Interferon has no protective effect during acute or persistent reovirus infection of mouse SC1 fibroblasts

Virus ELSEVIER

Virus Research 51 (1997) 139-149

Interferon has no protective effect during acute or persistent reovirus infection of mouse SC1 fibroblasts C. Danis, T. Mabrouk, M. Faure, G. Lemay * DOpartement de Microbiologie et Immunologic, Universit~ de Montreal, P.O. Box 6128, Station Centre-ville, Montreal, Quebec, H3C 3J7, Canada

Received 31 January 1997; received in revised form 15 July 1997; accepted 17 July 1997

Abstract

Mouse SCI fibroblasts can support reovirus multiplication although they exhibit a partial resistance to viral-induced cytopathology; a significant percentage of infected SC1 cells can remain viable while becoming persistently infected by the virus. In the present study, the possible role of interferon on the fate of reovirus-infected cells was investigated. Treatment of mouse L fibroblasts with fl-interferon resulted in a reduced viral efficiency of plating while essentially no effect was observed on SC1 cells; the results were similar with the unrelated encephalomyocarditis virus. This suggests that the interferon-regulated pathways are somehow deficient in SC1 cells even though these cells do respond to interferon treatment, as evidenced by an increase in the level of active interferon-inducible protein kinase double-stranded RNA-dependent (PKR) enzyme. Persistently infected SC1 cells constitutively release interferon even though treatment with anti-interferon antiserum suggests that interferon presence is unrelated to maintenance of the persistent state. The possible significance of the correlation between the lack of interferon-induced antiviral effect and relative resistance of SC1 cells to viral-induced cytopathology is briefly discussed. © 1997 Elsevier Science B.V. Keywords: Interferon; Protein kinase double-stranded RNA-dependent (PKR) enzyme; Reovirus

1. Introduction

M a m m a l i a n reoviruses generally act as cytolytic viruses. A l t h o u g h the exact mechanism o f cell killing is unclear, it is k n o w n that different cellular functions are inhibited during viral multiplica* Corresponding author. Tel.: + 1 514 3432422; fax: + 1 514 3435701; e-mail: [email protected].

tion. Ultimately, reovirus-infected cells are killed and newly m a d e viral particles are released following cellular disruption (for a review o f the reovirus multiplication cycle, see Nibert et al., 1996). However, a certain percentage o f reovirus-infected cells in a given culture can survive. These cells become persistently infected and permanently release virus without being killed. W i t h m o s t cell

0168-1702/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0168- 1702(97)00088-9

140

C. Danis et a l . / Virus Research 51 (1997) 139-149

lines, cell survival and establishment of persistence is solely observed when the cells are infected with high-passage reovirus stocks containing defective and mutant viruses (Ahmed and Graham, 1977; Ahmed et al., 1981; Dermody et al., 1993). Most cell lines persistently infected with reovirus can also be cured by antiviral antibody treatment (Taber et al., 1976; Ahmed et al., 1981; Dermody et al., 1993; Wetzel et al., 1997). This is interpreted as an indication that constant reinfection is needed to maintain the virus in the culture, a situation defined as a special case of carrier culture (Mahy, 1985), in which most or all cells are infected (Dermody et al., 1993). Mammalian reoviruses can infect a wide range of host species and cell types. In recent studies, it was established that the kinetics of the different stages of the viral multiplication cycle are quite different when two cell lines of mouse fibroblasts, L cells and SC1 cells, were compared (Danis and Lemay, 1993; Danis et al., 1993). The SC1 cells are much more resistant to viral-induced cytopathic effects and a significant percentage of the cells remained viable, and persistently infected, even when low-passage viral stocks were used to infect the cells. Persistently infected SC1 cells also express a high level of the double-stranded RNAactivated protein kinase (Danis et al., 1993). The expression of this enzyme, referred to as P K R (protein kinase double-stranded RNA-dependent), is normally induced by interferon. The high level of P K R observed in persistently infected cells is of interest since P K R was suggested to be the main factor involved in the anti-reovirus effect of interferon (Miyamoto and Samuel, 1980; Nilsen et al., 1982; reviewed in Samuel, 1988). This prompted us to examine the possible involvement of the interferon-regulated antiviral response in the regulation of viral multiplication in SC1 cells. In the present study, it was observed that interferon does not confer protection to either acute reovirus or encephalomyocarditis virus infection in SC1 cells. It also appears that constitutive interferon release by persistently infected SC1 cells is not directly involved in the maintenance of persistent reovirus infection in these cells since an anti-interferon antiserum had no effect on maintenance of viral persistence. However, SC1 cells can

respond to interferon treatment as evidenced by the increased expression of active P K R in the treated cells. This strongly suggests that the interferon-regulated pathways must be somehow deficient in SC1 cells, hence the lack of antiviral activity. These observations also raise the possibility that host-cell protein synthesis inhibition and cytopathic effects in reovirus infection may be at least partly mediated through an interferon-regulated pathway.

2. Materials and methods

2.1. Cells and viruses SC1 feral mouse embryo fibroblasts, mouse L cells (clone 929), as well as initial inoculum of reovirus serotype 3 (strain Dearing), were originally obtained from the American Type Culture Collection (Rockville, MD). Cured SC1 cells were a generous gift from Dr. Terence Dermody (Vanderbilt University) and were obtained by antireovirus antiserum treatment of persistently infected SC1 cells. Initial inoculum of mouse encephalomyocarditis virus (EMC) was a generous gift from Dr. Serge Dea (Institut Armand-Frappier, Laval, Canada). Viruses were propagated at low multiplicity of infection on monolayers of L cells. All viral infections were performed according to standard procedures. Viral adsorption was allowed to proceed in a minimal volume of serumfree medium for 1 h at 4°C; following viral adsorption, cells were refed with fresh medium containing 2% heat-inactivated fetal bovine serum.

2.2. Determ&ation of viral ejficiency of plating Relative viral efficiency of plating (EOP) was determined by performing virus titration onto serial dilutions of a given viral stock in 96-well microtiter plates using the 'tissue culture infectious dose 50%' (TCID5o) method, essentially as previously described (Danis and Lemay, 1993). To facilitate reovirus titer determination, a 50 /tl aliquot was collected from each well after 6 days of incubation and used to reinfect L cells in a

C. Danis et al. / Virus Research 51 (1997) 139-149

duplicate microtiter plate; these duplicate plates were examined for cytopathic effects after 2 days of incubation. This procedure was judged necessary due to the poor stability of SC1 cell monolayers, which rendered difficult the observation of cytopathic effects in some wells. In the case of EMC virus, this procedure was not necessary since the replication cycle of this virus is much shorter; in these viral infections, the plates were examined after 3 days of incubation.

2.3. Interferon treatment of cellular monolayers Subconfluent cellular monolayers were pretreated for 15 h with 250 international units (IU)/ml of mouse fl-interferon (Lee Biomolecular). These monolayers were then used to determine the relative efficiency of plating in the continuous presence of interferon. The TCIDso value of a given viral stock onto interferontreated cells was compared with the results obtained with the same viral stock onto untreated cellular monolayers.

2.4. Recovery of "conditioned" medium for interferon detection Subconfluent cellular monolayers were fed with flesh medium containing 2% heat-inactivated fetal bovine serum and incubated for 24 h. Medium was recovered, brought to pH 2.0 by addition of 1 N HC1, let at 4°C for 3 days and neutralized by addition of 1 N NaOH; Hepes-NaOH (pH 7.5) buffer was further added to a final concentration of 50 mM. Buffered medium was then placed in a large petri dish (100 mm diameter petri dish for 5 ml of medium) and ultraviolet irradiated for 30 min using built-in germicidal lamps of the tissue culture hood. Irradiated medium was finally filtered onto a sterile 0.2 pm nitrocellulose filter before being used. To assess interferon presence, L cells in microtiter plates were pretreated with this conditioned medium and used for titration of encephalomyocarditis virus. The titer of the same virus stock was determined in parallel on untreated cells.

141

2.5. Anti-interferon treatment Anti-mouse interferon (~ + fl) was purchased from Lee Biomolecular. This antibody was used to treat persistently infected cells by replacing their medium with fresh medium containing the antibody at various concentrations. Synthesis of proteins was then examined using metabolic radiolabeling for 1 h in methionine-free medium with addition of 100 pCi/ml of Tran35S-Label (ICN; 1000 Ci/mmol) followed by S D S - P A G E analysis, essentially as previously described (Danis et al., 1993).

2.6. Detection of PKR induction and activity Subconfluent cells (2.5 x 105 cells in 60 mm diameter petri dishes) were either left untreated or treated for 24 h with 250 IU/ml of mouse fl-interferon (Lee Biomolecular). Cells were recovered by centrifugation and lysates prepared by resuspension in 200 pl of 0.5% NP-40. Protein samples (30 gl) were then diluted 3-fold and treated with 10 units of calf intestine alkaline phosphatase (Boehringer Mannheim) for 1 h at 37°C in the buffer provided by the manufacturer. Control samples were similarly treated without addition of alkaline phosphatase. Proteins were then resolved by SDS-PAGE followed by electrotransfer onto nitrocellulose filter. Detection of PKR was achieved using a rabbit monospecific anti-PKR antiserum followed by a secondary antibody conjugated to alkaline phosphatase; detection of antigen-antibody complexes was performed using NBT-BCIP substrate using standard procedures (Mabrouk and Lemay, 1994). Alternatively, interferon-treated or control cells were incubated in phosphate-deficient medium containing 50 pCi/ml of [32p]orthophosphoric acid; this labeling was performed following a preincubation of 5 h in phosphate-deficient medium. In the case of interferon treatment, mouse fl-interferon was added overnight (250 IU/ml) and during pre-incubation in phosphatb-deficient medium. Following labeling, cells were recovered by scraping and resuspended in binding buffer (Hepes-KOH 20 mM (pH 7.5); NaC1 0.3 M; dithiothreitol 1 mM; PMSF 1 raM; 0.5% NP-40;

142

C. Danis et al. / Virus Research 51 (1997) 139-149

10% glycerol). The supernatant from 106 cells in 100/ll was then mixed with 100/zl of poly(I) poly(C) agarose (Pharmacia) previously washed in binding buffer. Following adsorption for 60 min at room temperature with occasional agitation, the agarose beads were washed three times with binding buffer containing an increased NaC1 concentration to 0.5 M, and twice in standard binding buffer. The beads were then resuspended in Laemmli's SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled in a water bath for 5 min, centrifuged, and the supernatant analyzed on 10% S D S - P A G E followed by autoradiography.

3. Results

3.1. Effect of interjeron treatment on viral multiplication In an effort to determine if interferon-regulated pathways could be responsible for the differences in the control of reovirus multiplication observed between L and SC1 cells the protective effect of interferon treatment against acute lytic reovirus infection in L cells and SC1 cells was first examined. The relative efficiency of plating (EOP) of reovirus in the two cell lines was determined in the presence of interferon or in control untreated cells. The EOP was decreased by almost 100-fold following treatment of L cells with a saturating concentration of mouse fl-interferon (250 IU/ml) while the same treatment had only a negligible, 2-3-fold, effect on reovirus EOP in SC1 cells (Fig. 1A). To determine if the lack of effect of interferon in SC1 cells is restricted to reovirus or is a property of the cell line, the multiplication of the unrelated mouse encephalomyocarditis virus (EMC) of the Picornaviridae' family was examined. Even though EMC virus was more sensitive than reovirus to interferon treatment in L cells, the same treatment had only a negligible antiviral effect in SC1 cells (Fig. IB).

3.2. Endogenous interferon production in persistently infected SC1 cells The lack of protective effect of exogenous interferon in SCI cells did not exclude the possibility that endogenous interferon could be active. This point was examined in persistent reovirus infection since reovirus infection is known to be an inducer of interferon production (Lai and Joklik, 1973; Gupta et al., 1982). Persistently infected SC1 cells were previously shown to express a high level of P K R without addition of exogenous interferon (Danis et al., 1993). It was thus verified if this increased PKR expression level may be due to constitutive interferon production by these cells, Medium was recovered from persistently infected cells and subjected to acid treatment and UV irradiation to inactivate infectious virus while sparing interferon, as described in Section 2. This 'conditioned' medium was then applied to L cells and these treated cells were challenged with EMC virus. It appears that medium from persistently infected SC1 cells can successfully protect L cells while the medium from normal L or SC1 cells, used as negative controls, has no effect (Fig. 2A). The amount of interferon produced was more precisely examined by comparing its protective effect with that of serial dilutions of the commercial preparation of fl-interferon (Fig. 2B). This allowed us to estimate the amount of released interferon to be around 500 IU/ml in the medium recovered from semi-confluent cells after 24 h (3 × 106 cells in a 100 mm diameter petri dish fed with 10 ml of medium). The induction of interferon production by reovirus in the persistently infected cells is a reversible phenomenon, apparently dependent upon continuous virus presence. Cured cells were obtained following an antireovirus antiserum treatment, as previously described for other persistently infected cell lines, since continuous reinfection is required for maintenance of persistent reovirus infection (Ahmed et al., 1981; Dermody et al., 1993). These cured cells did not release interferon (Fig. 2A).

C. Danis et al. / Virus Research 51 (1997) 139-149

143

;BOVZRUS

A

~4 A

--4 ~ .

• Control []Interferon

~o u~ m~

~H

-,.'t

L

B

SCI

IE+08

A

-r4 ~ .

~o u~ m~

•

~H

Control

[]Interferon

L

SCI

Fig. 1. Effect of interferon treatment on viral efficiency of plating. L or SC1 cells were either left untreated (black bars) or pre-treated with interferon (hatched bars) as described in Section 2. The titer of the same viral stock was then determined on both cell types. Results from a representative experiment are expressed in TCIDso units/ml and are presented on a logarithmic scale. Panel A presents results obtained with reovirus serotype 3 Dearing and panel B are results obtained with EMC virus.

144

C. Danis et al.

Virus Research 51 (1997) 139 149

A

o

~ ou~ ~H

Cont

~"

_

L

SCI

Pers. C u r e d

Interf.

1.2

300

[2o0 1-150 o

1

I: °

0 1 +

+

+

+

Virus liter (T C I D 5 0 / m L )

Fig. 2. Production of interferon by persistently infected SCI cells. Medium was recovered from cultures of the different cell lines (conditioned medium) and treated as described in Section 2. 'Pers.' and 'cured' refer, respectively, to the medium recovered from persistently infected SCI cells or cured cells obtained by anti-reovirus antiserum treatment of such persistently infected cells. L cells were either left untreated (Cont) or pre-treated with these different conditioned media, as indicated; TCIDso of the same E M C virus stock was then determined on these cells in the continuous presence of conditioned medium (panel A). The effect of the commercial preparation of interferon (250 international units/ml) is shown as a positive control (lnterf.). The a m o u n t of interferon released by persistently infected cells was then better quantitated (panel B). L cells were pre-treated with different dilutions of a one-tenth dilution of conditioned medium from persistently infected cells ( l l) or dilutions of commercial mouse fl-interferon with an initial concentration of 250 IU/ml (©). Cells were then used to titrate the same stock of E M C virus. Titers are expressed as T C I D s , units/ml and are presented on a logarithmic scale.

C. Danis et al. / Virus Research 51 (1997) 139-149

3.3. Effect of anti-interferon treatment Having established that SC1 cells persistently infected by reovirus permanently release interferon, the possibility that this continuous interferon presence plays a role in maintenance of the persistent state was examined. Anti-interferon antiserum treatment was used to block the effect of interferon. Persistently infected cells were treated with 500 neutralizing units/ml of anti-interferon antiserum; according to the manufacturer, 1 unit of this antiserum is sufficient to neutralize 3 - 1 0 units of interferon (Lee Biomolecular). It was verified that 1 unit was at least able to completely abolish the anti-EMC protective ability of 1 unit of commercial mouse fl-interferon (Lee Biomolecular) when added to L cells (data not shown). Anti-interferon was thus added to persistently infected SCI cells at either 500 or 2500 units/ml, a concentration that should thus be largely sufficient to abolish the activity of the interferon present in the medium of these cells. There was no apparent increase in cytopathic effects at either concentration of antiserum even when cells were kept for more than 3 days. Similarly, viral production was not affected by treatment with the antiserum (data not shown). Radiolabeling was finally performed and indicated that synthesis of viral proteins in the persistently infected cells was not affected by the treatment, as most easily visualized with the major a3 capsid protein (Fig. 3).

145

increased resistance to viral infection following interferon treatment, it was of importance to reassess more directly the status of P K R in these cells. In order to determine total amounts of PKR, immunoblotting analysis was performed using a monospecific anti-PKR antiserum. Small amounts of P K R were observed in L cells and SC1 cells; an increased amount was also observed following interferon treatment in both cell lines (Fig. 4a). Interestingly, the protein was resolved in two species; the amount of slower-migrating protein was very low in the absence of interferon treatment and was increased in the presence of interferon. Upon alkaline phosphatase treatment, the two protein species co-migrate, consistent with the removal of phosphate residue responsible for S('I -

PERSIST. +

+

+

+

ANTISERUM

cr3

3.4. PKR levels in L and SCI cells (Fig. 4) It was previously reported that both L and SC1 cells can express the cellular interferon-inducible double-stranded RNA-activated protein kinase (PKR) (Danis et al., 1993). The expression of this protein can have important consequences and is often viewed as the main factor involved in the antiviral activity of interferon. Both the autophosphorylation and phosphorylation of histones added as an exogenous substrate were similar in vitro using cell-free lysates from either L or SC1 cells (Danis et al., 1993). However, this might not accurately reflect the level of active P K R present in vivo. Since SC1 cells do not demonstrate an

I !

I

I

I

I

I

I

I

2

3

4

5

6

7

8

Fig. 3. Effect of anti-interferon treatment on persistently infected cells. Uninfected SC 1 cells (lanes 1 and 2) or persistently infected SC1 cells (lanes 3-8) were either left untreated (lanes l, 3, 5, 7) or pre-treated for 3 h (lane 4), 15 h (lane 6) or 48 h (lanes 2 and 8) with the anti-interferon antiserum at 500 neutralizing units/ml as described in Section 2. Corresponding untreated control cells were recovered at 3 h (lane 3), 15 h (lane 5) or 48 h (lanes l and 7). Metabolic radiolabeling was then performed for 1 h as described in Section 2 and proteins were analyzed by SDS-PAGE. Identification of viral proteins expressed in persistently infected cells is facilitated by comparisons with uninfected SC1 cells. The position of the abundant ~3 viral protein is indicated.

146

C. Danis et al. , Virus Research 51 (1997) I39

®

L cells +

Interf.

ClAP

SCI cells

-~

I

+ I

... I

-, +

-~

I,

!

+

+ i

i

+

i ~-97

66

- 55 -- 43

i

i

i

I

!

i

~

i

i

2

3

4

5

6

7

8

149

L and SC1 cells and there was no evidence for differences in molecular mass of the polypeptides in the two cell lines. An alternative approach was also used to confirm the induction of P K R following interferon treatment. P K R was labeled with radioactive phosphate and recovered by [poly(I)poly(C)]-agarose affinity chromatography; this procedure takes advantage of P K R affinity for double-stranded R N A (dsRNA). This approach also showed that a low level of active P K R is constitutively expressed in L and SC1 cells while its expression is induced by interferon treatment in both cell lines (Fig. 4b).

4. D i s c u s s i o n

® i

L lntcrf.

-" I

+ I

SCI -" i

-r I

97 66 55

43

t

I

1

I'

9

Ill

!1

12

Fig. 4. P K R expression in L and SCI cells. Panel A: proteins from cells treated with 250 IU/ml of interferon ( + ) or control untreated cells ( - ) were analyzed by immunoblotting using monospecific anti-PKR antiserum, as described in Section 2. When indicated, proteins were pre-treated with calf intestine alkaline phosphatase (CIAP) before electrophoresis. Panel B: labeling with radioactive phosphate was performed on either control cells ( - ) or cells treated with mouse fl-interferon ( + ) . P K R was next purified by affinity chromatography, as described in Section 2, and analyzed by SDS-PAGE followed by autoradiography. Positions of molecular mass standards are indicated. The expected molecular mass of P K R in mouse cells is 65 kDa.

the difference in electrophoretic mobility between the two polypeptides. The amount of both fast and slow-migrating forms of PKR were similar in

In the present study, the antiviral effect of interferon in SC1 cells was examined. In the course of our ongoing work concerning the control of protein synthesis during reovirus multiplication, it was previously observed that mouse SC1 cells exhibit resistance to "the inhibition in the synthesis of cellular proteins observed in most cell lines upon infection by reovirus (Danis and Lemay, 1993). The SC1 cells were also relatively resistant to cell killing; about 5-10% of infected cells in any given culture will survive and become persistently infected (Danis et al., 1993). Differences in the interferon-regulated antiviral response is one possible mechanism that might explain differential susceptibility of cell lines to the same viral infection. In the present study, it was established that SCI cells can respond to interferon and that the level of active PKR is increased during this response. It has been suggested that the main factor mediating the interferon-mediated resistance to reovirus multiplication is the induction of PKR (Miyamoto and Samuel, 1980; Gupta et al., 1982; Nilsen et al., 1982; De Benedetti et al., 1985). However, this point is still discussed and it is possible that P K R is only one of the factors required to affect reovirus multiplication (Samuel and Knutson, 1981; discussed in Samuel, 1988; Staehli, 1990). The situation is also unclear for

C. Danis et al. / Virus Research 51 (1997) 139-149

EMC: there is much evidences to suggest that PKR is not the main factor involved (reviewed in Samuel, 1988; Staehli, 1990). However, recent work showed that overexpression of PKR is sufficient to confer protection to EMC (Meurs et al., 1992); the question is still open. Previous investigators have noticed that cytosolic PKR exists in a partially phosphorylated form in interferon-treated L cells, resulting in a protein species of reduced electrophoretic mobility (Langland and Jacobs, 1992). In the present study, a similar protein was also observed in SC1 cells; removal of phosphate groups by alkaline phosphatase treatment decreases its apparent molecular mass, consistent with phosphorylation. This supports the idea that PKR activity per se is not deficient in SC1 cells. Although it cannot be completely ruled out that subtle differences in the in vivo activation and/or catalytic activity of PKR were not detected, various criteria have been used without any evidence of impaired PKR response in SC1 cells: induction by interferon, activation by dsRNA, phosphorylation of an exogenous substrate (histones), are similar to the situation observed in L cells (Danis et al., 1993; and this study). This suggests that the PKR induction/activation pathway is not sufficient to confer protection to either reovirus or EMC. The fact that interferon treatment fails to confer protective antiviral activity against either reovirus or EMC virus is most easily interpreted if interferon-dependent events, other than PKR induction or activation, are somehow deficient in SC1 cells. Such incomplete interferon response in some cultured cell lines has been previously suspected (see, for example, Samuel and Knutson, 1981; reviewed by Samuel, 1988; Sen and Ransohoff, 1994). The role of interferon during establishment and/or maintenance of persistent infection has been examined in multiple different examples of virus-cell systems. Although in many cases it appears that the interferon response was not involved in limiting cytopathic effect (see, for example, Stohlman and Weiner, 1978; Andzhaparidze et al., 1981; Dawson et al., 1984; Verani et al., 1984), some clear examples of interferon involvement have been reported (see, for example, Jacob-

147

son and McFarland, 1982; Rager-Zisman et al., 1984; Ito et al., 1985; Mannini-Palenzona et al., 1985). In the case of reovirus, it has been shown that persistently infected CHO cells do not produce interferon and this factor is thus unlikely to be involved in the maintenance of persistence (Taber et al., 1976). Since persistently infected SC1 cells were in fact releasing interferon, in contrast to persistently infected CHO cells, we therefore examined if this factor may be important during persistence, although it did not appear to be the case during acute infection. It turned out that neutralization of interferon using an anti-interferon antiserum treatment could not abolish viral persistence or increase synthesis of viral proteins. Finally, it is interesting to mention the correlation between this absence of anti-reovirus interferon action in SC1 cells with the lack of protein synthesis inhibition reported in these cells (Detjen et al., 1982; Danis and Lemay, 1993). Two interesting parallels can be drawn from this observation. (1) It has been shown that reovirus serotype 1 possesses a stronger mechanism of resistance to interferon (Jacobs and Ferguson, 1991); coincidentally, infection with this serotype does not result in inhibition of protein synthesis (Munemitsu and Samuel, 1984). (2) The viral ~r3 protein, shown to have an anti-PKR action via its affinity for double-stranded RNA (Imani and Jacobs, 1988; Giantini and Shatkin, 1989; Seliger et al., 1992; Mabrouk et al., 1995), has also been proposed to be responsible for different levels of cellular protein synthesis inhibition induced by different serotypes of reovirus (Sharpe and Fields, 1982). In conclusion, the possible involvement of interferon-mediated pathways in inhibition of cellular protein synthesis during reovirus multiplication certainly deserves further study; in addition to the role of PKR and its viral antagonist ~r3, other interferon-regulated factors may well be involved in the control of protein synthesis and viral multiplication during reovirus infection. Comparisons between L cells and SC1 cells appear as an especially interesting approach for such studies.

148

C. Danis et al.

Virus Research 51 (1997) 139-149

Acknowledgements We thank Denise Wetzel and Terence Dermody (Vanderbilt University, Nashville, TN), Serge Dea (Institut Armand-Frappier, Laval, Canada), and J o h n Bell ( U n i v e r s i t y o f O t t a w a , C a n a d a ) f o r t h e i r g e n e r o u s gift o f t h e ' c u r e d ' SC1 cell line, initial inoculum of EMC virus and anti-PKR antiserum, respectively. This work was supported by a grant to G.L. from the Medical Research C o u n c i l o f C a n a d a . G . L . is a l s o t h e r e c i p i e n t o f a ' C h e r c h e u r - B o u r s i e r ' a w a r d f r o m t h e ' F o n d s d e la Recherche en Sant6 du Qu6bec'. T.M. was the recipient of a studentship from the 'Agence Canadienne de D6veloppement

International'.

References Ahmed, R., Graham, A.F., 1977. Persistent infections in L cells with temperature-sensitive mutants of reovirus. J. Virol. 23, 250 262. Ahmed, R., Canning, W.M., Kauffman, R.S., Sharpe, A.H., Hallum, J.V., Fields, B.N., 1981. Role of the host cell in persistent viral infection: coevolution of L cells and reovirus during persistent infection. Cell 25, 325 332. Andzhaparidze, O.G., Bogomolova, N.N., Boriskin, Y.S., Bektemirova, M.S., Drynow, I.D., 1981. Comparative study of rabies virus persistence in human and hamster cell lines. J. Virol. 37, 1 6. Danis, C., Lemay, G., 1993. Protein synthesis in different cell lines infected with orthoreovirus serotype 3: inhibition of host-cell protein synthesis correlates with accelerated viral multiplication and cell killing. Biochem. Cell Biol. 71, 81 85. Danis, C., Mabrouk, T., Garzon, S., Lemay, G., 1993. Establishment of persistent reovirus infection in SCI cells: absence of protein synthesis inhibition and increased level of double-stranded RNA-activated protein kinase. Virus Res. 27, 253 265. Dawson, G.J., Mowshowitz, S.L., Cohen, R., Elizan, T.S., 1984. Herpes simplex virus persistence in mouse neuroblastoma cell cultures: role of interferon. J. Neural Transmission 59, 309 317. De Benedetti, A., Williams, G.J., Baglioni, C., 1985. Inhibition of binding to initiation complexes of nascent reovirus mRNA by double-stranded RNA-dependent protein kinase. J. Virol. 54, 408 413. Dermody, T.S., Nibert, M.L., Wetzel, J.D., Tong, X., Fields, B.N., 1993. Cells and viruses with mutations affecting viral entry are selected during persistent infections of L cells with mammalian reoviruses. J. Virol. 67, 2055 2063.

Detjen, B.M., Walden, W.E., Thach, R.E., 1982. Translational specificity in reovirus-infected mouse fibroblasts. J. Biol. Chem. 257, 9855 9860. Giantini, M., Shatkin, A.J., 1989. Stimulation of chloramphenicol acetyltransferase mRNA translation by reovirus capsid polypeptide a3 in co-transfected COS cells. J. Virol. 63, 2415 2421. Gupta, S.L., Holmes, S.L., Mehra, L.L., 1982. Interferon action against reovirus: activation of interferon-induced protein kinase in mouse L929 cells upon reovirus infection. Virology 120, 495-499. Imani, F., Jacobs, B.L., 1988. Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 a3 protein. Proc. Natl. Acad. Sci. USA 83, 7887 7891. lto, Y., Tsurudome, M., Hishiyama, M., 1985. Persistence of mumps virus in mouse L929 cells. Microbiol. Immunol. 29, 847 857. Jacobson, S., McFarland, H.F., 1982. Measles virus persistence in human lymphocytes: a role for virus-induced interferon. J. Gen. Virol. 63, 351 357. Jacobs, B.L., Ferguson, R.E., 1991. The Lang strain of reovirus serotype 1 and the Dearing strain of reovirus serotype 3 differ in their sensitivities to beta interferon. J. Virol. 65, 5102 5104. Lai, M.-H.T., Joklik, W.K., 1973. The induction of interferon by temperature-sensitive mutants of reovirus, UV-irradiated reovirus, and subviral reovirus particles. Virology 51, 191 204. Langland, J.O., Jacobs, B.L., 1992. Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated M r = 66 000 subunits. J. Biol. Chem. 267, 10729 10736. Mabrouk, T., Lemay, G., 1994. The sequence similarity of reovirus ~r3 protein to picornaviral proteases is unrelated to its role in /tl viral protein cleavage. Virology 202, 615-620. Mabrouk, T., Danis, C., Lemay, G., 1995. Two basic motifs of reovirus a3 protein are involved in double-stranded RNA binding. Biochem. Cell Biol. 73, 137 145. Mahy, B.W.J., 1985. Strategies of viral persistence. Br. Med. Bull. 41, 50-55. Mannini-Palenzona, A., Bartoletti, A.M., Foa'-Tomasi, L., Baserga, M., Tognon, M., Manservigi, R., 1985. Establishment and characterization of a persistent infection of MDCK cells with herpes simplex virus. Microbiologica 8, t65--180. Meurs, E.F., Watanabe, Y., Kadereit, S., Barber, G.N., Katze, M.G., Chong, K., Williams, B.R.G., Hovanessian, A.G., 1992. Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth. J. Virol. 66, 5805 5814. Miyamoto, N.G., Samuel, C.E., 1980. Mechanism of interferon action: interferon-mediated inhibition of reovirus mRNA translation in the absence of detectable mRNA

C. Danis et al. / Virus Research 51 (1997) 139-149

degradation but in the presence of protein phosphorylation. Virology 107, 461-475. Munemitsu, S.M., Samuel, C.E., 1984. Biosynthesis of reovirus-specified polypeptides: multiplication rate but not yield of reovirus serotypes 1 and 3 correlates with the level of virus-mediated inhibition of cellular protein synthesis. Virology 136, 133-143. Nibert, M.L., Schiff, L.A., Fields, B.N., 1996. Reoviruses and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M. (Eds.), Fundamental Virology, 3rd ed. Raven Press, New York, pp. 691-730. Nilsen, T.W., Maroney, P.A., Baglioni, C., 1982. Inhibition of protein synthesis in reovirus-infected HeLa cells with elevated levels of interferon-induced protein kinase activity. J. Biol. Chem. 257, 14593-14596. Rager-Zisman, B., Egan, J.E., Kress, Y., Bloom, B.R., 1984. Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells. J. Virol. 51, 845-855. Samuel, C.E., 1988. Mechanisms of the antiviral action of interferons. Progr. Nucleic Acid Res. Mol. Biol. 35, 27-72. Samuel, C.E., Knutson, G.S., 1981. Mechanism of interferon action: cloned human leukocyte interferons induce protein kinase and inhibit vesicular stomatitis virus but not reovirus replication in human amnion cells. Virology 114,

149

302-306. Seliger, L.S., Giantini, M., Shatkin, A.J., 1992. Translational effects and sequence comparisons of the 3 serotypes of the reovirus $4 gene. Virology 187, 202-210. Sen, G.C., Ransohoff, R.M., 1994. Interferon-induced antiviral actions and their regulation. Adv. Virus Res. 42, 57102. Sharpe, A.H., Fields, B.N., 1982. Reovirus inhibition of cellular RNA and protein synthesis: role of the $4 gene. Virology 122, 381-391. Staehli, P., 1990. Interferon-induced proteins and the antiviral state. Adv. Virus Res. 38, 147-200. Stohlman, S.A., Weiner, L.P., 1978. Stability of neurotropic mouse hepatitis virus during chronic infection of neuroblastoma cells. Arch. Virol. 57, 53-61. Taber, R., Alexander, V., Whitford, W., 1976. Persistent reovirus infection of CHO cells resulting in virus resistance. J. Virol. 17, 513-524. Verani, P., Nicoletti, L., Marchi, A., 1984. Establishment and maintenance of persistent infection by the phlebovirus Toscana in Vero cells. J. Gen. Virol. 65, 367-375. Wetzel, J.D., Chappell, J.D., Fogo, A.B., Dermody, T.S., 1997. Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses. J. Virol. 71,299-306.