Inhibition by interferon of the uncoating of vaccinia virus

36, 505-511 (1968)

VIROLOGY

Inhibition WAYNE

by Interferon

E. MAGEE,

Virology

SEYMOUR

Research,

of the Uncoating LEVINE, OLGA HAMILTON

The Upjohn

Company

Accepted April

of Vaccinia V. MILLER,

Kalamazoo,

Michigan

AND

Virus RAMON

D.

@OOl

29, 2968

The effect of interferon was determined on those steps in vaccinia virus infection that precede release of viral DNA into the cytoplasm. These include loss of the outer protein coat of the particle which exposes the “core” and release of t)he DNA from the core (uncoating). Primary cult,ures of chicken embryo fibroblasts were treated with interferon and infected with radioactive vaccinia virus prepared with thymidine-3H in the DNA. At various times after infection the cells were ruptured, and the amounts of virus, cores, and viral DNA were determined after separation of these components by sucrose density centrifugation. Uncoating also was measured by DNase sensitivity of the viral DNA. Interferon did not alter either the rate of disappearance of virus particles or the rate of formation of cores. However, uncoating was inhibited strongly so that very little viral DNA was liberated and cores tended to accumulate. The response of uncoating to increasing concentrations of interferon was similar to that determined previously for the synthesis of viral DNA-polymerase and viral DNA. These observations suggest that uncoating is a viral function. Not enough cores accumulated in the interferon-treated cells to account for all the virus that disappeared. Experiments with heat-inactivated virus, and with normal virus in the presence of cycloheximide, showed that chicken embryo fibroblasts can digest both virus and cores to acid-soluble materials without accumulating acidinsoluble intermediates. The inhibition of uncoating obtained with cycloheximide closely resembled that seen with interferon. INTRODUCTION We previously reported that synthesis of vaccinia virus DNA-polymerase and DNA is inhibited in interferon-treated cells (Levine et a&., 1967). These steps in vaccinia virus infection are preceded by core formation and uncoating. Enzymes already available in the cell remove the outer protein coat to expose the core, but liberation of the viral DNA requires newly synthesized enzymes, since it does not occur in the presence of inhibitors of protein synthesis (Joklik, 1965; Dales, 1965a). We have now examined the effects of interferon on these two earlier steps. MATERIALS

AND METHODS

Chicken embryo fibroblast monolayers and partially purified interferon were 505

prepared and used as previously described (Levine et al., 1967). Radioactive vaccinia was prepared in HeLa cells that were inoculated with a high multiplicity of virus and incubated for 20 hours at 37” in medium containing thymidine-3H. At this time the cells were scraped into the medium, centrifuged, and resuspended in McIlvain’s buffer (1.8 X low4 A1 citric acid, 3.6 X 10V3 M N%HP04, pH 7.4) to give a concentration of approximately 2 X 10’ cells/ml. These were disrupted in lo-ml aliquots by prolonged sonication (40 minutes, including frequent stops for cooling), using a Raytheon lo-kc sonic vibrator (MacPherson, 1958). Large particles of cell debris were removed from the suspension by 2 minutes of centrifugation at 600 8. The supernatant fluid was

506

MAGEE,

treated with crystalline trypsin (0.01% final concentration) at 37” for 30 minutes. An aliquot of 10 ml was divided between 3 tubes, each containing layers of sucrose in RcIlvain’s buffer consisting of 10 ml of 60 % sucrose (weight/volume), 5 ml of 50%, 5 ml of 40%, and 5 ml of 30 %. The tubes were centrifuged in the SW 25.1 swingingbucket rotor on a Spinco preparative centrifuge for 40 minutes at 25,000 rpm (Zwartouw, et al., 1962). The virus, which banded at the interface between the 50% and the 60 % sucrose layers, was removed from the gradient tubes by means of a hypodermic needle inserted just below the band. It was then diluted in Mcllvain’s buffer and centrifuged 2-3 hours at 30,000 rpm in a No. 30 centrifuge head. The pellets were resuspended in 0.05 M Tris buffer (4 ml) by brief sonication and DNase (40 pg/ml final concentration) and MgClz (3 PM/ml) were added, after which the virus suspensions were incubated at 37” for 30 minutes. McIlvain’s buffer was added to stop DNase action, and the virus The was recovered by centrifugation. pellets were resuspended in the McIlvain’s buffer (4 ml) and hyaluronidase was added to a final concentration of 0.1 mg/ml. At this point in the purification procedure the virus suspension was stored overnight at O-5”. It was then incubated at 37” for 30 minutes, diluted with the McIlvain’s buffer, and centrifuged? At this stage the virus was almost free of contaminants (judging by electron microscopy), but was further purified by centrifugation through a sucrose density gradient (Magee and Miller, 1962; Joklik, 1962). The virus preparation used for most of the experiments contained 3.6 X log physical particles/ml, 1.21 X lo5 cpm/ml and, when assayed on two separate occasions on monolayers of chicken embryo fibroblasts, S.5 X lo8 and 1.5 X log PFU/ml. The suspensions were sonicated briefly just before use to disperse virus clumps. The monolayers (containing approximately 10’ cells per 60-mm petri dish) were

ET AL.

exposed to the interferon overnight, then washed, chilled, and inoculated with the virus. To allow the virus t’o attach to the cells, the dishes were kept at 4” for 2 hours, and during this period, to facilitate the attachment, were centrifuged (in the cold) at SO0 rpm for 10 minutes in a swingingbucket rotor on an International PR-2 centrifuge. To remove unattached virus, the monolayers were washed twice with cold growth medium; then 5 ml of warm growth medium was added and the cells were incubated at 37” (zero time). The adsorbed multiplicity was 2-12 physical particles per cell. At various times after the start of the incubation period, representative monolayers were washed once with T&saline buffer and cells were scraped into 0.05 II1 Tris buffer (pH 7.4) and ruptured by sonication for 3 minutes. Virus, cores, and viral DNA were separated by centrifugation at 15,000 rpm for 40 minutes in the SW 25.1 rotor in linear sucrose density gradients (Joklik, 1964b) of 20-40 % sucrose in 0.01 M Tris, pH 7.4. The breakdown of virus particles into cores and the release of DNA from the cores could be followed by determining the distribution along the gradients of the acidinsoluble radioactivity at, different times after infection. Some radioactivity was found in the pellet fractions from gradients of both the control and the interferontreated cells. This was not further analyzed because there was little difference between the amounts found for the control and the treated cells. Because there was some variation in the quantity of radioactivity adsorbed to cells after different treatments, the data were normalized to make comparisons possible. In the cold, less virus was adsorbed to cells treated with high levels of interferon than to nontreated cells. This difference varied from experiment to experiment but never fell below one-half of that adsorbed for the control. In addition, more heated virus than nonheated virus adsorbed to the monolayers, presumably as a result of clumping of the particles. Therefore, t’he 1 For the conditions of hyaluronidase treatmeasurements from all the ment, which greatly reduced clumping, we are ‘1radioactivity samples from the experimental sets were indebted to Dr. D. A. Buthala and J. Mathews.

INTERFERON

multiplied factor:

by

the

EFFECT

following

ON’ VACCINIA

correction

correction factor = cpm adsorbed to control cells (0 hr) cpm adsorbed to expt. cells (0 hr) Virus uncoating also was followed’ by measuring the quantity of viral DNA that was susceptible to DBase at different times after infection (Joklik, 1964a). Aliquots (0.4 ml) containing the homogenate from 3 X 10s cells were treated with 0.1 mg of crystalline pancrea.tic DNase for 30 minutes at 37” and then were precipitated with 0.1 ml of cold 2.5 N HCIO1. The pellets were washed once with 0.5 N HCIOJ, hydrolyzed, and counted. Duplicate untreated samples weie processed in order to determine the total acid-precipitable radioactivity in the homogenates before DNase t’reatment. RESULTS

The formation of cores and the subsequent liberation of the viral DXA was compared in control and interferon-treated cells (Fig. 1). In the controls, intact virus disappeared rapidly as cores appeared so that by 45 minutes after infection there was 1.5 times as much radioactivity in the core

WRUS

fraction of the density gradietits as in the virus fraction. With time, cores disappeared and DNA appeared at the top of the gradient, where it accumula.ted (Fii. 1A). In the interferon-treated cells, virus disappeared at the same rate as in control cells, but cores tended to accumulate and only small amounts of DNA were’ released (Fig. 1B). Changes with time in the percentage of the radioactivity associated with virus and cores are shown in Fig. 2. The data were obtained by determining the areas under the gradient curves. The persistence of cores in the interferon-treated cells suggested that uncoating wars inhibited in these cells. In another similar experiment the release of DNA from cores in control and interferon-treated cells was followed by both sucrose density gradient centrifugation and DNase susceptibility measurements (Fig. 3, A and B). The results obta.ined with the two methods agreed closely. Only small amounts of viral DNA were released from cores in interferon-treated cells, and then only after a delay of about 2 hours. The relationship between the dose of interferon and inhibition of uncoating was determined using the DNase assay (Fig. 4).

A

B

CONTROL

300

VIRUS

i

0

507

UNCOATING

INTERFERON

300

II

5

IO

I5

20

FIG. 1. Separation of virus, cores, and viral the infection of control and interferon-treated per petri dish in the pretreatment.

25 0 TUBE NO.

5

IO

15

20

25

DNA on sucrose density gradients at various cells. Three hundred (300) units of interferon

times after were used

508

MAGEE,

ET AL.

‘;:i,_l 0.1

FIG. 2. Time course for the changes in virus and cores in control (circles) and interferon-treated (triangles) cells expressed as percentage of cellassociated radioactivity at zero time.

I

0

2

0+---

4

4

6

HOURS

FIG. 3. Inhibition of uncoating by interferon. (A) The radioactivity in the DNA region of the sucrose gradients. (B) The radioactivity that became acid soluble after the homogenates were treated with DNase (expressed as total for 4 X lo7 cells). The experiment was a duplicate of the one shown in Fig. 1.

Previously published values for the response of the rate of viral DNA synthesis to various levels of interferon (Levine et cd., 1967) are plotted on the same chart. Virus uncoating and viral DNA synthesis evidently have very similar sensitivities to interferon, except that the response of uncoating levels off at high concentrations of interferon. These experiments showed that interferon can act by inhibiting uncoating. However, cores did not accumulate in sufficient quantity to account for all the virus that disappeared. Total acid-insoluble radioactivity associated with both interferontreated and control cells declined with time after infection. The average loss calculated from several experiments was 20% for control and 48% for interferon-treated

0.3

3 IO UNITS INTERFERON

30

100

300

FIG. 4. The effect of the concentration of interferon on the uncoating of vaccinia virus. The net amount of viral DNA which became susceptible to DNase digestion by 4-hour post-infection equaled 100% (see Fig. 3B). Average values for viral DNA synthesis were determined previously (Levine et al., 1967).

cells by 2-4 hours postinfection. The missing radioactivity was found in the medium and washes and consisted chiefly of acid-soluble materials. These losses were not due to cellular damage since recovery of protein remained unchanged with time, and signs of cellular injury were not evident by microscopic examination for at least 8 hours. These data suggested that when uncoating was blocked, the cores did not accumulate because they were digested. If this were not the case, t’hen a more complicated mode of action for interferon would have to be proposed. For example, uncoating might be only partially inhibited and a DNase might be activated in the interferontreated cells which rapidly hydrolyzed viral DNA as it was liberated from the core. The next experiments were run to see whether virus and cores could be digested without the intracellular appearance of viral DNA. The fate of heat-inactivated virus was examined because these particles are degraded without being uncoated (Joklik, 1964c; Dales, 1965a; Kates and McAuslan, 1967a). We found that virus disappeared rapidly without formation of cores or liberation of DNA (Fig. 5). At the same time there was a continuous loss of radioactivity from the cells, even greater than that which occurred with unheated virus, so that by 4 hours, 60% of the initially adsorbed radio-

ISTERFERON

EFFECT

ON VACCINIA

FIG. 5. The breakdown of heat-inactivated virus (GO”, 15 minutes) by chicken embryo fibroblasts. The sucrose density gradients were centrifuged simultaneously with those shown in Fig. 1.

VIRUS

UNCOATING

509

hibitor of protein synthesis could be compared to those of interferon on the accumulation and disappearance of cores. The radioactive virus preparation and the experimental conditions used were identical to those employed for the experiment shown in Fig. 1. It may be seen that the curves in Fig. 6 are nearly superimposable on those for the interferon-treated cells (Fig. 1B). Not enough cores accumula8ted to account for all the virus that disappeared, and fewer cores were present at 4 hours than at 2 hours post-infection. Thus, blocking of uncoating was followed by digestion of the cores regardless of whether the block was produced by inhibition of total protein synthesis or by interferon. In view of these results, it would seem very unlikely that the effect of interferon could be other than an inhibition of uncoating. DISCUSSION

The outer protein coat of vaccinia virus was removed at a normal rate in interferontreated cells to expose the cores. The absence of DNA and the persistence of cores 2 HR in the interferon-treated cells suggested . that uncoating was inhibit)ed. An objection 00 this hypothesis was the absence of a 4 HR quantitative accumulation of cores, since the only known pathway until now for disappearance of cores was uncoating. The demonstration that there exists a mecha0 5 IO 15 20 25 nism in these cells for digestion of cores TUBE NO. when uncoating is blocked by cyclohexiFIG. 6. Sucrose density gradients showing inmide explains why cores did not accumulate hibition of uncoating by cycloheximide. Cyclocells and removes t,he heximide (25 pg/ml) was added to the cultures 30 in interferon-treated objection to the proposa.1 that interferonminutes prior to infection and was present thereafter. treatment inhibits uncoating. Melnikova et al. (1967) claimed an inhibitory effect of interferon on a “factor that deproteinizes activity was no longer cell-associated. Most of it was found in the medium in acid-soluble the viral nucleocapsid.” form. In a second, similar experiment with The response of virus uncoating to ina different preparation of heated virus, creasing concentrations of interferon was essentially identical to the responses resmall amounts of radioactivity were found in the core region of the gradients. We conported for the synthesis of viral DNAcluded that the nucleic acid from these polymerase and viral DNA (Levine et al., virus particles can be degraded and lost 1967) through a range of interferon confrom the cells without the intracellular accentrations from 3 to 100 units. Increasing cumulation of acid-insoluble fragments. the interferon concentration above 100 Uncoating was inhibited by cyclohexiunits did not result in any further inhibition mide (Fig. 6) so that the effect’s of an in- of uncoating. About 20% of the normal zoo-

510

MAGEE,

amounts of released viral DNA were found 4 hours after infection in cells treated with 100-300 units of interferon. We do not know whether all this DNA is functional, but its presence is consistent with our earlier observation that such a level of interferon did not completely prevent the initiation of new DNA synthesis at discrete cytoplasmic sites. The small amount of synthesis that took place under these conditions suggests tha.t virus-specific steps subsequent to uncoating also are inhibited. This agrees with current theories that interferon acts by blocking the synthesis of virus-specific proteins (Marcus and Salb, 1966; Joklik and Merigan, 1966). Since interferon is known to inhibit only viral functions, and not those of the host, our data suggest that the information for uncoating enzyme must reside in the viral nucleic acid. The recent experiments of Kates and McAuslan (1967b) provide a mechanism by which a portion of the viral nucleic acid code can be read before the bulk of the genome is exposed by uncoating. They demonstra.ted that vaccinia virus carries its own DNA-primed RNApolymerase and that this enzyme synthesizes RNA in vitro once the outer protein coat of the virus is removed. The RNA contains the message for thymidine kinase, and our results suggest that it also contains the message for the uncoating enzyme. Inside the cell, virus-specific RNA accumulated when uncoating was blocked by streptovitacin A (Kates and McAuslan, 1967a). Joklik and Merigan (1966) already had observed a similar, early accumulation of virus-specific RNA during interferon inhibition, and on this basis, Kates and McAuslan (1967a) predicted that interferon would block uncoating. Virus and cores apparently were digested to acid-soluble materials whenever uncoating was delayed or prevented. Digestion of virus particles probably takes place in the vacuoles associated with virus penetration (Dales, 1965a). In fact, the only known mechanism for digestion of intracellular particles is by the secretion of lysosomal enzymes into vacuoles (Novikoff et al., 1964). We were surprised, therefore, to find

ET AL.

that cores were digested, because Dales (1965b), in his study of vaccinia virusinfected HeLa cells, did not find cores in vacuoles. Rather, they were free in the cytoplasm where they accumulated when protein synthesis was inhibited. Perhaps when uncoating is delayed in infected chicken embryo fibroblasts the cores are again engulfed by vacuoles in the same way that cellular organelles are taken into autopha.gic vacuoles in starving cells (Novikoff et al., 1964). Such a mechanism might provide an alternative to cell death in certain abortive infections. REFERENCES DALES, S. (1965a). Penetration of animal viruses into cells. “Progress in Medical Virology” (J. L. Melnick, ed.), Hafner, vol. 7, pp. 143. New York. DILES, S. (196513). Effects of streptovitacin A on initial events in the replication of vaccinia and reovirus. Proc. Natl. Acad. Sci. U.S. 54, 462468. JOKLIK, W. K. (1962). The preparation and characteristics of highly purified radioactivelylabeled poxvirus. Biochim. Biophys. Acta 61, 290-301. JOKLIK, W. K. (1964a). The intracellular uncoating of poxvirus DNA. I. The fate of radioactively-labeled rabbitpox virus. J. Mol. Biol. 8, 263-276. JOKLIK, W. K. (196413). The intracellular uncoating of poxvirus DNA. II. The molecular basis of the uncoating process. J. Mol. Biol. 8, 277288. JOKLIK, W. K. (1964c). The intracellular fate of rabbitpox virus rendered noninfectious by various reagents. Virology 22, 620-633. JOKLIK, W. K. (1965). The molecular basis of the viral eclipse phase. “Progress in Medical Virology” (J. L. Melnick, ed.), vol. 7, pp. 44-96. Hafner, New York. JOKLIK, W. K., and MERIGSN, T. C. (1966). Concerning the mechanism of action of interferon. Proc. Xatl. Acad. Sci. U.S. 56, 558-565. KATES, J. R., and MCAUSLAN, B. R. (1967a). Messenger RNA synthesis by a “coated” viral genome. Proc. Natl. Acad. Sci. U.S. 57, 314-320. KATES, J. R., and MCAUSLAN, B. R. (1967b). Poxvirus DNA-dependent RNA polymerase. Proc. Natl. Acad. Sci. U.S. 58, 134-141. LEVINE, S., MAGEE, W. E., &MILTON, R. D., and MILLER, 0. V. (1967). Effect of interferon on early enzyme and viral DNA synthesis in vaccinia virus infection. Virology 32, 33-40.

INTERFERON

EFFECT

ON VACCINIA

I. A. (1958). Liberation of cellbound vaccinia virus by ultrasonic vibration. J. Hyg. 56, 29-38. M.~GEE, W. E. and MILLER, 0. Y. (1962). Dissociation of the synthesis of host and viral DNA. Biochim. Biophys. Acta 55, 818-826. M.~RCUS, P. I., and &LB, J. M. (1966). Molecular basis of interferon action: inhibition of viral RNA translation. Virology 30, 502-516. h~ELNlKov.4, L. A., Koz~ov.4, I. A., PETERSON, 0. P., BOSTANDZHYAN, M. G., FADEYEVA, L. L., MACPHERSON,

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and ZHDANOV, I-. M. (1967). On the mechanism of action of interferon on the early stage of vaccinia virus reproduction. A& Viral. (Prague) 11, 293-296. NOVIKOFF. A. B., ESSNER, E., and &UINTA4NA, N. (1964). Golgi apparatus and lysosomes. Federation Proc. 23, 1010-1022. ZWARTOU\V, H. T., WEST~V~~D, J. C. N., and APPLEYARD, G. (1962). Purification of pox viruses by density gradient centrifugation. J. Gen. Microbial. 29, 523-529.