Viral messenger RNA unmethylated in the 5′-terminal guanosine in interferon-treated HeLa cells infected with vesicular stomatitis virus

VIROLOGY112.426-435 (1981)

Viral Messenger Interferon-Treated

RNA Unmethylated in the 5’-Terminal Guanosine in HeLa Cells Infected with Vesicular Stomatitis Virus

FRANCESCA DE FERRA AND CORRADO BAGLIONI’ Department of Biological Sciences, State University of New York at Albany, Albany, New York l.%%% Accepted February 10, 1981 The distribution of viral mRNA between polysomal and nonpolysomal fractions was investigated in interferon-treated cells infected with vesicular stomatitis virus (VSV). More than half of the viral mRNA synthesized by these cells is in the nonpolysomal fraction, whereas less than one-third of the mRNA synthesized by control cells is found in this fraction. Polysomal and nonpolysomal mRNA sediment identically on sucrose density gradients, but the nonpolysomal mRNA from interferon-treated cells is undermethylated, as shown by labeling experiments with [methyL3H]methionine. Analysis of VSV mRNA after digestion with nucleases shows that all molecules are capped but about 60% of the nonpolysomal mRNA isolated from interferon-treated cells is unmethylated in the 5’-terminal G. When tested in an assay for initiation of protein synthesis, only 45% of this mRNA binds to ribosomes, as compared to 80% binding for polysomal mRNA from either control or interferon-treated cells.

Joklik and Merigan (1966) reported that in interferon-treated cells the translation of viral mRNA is inhibited. These authors clearly showed that less vaccinia mRNA is associated with polyribosomes in interferon-treated cells than in control cells, but could not distinguish between an effect of interferon on the viral mRNA or on the translational apparatus. A similar observation was recently made by Simili et al. (1980), who found a larger proportion of vesicular stomatitis virus (VSV) mRNA not associated with polyribosomes in interferon-treated cells than in control cells. Moreover, synthesis of all VSV proteins was inhibited in interferon-treated cells, suggesting that VSV mRNA was not efficiently translated in these cells. The present investigation was undertaken with the aim of explaining these observations in view of the recent advances in our understanding of the mechanism of action of interferon (Baglioni, 1979). Interferon induces the synthesis of several new proteins in animal cells (Gupta et al., 1979; Knight and Korant, 1979). Among these proteins are a protein kinase that phosphorylates the cysubunit of ini1To whom reprint requests should be addressed. 0042-6822/81/100426-10$02.00/O Copyright0 1981 by Academic Press, Inc. All rights

of reproduction

in any form

reserved.

426

tiation factor eIF-2 and the 2’,5’-oligo(A) polymerase or synthetase, that polymerizes ATP into a series of oligoadenylates (see for references, Baglioni, 1979). These interferon-induced enzymes require double-stranded RNA (dsRNA) for activation (Roberts et al., 1976; Minks et al., 1979). The 2’,5’-oligo(A) polymerase has been implicated in the inhibition of replication of picornaviruses because 2’,5’-oligo(A), which activates an endonuclease (Baglioni et al., 1978; Clemens and Williams, 1978), is found in elevated levels in interferontreated L cells infected with encephalomyocarditis virus (EMCV) (Williams et al., 1979). The 2’,5’-oligo(A) is presumably formed upon activation of the polymerase by viral replicative complexes containing dsRNA. Nilsen et al. (1981) demonstrated the formation of viral dsRNA in infected cells by crosslinking complementary strands of EMCV RNA in intact cells with a psoralen derivative. Viral dsRNA, however, could not be detected in cells infected with VSV by the same experimental approach (Nilsen et al., 1981). It seems possible, therefore, that some interferoninduced activity other than the dsRNAactivated enzymes may be involved in the inhibition of VSV replication.

VSV mRNA UNDERMETHYLATION

IN INTERFERON-TREATED

CELLS

427

sine, and 20 PM guanosine. After 20 min, 40 &i/ml of [methy2-3H]methionine was added for the times indicated in figure legends. Viral RNA was doubly labeled in some experiments by including 0.2 &i/ml of [14C]uridine. Cell fractionation and analysis of VSV RNA. The cells were broken by homogenization as described by Weber et al. (1975) and centrifuged for 5 min at 30,000 g. The supernatant fraction obtained was centrifuged for 90 min at 40,000 rpm in the SW41 rotor to display polysomes as described by Simili et al. (1980). Gradient fractions were precipitated with ethanol after adjusting the NaCl concentration to 0.2 M. Alternatively, polysomes and nonpolysoma1 fractions were separated by a modification of the method of Rose and Lodish (1976). This procedure, which involves centrifugation through sucrose gradients containing 0.5 M NaCl and 30 mM Mg(OAc)z, has been previously described MATERIALS AND METHODS by Simili et al. (1980). Pellets were disCell infection. HeLa cells were grown in solved in 0.5% sodium dodecyl sulfate and suspension culture in Joklik’s modified poly(A)+ RNA was isolated by chromatography on oligo(dT)-cellulose columns minimum essential medium as previously as described by Weber et al. (1979). described (Simili et al., 1980). Wild-type VSV (Indiana serotype) was grown in L Poly(A)+ RNA labeled with [metI@was chromatographed cell monolayer cultures infected at a m.o.i. 3H]methionine, of 0.02 and assayed as previously described twice on oligo(dT)-cellulose and heated for (Simili et al., 1980). Stock titers were l-5 2 min at 70” before the second chromaX 10’ PFU/ml. HeLa cells were treated for tography. The poly(A)+ RNA was precipitated with ethanol after addition of 25 18 hr with 40 units/ml of human fibroblast interferon (HuIFN-8; 3 X lo5 reference pg/ml of carrier tRNA and 0.25 M LiCl. units/mg of protein, obtained from the The RNA was dissolved in water and O.lml aliquots were taken for digestion with Interferon Working Group of the National Cancer Institute, NIH). Control and inter- nucleases at 37”. The digestion with 1 unit feron-treated HeLa cells were infected at of T2 RNase was in 10 mM acetate buffer, a m.o.i. of 10 after washing with serum- pH 5.2, for 16 hr. At the end of this digesfree medium and resuspending the cells tion, the pH was raised to 7 by the addition at 4 X 106/ml. After 45 min the cells were of 2 M Tris; 0.1 M MgClz was added to 1.5 diluted with an equal volume of medium mM and the samples were incubated with containing 10% fetal calf serum. The cells 0.5 unit of bacterial alkaline phosphatase (BAP) for 1 hr. For Pl nuclease digestion, were treated with 5 bg/ml of actinomycin D for 15 min prior to labeling with 10 pCi/ the pH was adjusted to 6.0 with 5 mM ml of [3H]uridine for the times indicated NaOAc and 3 units of Pl was added for in figure legends. To label methylated nu- 90 min; the subsequent digestion with cleotides, the cells were washed and re- BAP was carried out as described above. suspended in medium containing 5% di- For digestion with nucleotide pyrophos-. phatase, 0.1 M MgClz was added to the Pl alyzed horse serum, 5 pg/ml actinomycin D, 20 mM sodium formate, 20 PM adeno- digest to a final concentration of 1.5 mM

An effect of interferon that may play a role in the inhibition of viral replication was reported by Sen et al. (1975,197’7) and by Shaila et al. (1977). These authors compared the methylation of capped but unmethylated reovirus mRNA added to control cell extracts or to extracts of interferon-treated cells. The methylation of the 5’-terminal guanosine was impaired in the latter cell extracts. This effect may be due to a macromolecular inhibitor of methylation that does not require dsRNA for activation (Sen et al., 1977). We have examined whether this or a similar effect of interferon may be responsible for the inefficient translation of VSV mRNA and report here that a significant proportion of the VSV mRNA not associated with polysomes in interferon-treated cells is not methylated in the 5’-terminal guanosine residue.

428

DE FERRA AND BAGLIONI

and the samples were incubated with 0.6 unit of pyrophosphatase for 1 hr. This digestion was followed by incubation with BAP as described above. An&&s of RNA digests. T2 RNase and Pl nuclease plus BAP digests were mixed with the unlabeled markers indicated in the figures and chromatographed on DEAE-cellulose columns (0.7 X 25 cm) equilibrated with 10 mM NaCl, 7 M urea, 20 mM Tris/HCl, pH 7.6. After washing the columns with this buffer, charged oligonucleotides were eluted with a gradient from 10 to 200 mM NaCl and counted as previously described (Minks et al., 1979). Nucleosides obtained by enzymatic digestion of poly(A)+ RNA were separated by paper chromatography in isopropanol/ water/ammonia (7/2/l). Unlabeled markers were run in parallel and visualized by uv absorption. The chromatograms were cut into l-cm strips which were transferred to scintillation vials, eluted with 1 ml of 1 M LiCl, and counted in Scintiverse (Fisher). Preparation of VSV mRNA for binding Control to reticulocgte ribosomes. cells were incubated with 10 &i/ml of r3H]uridine from 2 to 4 hr after infection; cells pretreated with 40 units/ml of interferon were incubated from 3 to 5 hr after infection. Cell extracts, polysomal and nonpolysomal fractions, poly(A)+ RNA and mRNA isolated by centrifugation on sucrose gradients, were prepared as described above. Fractions corresponding to the 12-16 S peak were combined and precipitated with ethanol. The binding of VSV mRNA to rabbit reticulocyte ribosomes was assayed as described by Weber et al. (1978). RESULTS

Distribution of VSV mRNA between PO& somal and Nonpol~somul Fractions Treatment of HeLa cells with high concentrations of interferon inhibited viral RNA synthesis upon infection with VSV. To label VSV mRNA in these cells it was therefore convenient to treat the cells with interferon concentrations that were not

markedly inhibitory for VSV RNA synthesis and to infect the cells at relatively high m.o.i. We chose an 18-hr treatment with 40 units/ml of HuIFN-8 and a m.o.i. of 10, on the basis of our previous work on interferon-treated HeLa cells (Simili et al., 1980). Viral RNA was labeled in control and interferon-treated cells infected in this way by incubation with [3H]uridine in the presence of actinomycin D (see Materials and Methods). Viral RNA synthesis peaked at 3.5 hr in control cells but about one hour later in interferon-treated cells (data not shown). The distribution of viral RNA between polysomal and nonpolysomal fractions was examined by fractionating extracts obtained from cells labeled from 2 to 4 hr postinfection by sucrose gradient centrifugation (Fig. 1). The poly(A)+ RNA was then isolated from the polysomal and nonpolysomal fractions by oligo(dT)-cellulose chromatography. A relatively larger fraction of poly(A)+ RNA sedimented in the nonpolysomal fraction in interferon-

Contml

,n+erferon-Trwfed

I

FIG. 1. Distribution of VSV RNA between polysomal and nonpolysomal fractions in control and interferon-treated cells. The treatment with HuIFN@ (40 units/ml) was for 18 hr. Cells infected with VSV were labeled from 2 to 4 hr postinfection with 10 &i/ml of PHjuridine in the presence of 5 pg/ml of actinomycin D. Cell extracts were prepared and fractionated as described under Materials and Methods. An aliquot of each fraction was counted and fractions 1-16 (I, polysomes) and 17-23 (2, nonpolysomal) were pooled. The peak sedimenting at about 110 S is viral nucleocapsid present in reduced amounts in interferon-treated cells.

VSV mRNA UNDERMETHYLATION

treated cells than in control cells (Table 1, expt. 1). The polysomal and nonpolysomal fractions were also fractionated on gradients containing 0.5 M NaCl (see Materials and Methods). These ionic conditions improved the resolution between polysomes and nonpolysomal RNA by promoting the dissociation of proteins complexed with mRNA in ribonucleoprotein particles (Greenberg, 1975). The distribution of VSV mRNA between polysomal and nonpolysomal fractions was thus examined at different times after infection and in cells labeled with both uridine and [methyl-3H]methionine (Table 1). Consistently, about one-third of the viral mRNA is not bound to polysomes in control cells, whereas more than one-half is not bound to polysomes in interferon-treated cells. The presence of VSV mRNA nonassociated with polysomes in control cells can be explained by the synthesis of an excess of viral mRNA. It seems unlikely, however, that a similar explanation accounts for the increased level of nonpolysomal ‘mRNA in interferon-treated cells, since TABLE

IN INTERFERON-TREATED

CELLS

429

these cells synthesize reduced amounts of viral mRNA. A possible explanation for this finding is that the viral mRNA synthesized by interferon-treated cells is defective. Simili et al. (1980) had previously shown that polysomal and nonpolysomal VW mRNA sediment identically on sucrose gradients and therefore ruled out major structural alterations of the viral mRNA synthesized in interferon-treated cells. In the following experiments we compared the methylation patterns of VSV mRNA in these cells and in control cells. Methylation of VW mRNA in InterferonTreated Cells VSV mRNA prepared from cells labeled with both [14C]uridine and [methyL3H] methionine was analyzed by sucrose gradient centrifugation (Fig. 2). The viral

1

DISTRIBUTION OF VIRAL mRNA BETWEEN POLYSOMAL AND NONPOLYSOMAL FRACTIONS IN CONTROL AND INTERFERON-TREATED CELLS’ Nonpolysomal poly(A)+ RNA (% ) Experiment 1 2

3

Labeling time (hr postinfection) 2-4.5 l-2.5 l-4.5 l-6.5 2-5

Control 39 29 31 23 34

Interferon treated 60 55 72 61 52

“The cells were treated with interferon and infected with VSV as described under Materials and Methods. The separation of polysomal and nonpolysomal fractions of expt 1 is shown in Fig. 1. These fractions were separated by centrifugation on gradients containing 0.5 M NaCl for expts 2 and 3. The sedimentation of poly(A)+ RNA analyzed in expt 3 is shown in Fig. 2. The nonpolysomal poly(A)f RNA is expressed as a percentage of the total poly(A)+ RNA recovered in each experiment.

Fractions

FIG. 2. Sedimentation analysis of VSV mRNA labeled with [methy63H]methionine and [“Cluridine. Interferon-treated and control cells were labeled from 2 to 5 hr postinfection as described under Materials and Methods. The poly(A)+ RNA was isolated by chromatogrphy on oligo(dT)-cellulose from the polysomal and nonpolysomal fractions obtained by centrifugation on gradients containing 0.5 M NaCl (see Materials and Methods). Centrifugation of poly(A)+ RNA was for 16 hr at 34,090 rpm in the SW41 rotor. The ‘H/“C ratio calculated for fractions 7-9 is indicated.

430

DE FERRA AND BAGLIONI

mRNA from both polysomal and nonpolysomal fractions sedimented in a 12-16 S peak. Some RNA sedimenting toward the bottom of the gradient was also observed in the polysomal fraction (Fig. 2). This RNA was not further investigated. The relative methylation of the RNA in the 12-16 S peak was determined by its 3H/‘4C ratio; the nonpolysomal RNA from interferon-treated cells was undermethylated relative to polysomal RNA or to nonpolysomal RNA from control cells (Fig. 2). VSV mRNA is methylated at two positions by viral transmethylase activities (see for references, Banerjee, 1980): in the 5’-terminal m7G and in the Z-OH of the adenosine linked to m7G (Moyer et al., 1975). Two further methyl groups are added by cellular transmethylases to about half the VSV mRNA molecules (Moyer and Banerjee, 1976). Since VSV mRNA is not internally methylated (Moyer and Banerjee, 1976), all the methyl groups are contained in the common 5’-terminal sequence m7Gppp(m6)AmpA~“~pCp (where the parentheses indicate methyl groups transferred by cellular enzymes in fractional amounts and the superscript m designates a 2’-0-methylation). The undermethylation in nonpolysomal VSV mRNA of interferon-treated cells could be either due to improper “capping,” resulting in molecules without 5’-terminal G or to reduced methylation at any of the three 5’-terminal nucleotides. To distinguish between these possibilities, we digested polysomal and nonpolysomal mRNA obtained from control and interferontreated cells with T2 RNase and bacterial alkaline phosphatase (BAP), as described under Materials and Methods. The products of digestion were fractionated by chromatography on DEAE-cellulose columns with a NaCl gradient that separates nucleotides according to their charge (Minks et al., 1979). No significant difference in the elution profile of the different RNA samples was observed (Fig. 3). Methylated oligonucleotides eluted with a nominal charge of about -3.5 and -4.5, as expected for digestion products containing a triphosphate bridge linking a terminal

FIG. 3. Chromatographic analysis of T2 RNase digest of VSV mRNA. Control and interferon-treated cells were incubated with 40 &i/ml of [metA& ‘Hlmethionine from 2 to 4 and 3 to 5 hr postinfection, respectively. Poly(A)+ RNA isolated from polysomal and nonpolysomal fractions was digested with T2 RNase and phosphatase, and the products of digestion were chromatographed on DEAE-cellulose columns (see Materials and Methods).

G to either two or three additional nucleotides (the presence of a 2’-O-methyl group in the A residues renders the phosphodiester bond resistant to T2 RNase digestion). This indicates that all VSV mRNA species labeled with [methyL3H]G; methionine contain a “capping” (p)p(m6)A”pA(“‘pC-terminated mRNAs would be digested by T2 and BAP to (m6)A”pA”pC (with charge -2) and (m’)A”pA (with charge -1). This chromatographic analysis also rules out that VSV mRNA is internally methylated in interferon-treated cells, since no methylated dinucleotides eluting with a -1 charge were observed. The 5’ termini of VSV mRNA were further analyzed after digestion with Pl nuclease, which cleaves phosphodiester bonds between methylated nucleotides, and BAP. The elution pattern of the resulting dinucleotides was compared with that of synthetic m7GpppAm and GpppA” (Fig. 4). These dinucleotides were resolved by DEAE-cellulose chromatography because the N-7’+’ group in m7G partially neutralizes the charge of one phosphate group (Hickey et al., 1977). The dinucleotide prepared from nonpolysomal mRNA of control cells was eluted with m7GpppA”,

VW mRNA UNDERMETHYLATION

FIG. 4. Chromatographic analysis of VSV mRNA digested with Pl nuclease, and bacterial alkaline phosphatase. VSV-infected cells were incubated as described in Fig. 3; poly(A)+ RNA was isolated and digested as described under Materials and Methods. Fractionation of unlabeled cap markers is shown in (A); the material in peak X is unidentified. The same markers were included in the analysis of the digests of nonpolysomal poly(A)+ RNA obtained from control (B) and interferon-treated cells (C). The position of the peaks absorbing at 260 nm is indicated by the arrows.

whereas that prepared from interferontreated cells eluted in two poorly resolved peaks corresponding to the markers. In all these chromatographic analyses a small peak eluted before the marker or labeled dinucleotide peaks. The material in this peak was not further studied, but it seems possible that it represents a degradation product of the dinucleotides. VSV mRNA samples were then digested with Pl nuclease, pyrophosphatase, and BAP to obtain complete enzymatic hydrolysis to nucleosides (see Materials and Methods). The nucleosides were separated by paper chromatography and identified by running in parallel m’G, m6Am, and A” markers (Fig. 5). When double-labeled mRNA was analyzed in this way, [r4C]uridine was resolved from the methylated nucleosides (Table 2). The amount of m7G recovered is calculated either relatively to the other methylated nucleosides or relative to [14C]uridine. In each case, there is a deficit of m7G methylation for the mRNA obtained from interferontreated cells, particularly for the nonpolysomal mRNA fraction, whereas the methylation pattern of viral mRNA from

IN INTERFERON-TREATED

CELLS

431

control cells is in agreement with that previously reported by Moyer and Banerjee (1976). The finding that polysomal mRNA from interferon-treated cells contains less m’G than mRNA from control cells may be explained by an incomplete resolution of polysomal from nonpolysomal fractions. An interesting difference between polysomal and nonpolysomal mRNA of both control and interferon-treated cells was observed in these experiments. The relative content of m6Am was higher in polysomal mRNA, suggesting that mRNAs containing this nucleoside may be preferentially translated and therefore associated with polysomes. To relate the inhibition of cap G methylation to interferon concentration, we treated HeLa cells with 40 to 120 units/ml of interferon and measured the relative labeling of methylated nucleosides of viral mRNA isolated from infected cells (Fig. 6). The results obtained show that treatment with higher interferon concentrations resulted in greater inhibition of cap G methylation. It should be pointed out, however, that the range of interferon concentrations tested in this experiment is necessarily limited, since not enough viral

FIG. 5. Chromatographic analysis of the nucleosides obtained by enzymatic hydrolysis of poly(A)+ RNA of control and interferon-treated cells. VSVinfected cells were incubated as described in Fig. 3, poly(A)+ RNA was isolated and digested as described under Materials and Methods. The nucleosides were fractionated by paper chromatography and unlabeled markers were run in parallel (see Materials and Methods). One-centimeter-long strips were cut and counted. The bars indicate the position of the markers.

432

DE FERRA

AND BAGLIONI

TABLE

2

METHYLATIONOF VSV mRNA INCONTROLANDINTERFERON-TREATEDCELLS"

Experiment/fraction 1. Control Control

polysomal nonpolysomal

1. Interferon-treated Interferon-treated 2. Control Control

polysomal nonpolysomal

polysomal nonpolysomal

2. Interferon-treated Interferon-treated

polysomal nonpolysomal

Estimated m7G (%)

m’G

A”

m6Am

U

m7G/Am + m6Am

m7G/U

3350 2410

2930 1670

4860 1790

5760 4850

0.43 0.69

0.58 0.50

100 86

1940 850

2750 2530

2210 1290

4590 4060

0.40 0.22

0.42 0.22

77 37

8840 3510

6980 2200

10630 3310

-

0.50 0.63

-

-

6920 2709

6070 4100

11050 4190

-

0.40 0.32

-

-

‘In experiment 1 doubly labeled RNA was analyzed and the percentage m7G is calculated from the mrG/U ratio, by assuming that 100% of the 5’-terminal G in polysomal mRNA of control cells is methylated.

mRNA is synthesized in cells treated with more than 120 units/ml of interferon to make such an analysis reliable.

tions provide direct evidence for a functional alteration of the viral mRNA synthesized in interferon-treated cells infected with VSV.

Binding of VSVmRNA to Reticulocyte RibOScmZeS

The functional significance of the inhibition of cap G methylation was investigated by measuring the formation of initiation complexes between VSV mRNA and rabbit reticulocyte ribosomes in the presence of sparsomycin, according to Shafritz et al. (1976) and Weber et al. (1978). Labeled viral poly(A)+ 12-16 S RNA was isolated from control and interferon-treated cells as shown in Fig. 2. This RNA was incubated with reticulocyte lysate and mRNA bound to 80 S ribosomes was separated from unbound mRNA by sedimentation in sucrose density gradients (Fig. 7). Most of the mRNA from control cells was bound to reticulocyte ribosomes, whereas the mRNA from interferon-treated cells was bound less efficiently. The viral mRNA was also isolated from the polysomal and nonpolysomal fractions and assayed for ribosome-binding activity. The mRNA from both fractions of control cells was efficiently bound, whereas the nonpolysomal mRNA from interferon-treated cells showed a much reduced binding activity. These observa-

DISCUSSION

Less VSV mRNA is associated with polysomes in interferon-treated cells than in control cells. This observation led us to analyze VSV mRNA synthesized by interferon-treated cells. The mRNA isolated from the polysomal and nonpolysomal fraction appears to be identical in size, but a large fraction of the nonpolysomal mRNA is not methylated in the 5’-terminal G. Since the m7G in the cap promotes binding of mRNA to ribosomes (see for references, Shatkin, 1976), it seems likely that the VSV mRNA unmethylated in the 5’4erminal G is poorly translated. The reasons for the inhibition of cap G methylation in interferon-treated cells are unclear. It seems possible that the inhibitor of methylation detected in extracts of interferon-treated ascites (Sen et al., 1975, 1977) and HeLa cells (Shaila et al., 1977) may be responsible for this effect of interferon. It is also possible that a decreased level of AdoMet in interferontreated cells may have a similar effect, since in vitro the 7-methylation requires a 50-fold higher concentration of AdoMet

VSV mRNA UNDERMETHYLATION

40

0

HuIFN-/3 (units/ml)

FIG. 6. Ratio of ‘I-methylguanosine to methylated adenosines in VSV mRNA isolated from cells treated with different interferon concentrations. The cells were treated for 18 hr with the indicated interferon (HuIFN-P) concentration. Six milliliters of control cells or cells treated with 40 units/ml, 12 ml of cells treated with 80 units/ml, and 24 ml of cells treated with 120 units/ml of interferon were infected with VSV as described under Materials and Methods. The cells were labeled with 40 rCi/ml of [methyl3H]methionine at 2 to 3.5 hr postinfection for control cells or at 3 to 4.5 hr postinfection for interferontreated cells. Extracts prepared at the end of the incubation were fractionated by sucrose density gradient centrifugation as shown in Fig. 2. An aliquot of the gradients fractions was counted and the fractions corresponding to 12-16 S RNA were pooled and chromatographed on oligo(dT)-cellulose. The poly(A)containing RNA was precipitated with ethanol, redissolved, and digested to nucleosides as described in Fig. 5. These were separated by paper chromatography and counted as described under Materials and Methods.

than the 2’-O-methylation (Testa and Banerjee, 1977). This requirement differs from those of the reovirus and vaccinia systems, in which the ‘7-methylation precedes the 2’-0-methylation even at low AdoMet concentrations (see for references, Banerjee, 1980). In agreement with this tentative explanation, Desrosiers and Lengyel (1977) showed that in interferontreated cells infected with reovirus the relative amount of viral mRNAs 2’-Omethylated in the third nucleotide is decreased. A similar decrease in 2’-O-methylation was observed in vaccinia mRNA of interferon-treated cells by Kroath el al. (1978). The inhibition of cap G methylation in VSV-infected cells may represent a significant component of the antiviral state.

IN INTERFERON-TREATED

CELLS

433

An impairment of viral mRNA translation is likely to produce a cascade effect, because viral macromolecular synthesis is dependent on a progressive increase in viral protein synthesis. The effect of even a modest inhibition of protein synthesis on VSV replication was shown by Yau et al. (1978). These authors treated infected cells with subinhibitory concentrations of cycloheximide and observed that a 40% inhibition of protein synthesis reduced virus yield by more than 95%. In the experimental conditions described in this manuscript, only translation of viral mRNAs unmethylated in cap G would be

FIG. 7. Binding of VSV mRNA to reticuloeyte ribosomes. VSV mRNA was obtained from control and interferon-treated cells. Poly(A)+ RNA was prepared by chromatography on oligo(dT)-cellulose from unfractionated cell extract or from polysomal and nonpolysomal fractions. The latter RNAs were fractionated by sedimentation on gradients as shown in Fig. 2. The fractions corresponding to the 12-16s peak were combined. The RNA was precipitated with ethanol and dissolved in water. Aliquots containing about 10,000 cpm of labeled RNA were added to 0.1 ml binding reactions prepared according to Weber et al. (1978); these were centrifuged on 15-30% sucrose gradients for 16 hr at 22,009 rpm. A control incubation kept at 0” is shown in the upper left panel (0 - - 0). All other incubations were for 5 min at 30”. The amount of VSV mRNA bound to 80 S ribosomes is indicated in each panel. The position of 80s ribosomes, 60 S, and 40 S ribosomal subunits is indicated by the arrows (left to right).

434

DE FERRA AND BAGLIONI

impaired, but the overall effect on virus yield may be similar to that observed by Yau et al. (1978). It should be pointed out that these conditions were chosen in order to allow synthesis of VSV mRNA in amounts sufficient for its analysis. In cells treated with high concentrations of interferon there is very little synthesis of viral mRNA (Friedman, 1977) and it is difficult to obtain enough RNA for an analysis of methylated nucleosides. In cells treated with high concentrations of interferon the inhibition of cap G methylation is more pronounced (Fig. 6) and viral protein synthesis may be inhibited at an early stage after infection. The sensitivity of VSV replication to inhibitors of methylation was shown by Caboche and La Bonnardiere (1979), who treated infected cells with cycloleucine. This compound inhibits methylation and reduces viral protein synthesis. A different effect of interferon treatment on VSV replication was reported by Maheshwari and Friedman (1979). Interferon-treated cells produce VSV particles with low infectivity and reduced G and M protein content (Maheshwari and Friedman, 1980; Maheshwari et al., 1980). These observations may in part be explained by the finding of Lodish and Porter (1980) that VSV particles formed at early times after infection, when the pool of viral proteins is relatively small, as it is in interferon-treated cells, contain only one-third the amount of G protein, relative to internal structural proteins. Still another mechanism that may reduce the infectivity of VSV released from interferontreated cells was recently suggested by Wallach and Revel (1980), who detected the enzyme 2’,5’-oligo(A) polymerase in virus particles. Activation of this enzyme upon infection may elicit endonuclease activity and degradation of viral templates. The antiviral state results from the induction of several proteins (Gupta et al., 1979; Knight and Korant, 1979), which may have specific inhibitory effects in infections by different viruses. Several antiviral mechanisms may thus be activated in interferon-treated cells (Baglioni, 1979).

It is not possible at the present time to evaluate the relative contribution of each effect of interferon to the inhibition of viral replication. The inhibition of cap G methylation reported here may play an important role in limiting synthesis of VSV proteins, but it remains to be established whether this is a major mechanism by which VSV yield is reduced in interferon-treated cells. ACKNOWLEDGMENT This research was supported by Grant AI-16076 from the National Institutes of Allergy and Infectious Diseases. REFERENCES BAGLIONI, C. (1979). Interferon induced enzymatic activities and their role in the antiviral state. CeU 17,255-264.

BAGLIONI, C., MINKS, M. A., and MARONEY, P. A. (1978). Interferon action may be mediated by activation of a nuclease by pppA2p5AZp5’A. Nature (London) 273, 684-687. BANERJEE, A. K. (1980). 5’-Terminal Cap Structure in Eukaryotic Messenger Ribonucleic Acids. ikficrobid Rev. 44,175-205. CABOCHE,M., and LABONNARDIERE,J. (1979). VSV mRNA methylation in tivo: effect of cycloleucine an inhibitor of S-adenosylmethionien biosynthesis on viral transcription and translation. Virology 93, 547-557.

CLEMENS, M. J., and WILLIAMS, B. R. G. (1978). Inhibition of cell-free protein synthesis by pppA2’p5’A2’p5’A: A novel oligonucleotide synthesized by interferon-treated L cell extracts. CeU 13, 565-572.

DESROSIERS,R. C., and LENGYEL, P. (1977). Impairment of reovirus mRNA cap methylation in interferon-treated L cells. Fed Proc 36, 812. FRIEDMAN, R. M. (1977). Antiviral activity of interferons. Bacterid Rev. 41,543-567. GREENBERG,J. R. (1975). mRNA metabolism of animal cells. J. CeU Biol. 64, 269-288. GUPTA, S. L., RUBIN, B. Y. and HOLMES,S. L. (1979). Interferon action: Induction of specific proteins in mouse and human cells by homologous interferons. Proc. Nat. Ad Sci. USA 76,4817-4821. HICKEY, E. D., WEBER,L. A., BAGLIONI,C., KIM, C. H., and SARMA, R. (1977). A relationship between inhibition of protein synthesis and conformation of 5’-phosphorylated ‘I-methylguanosine derivatives. J. MoL Bid 109,173-183. JOKLIK, W. K., and MERIGAN,T. C. (1966). Concerning the mechanism of action of interferon. Proc Nat. Acd Sci. USA 56, 558-565.

VSV mRNA UNDERMETHYLATION KNIGHT, E. JR., and KORANT,B. D. (1979). Fibroblast interferon induces synthesis of four proteins in human fibroblast cells. Proc Nat. Acad Sci. USA 76,1824-182’7. KROATH, H., GROSS,H. J., JUNGWIRTH,C., and BODO, G. (1978). RNA methylation in vaceinia virusinfected chick embryo fibroblasts treated with homologous interferon. Nucleic Acid Res. 5, 24412454. LODISH, H. F., and PORTERM. (1980). Heterogeneity of vesicular stomatitis virus particles: Implications for virions assembly. J. Viral. 33, 52-58. MAHESHWARI, R. K., and FRIEDMAN, R. M. (1979). Production of vesicular stomatitis virus with low infectivity by interferon-treated cells. J. Gen Viral. 44,261-264. MAHESHWARI, R. K., and FRIEDMAN, R. M. (1980). Effect of interferon treatment on vesicular stomatitis virus (VSV): Release of unusual particles with low infectivity. Virology 101, 399-407. MAHESHWARI,R. K., DEMSEY,A. E., MOHANTI, S. B., and FRIEDMAN, R. M. (1980). Interferon-treated cells release vesicular stomatitis virus particles lacking glycoprotein spikes: Correlation with biochemical data. Proc. Nat. Acad Sci. USA 77,22842287. MINKS, M. A., BENVIN, S., MARONEY, P. A., and BAGLIONI, C. (1979). Synthesis of 2’5’-oligo(A) in extracts of interferon-treated HeLa cells. J. Biol. Chem. 254,5058-5064.

MOYER, S. A., and BANERJEE, A. K. (1976). In viva methylation of VSV and its host-cell mRNA species. Virology 70, 339-347. MOYER,S. A., ABRAHAM, G., ADLER, R., and BANERJEE, A. K. (1975). Methylated and blocked 5’ termini in VSV in viva mRNAs. CeU 5.59-67. NILSEN, T. W., WOOD, D., and BAGLIONI, C. (1981). Cross-linking of viral RNA by I’-aminomethyl4,5’,8-trimethylpsoralen in HeLa cells infected with Encephalomyocarditis virus and the tsGll4 mutant of Vesicular Stomatitis virus. Virology 109, 82-93. ROBERTS,W. K., HOVANESSIAN,A., BROWN,R. E., CLEMENS,M. J., and KERR, I. M. (1976). Interferonmediated protein kinase and low molecular weight inhibitor of protein synthesis. Nature (London) 264, 477-480. ROSE,J. K., and LODISH, H. F. (1976). Translation in vitro of vesicular stomatitis virus mRNA lacking 5’terminal ‘I-methylguanosine. Nature (London) 262, 32-37. SEN, G. C., LEBLEU,

B., BROWN, G. E., REBELLO,

M. A.,

FURUICHI, H., MORGAN, M., SHATKIN, A. J., and LENGYEL,P. (1975). Inhibition of reovirus messenger RNA methylation in extracts of interferon-

IN INTERFERON-TREATED

CELLS

treated Ehrlich aseites tumor cells. B&hem phys. Res. Commun

435 Bit+

65,427-434.

SEN, G. C., SHAILA, S., LEBLEU, B., BROWN,G. E., DESROSIER,R. C., and LENGYEL, P. (1977). Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrlich ascites tumor cells: Further characteristics of the phenomenon. J. Virol 21, 69-83. SHAFRITZ,D. A., WEINSTEIN, J. A., SAFER, B., MERRICK,W. C., WEBER,L. A., HICKEY, E. D., and BAGLIONI, C. (1976). Evidence for role of m’G’-phosphate group in recognition of eukaryotic mRNA by initiation factor IF-MS. Nature (London) 261,291294. SHAILA, S., LEBLEU, B., BROWN,G. E., SEN, G. C., and LENGYEL, P. (1977). Characteristics of extracts from interferon-treated HeLa cells: Presence of a protein kinase and endoribonuclease activated by double-stranded RNA and of an inhibitor of mRNA methylation. J Gen. Viral 37, 535-546. SHATKIN, A. J. (1976). Capping of eukaryotic mRNAs. Cell 9, 645-653. SIMILI, M., DE FERRA, F., and BAGLIONI, C. (1980). Inhibition of RNA and protein synthesis in interferon-treated HeLa cells infected with VSV. J. Gen. Viral. 47, 373-384.

TESTA, D., and BANERJEE,A. K. (1977). Two methyltransferase activities in the purified virions of vesicular stomatitis virus. J. Virol. 24, 786-793. WALLACH, D., and REVEL, M.(1980). An interferon induced cellular enzyme is incorporated into virions. Nature (London) 287, 68-70. WEBER, L. A., FEMAN, E., and BAGLIONI, C. (1975). A cell free system from HeLa cells active in initiation of protein synthesis. Biochemistry 14,53155321. WEBER,L. A., HICKEY, E. D., and BAGLIONI,C. (1978). Influence of potassium salt concentration on inhibition of mRNA translation by ‘I-methylguanosine-B-monophosphate. J. Biol. Chem. 253,178-183. WEBER, L. A., SIMILI, M., and BAGLIONI, C. (1979). Binding of viral and cellular messenger RNAs to ribosomes in eukaryotic cell extracts. In “Methods in Enzymology” (K. Moldave and G. Grossman, eds.), Vol. 60, pp. 351-360. Academic Press, New York. WILLIAMS, B. R. G., GOLGHER,R. R., BROWN,R. E., GILBERT, C. S., and KERR, I. M. (1979). Natural occurrence of 2-5A in interferon-treated EMC virus-infected L cells. Nature (London) 282.582-586. YAU, P. M. P., GODEFROY-COLBIJRN, T., BIRGE, C. H., RAMABHADRAN, T. V., and THA(:H, R. E. (1978). Specificity of interferon action in protein synthesis. J. Viral. 27, 648-658.