverine has complex pharmacological and biochemical properties which affect other enzyme systems besides CAMP phosphodiesterase (see Ferrari, 1974) it is entirely possible that the viral inhibitory effect obtained in this study may entail additional cellular targets.The fact that papaverine was found to inhibit both cellular DNA and RNA synthesis without adversely affecting production of host proteins would suggest that translation of existing cellular mRNA species is not blocked by this metabolic antagonist.These experiments also revealed the interesting finding that VSV causes an early stimulation of host DNA synthesis immediately after infection (Fig. 3) . This phenomenon may relate, in part, to the association of VSV-specific leader RNA species with the nuclear compartment recently demonstrated by other workers (Kurilla et al.. 1982) and may possibly represent an obligatory event that occurs prior to the shutoff of host macromolecular synthesis postulated to be caused by these early viral transcripts (Week et al., 1979; McGowan et al., 1982) .