Interferon production in L cells persistently infected with hemagglutinating virus of Japan (HVJ)

VIROLOGY

71, 463-470 (1976)

Interferon

YASUHIKO Germfree

Production in L Cells Persistently Infected Hemagglutinating Virus of Japan (HVJ)

ITO,’ YUKIHIRO NISHIYAMA, KIMURA, IKUYA NAGATA,

Life Research

institute,

AND

of Internal

and 21st Department Medicine, Showa-Ku, Accepted

KAORU

Nagoya,

January

SHIMOKATA, AKIRA KUNIP

Medicine, Japan

Nagoya

with

YOSHINOBU University

School

of

26,1976

The present study shows that when L cells persistently infected with hemagglutinating virus of Japan (HVJ) (L-HVJ cells) were incubated at 32”, the interferon-producing capacity of the cells was suppressed and was restored by the temperature shift-up to 38”. Synthesis of envelope protein antigen was not detected in the L-HVJ cells incubated at 38” which could produce interferon. Moreover, glutamine-starved L-HVJ cells did produce interferon even at 32”. The relationship between the suppressed state of interferon production and synthesis of viral envelope protein is discussed. INTRODUCTION

Interferon production in virus-infected cells has been studied by many investigators, including Hermodsson (1963) who reported that parainfluenza virus type 3 enhanced the growth of superinfecting Newcastle disease virus (NDV) in calf kidney cells, and suggested that this was due to the inhibition by the former virus of the production and antiviral action of interferon. Similar experimental results have also been reported in swine testis cells infected with hog cholera virus (Toba and Matumoto, 1971) and in HeLa cells persistently infected with HVJ (Maeno et al., 1966). At present, however, little is known about the mechanism of suppression of interferon production in virus-infected cells. The present study was performed to clarify the mechanism of suppression of interferon production in virus-infected cells by using a virus-carrier cell line which was established in L cells with a temperature-sensitive strain of HVJ isolated from BHK-HVJ cells (Nagata et al., 1972). MATERIALS

AND

METHODS

Cell cultures. Cells used in the present

study were mouse L cells, L-HVJ ’ Author addressed.

to whom

reprint

requests

cells (L should

be

cells persistently infected with HVJ), and chick embryo cells. Growth medium for these cells was Eagle’s minimum essential medium (MEM) supplemented with 10% bovine serum, 10% tryptose phosphate broth, and antibiotics. The maintenance medium contained no bovine serum supplement. A carrier culture of HVJ-infected L cells designated as L-HVJ cells was established with a temperature-sensitive strain of HVJ isolated from BHK-HVJ cells (BHK cells persistently infected with HVJ) (Nishiyama, unpublished data). The cells used in the present study were derived from 2040th subculture, and passages of the carrier culture were made routinely at 35”. In more than 99% of the cells, the viral antigens were present. Immunofluorescence staining. For immunofluorescence staining of intracellular antigens, cells were grown on cover slips in bottles, air-dried, and fixed in acetone for 20 min at room temperature. The cover slips were covered with fluorescein-labeled rabbit anti-envelope protein (V) or antinucleocapsid (S) antiserum, and were incubated for 30 min at 37”. They were then washed three times, rinsed, and mounted in buffered glycerol. Microscopy was carried out with an Olympus fluorescence microscope. 463

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

464

IT0

Preparation method of anti-envelope and anti-nucleocapsid antisera was described previously (Nagata et al ., 1972). Measurement of radioactivity incorporated into macromolecules. For determination of protein or RNA synthesis, cells were directly grown in glass counting vials of a liquid scintillation spectrometer. Then 0.5 $i of [14C]leucine per ml instead of unlabeled leucine and 1 PCi of [3H]uridine per ml were added to culture media. After 1 hr of incubation, incorporation was terminated by removal of the radioactive medium followed by two gentle washings with cold phosphate buffered saline (PBS). The cells were then gently washed three times with cold 5% trichloroacetic acid (TCA). The residues were dissolved in 0.1 ml of ammonia water (28%), and their radioactivity was determined in a liquid scintillation spectrometer (Alokal after the addition of 10 ml of Bray scintillation fluid. Interferon titration. Interferon samples were dialyzed overnight at pH 2.0, and redialyzed to neutrality against Hanks’ solution. Interferon was assayed by the plaque reduction method with mouse L cells and vesicular stomatitis virus (VSV), as challenge virus. Briefly, an aliquot (3.0 ml) of each specimen was placed on L cell culture in a petri dish and, 24 hr later, the culture fluid was aspirated, and challenged by about 100 plaque-forming units (PFU) of VSV. After 1 hr of incubation at 35”, the culture was overlayed with 1% agar in a medium consisting of 0.5% lactalbumin hydrolyzate and 0.1% yeast extract in Earle’s balanced salt solution (YLE). On Day 2 of virus challenge, the L cell monolayer was stained with neutral red and plaques were counted. Titers of interferon were expressed as the reciprocals of the dilutions causing 50% plaque-count reduction.

ET

AL.

fected with NDV (m.o.i. about 50). After a l-hr adsorption period at 35”, the virus inoculum was removed, the cells were washed three times with MEM, and then 3 ml of maintenance medium was added. After 20 hr of incubation at 35”, the culture fluid was collected and assayed for interferon activity. The results are shown in Table 1. L-HVJ cells incubated at 35” did not produce interferon, and the amount of interferon produced by L-HVJ cells superinfected with NDV was about 10% of that produced by L cells. This result shows that the viral-carrier cells used in this study have a low interferon-producing capacity in comparison with the noninfected control L cells. Interferon-Producing Cells Incubated

Capacity of L-HVJ at 38 or 32”

Confluent L and L-HVJ cells grown at 35” were incubated with maintenance medium at 38 or 32” for 24 hr, and then the cells were inoculated with NDV (m.o.i. about 50). After a 1-hr adsorption period at 38 and 32”, respectively, the virus inoculum was removed, the cells were washed, and then 3 ml of maintenance medium was added. After 20 hr of incubation at 38 or 32”, respectively, the culture fluid was assayed for interferon activity. As shown in Table 2, L-HVJ cells incubated at 38” produced a small amount of interferon without the stimulus of NDV (spontaneous interferon production), although the cells incubated at 32” produced no detectable interferon. NDV-superinfected L-HVJ cells incubated at 38” produced as much interferon as L cells inTABLE INTERFERON

Cell

PRODUCTION IN CELLS INCUBATED

Treatment (Inducer)

1

L CELLS

AND

L-HVJ

AT 35”

Interferon Expt.

1

titeP Expt.

2

RESULTS NDV None NDV

1200

3100
Interferon-Producing Capacity of LHVJ Cells Incubated at 35”

L Cell L-HVJ L-HVJ

L cells either uninfected or persistently infected with HVJ (L-HVJ cells) were grown to confluence at 35”, the culture fluid was removed, and the cells were in-

II Interferon titers were assayed by the plaquereduction method using L cells and WV. Titers of interferon were expressed as the reciprocals of the dilutions causing 50% plaque-count reduction.

Cell Cell


INTERFERON TABLE INTERFERON PRODUCTION IN CELLS INCUBATED peraure (9

L-HVJ Cells

AND VIRUS-INFECTED

2 L CELLS AND L-HVJ AT

32 or 38” feron titef

Yield of NDV

(PFUP

32

None

32 32

6.1 x 104 NDV
38 38 38

L Cells

PRODUCTION


2.2 x 105 2.4 x lo5

’ Interferon titers were assayed by the plaquereduction method using L cells and VSV. Titers of interferon were expressed as the reciprocals of the dilutions causing 50% plaque-count reduction. ’ Infectivity titers of NDV in the culture fluids were determined by the plaque method using monolayers of chick embryo cells. ’ Means “not tested.”

fected with NDV did, whereas in the culture fluid of L-HVJ cells incubated at 32 and superinfected with NDV, interferon activity was not detectable. When stimulated with NDV, the interferon-producing capacity of L cells incubated at 32” did not vary markedly as compared with the cells at 38”. The growth rates of NDV at 32 and 38” were not much different in L cells and L-HVJ cells, although only a small amount of infectious NDV was produced in L cells (Hecht and Summers, 1974). The results show that the interferonproducing capacity of L-HVJ cells was apparently suppressed at 32”.

CELLS

465

while none was formed at 32”. When LHVJ cells incubated for 24 hr at 32” were transferred to 38”, interferon production became apparent. This finding shows that the repressed state of interferon production in L-HVJ cells at 32” was derepressed by temperature shift-up to 38”. In another experiment, L-HVJ cells, grown out at 35”, were first incubated at 38” for 24 hr and then superinfected with NDV. After a 1-hr adsorption period at 4”, these cells were further incubated at 38”, and after various times were transferred to 32”. Twenty hours after the beginning of the second incubation, the culture fluid was assayed for interferon activity. The results are shown in Fig. 2. When temperature shift-down from 38 to 32” was done at 0 time (at the end of NDV adsorption) or at 1 hr after the incubation, no interferon could be detected in the culture fluid. If the shift-down was delayed till 3 hr after incubation, the final yields of interferon were markedly reduced. However, temperature shift-down at 6 hr after the incubation resulted in a full synthesis of interferon. These data suggest that the suppression of interferon production in L-HVJ cells is brought about within a short time after temperature shift-down from 38 to 32” and

Effects of Temperature Shift from 32 to 38” and from 38 to 32” on Interferon Production Confluent L-HVJ cells grown at 35” were incubated in the maintenance medium at 38 or 32”. After 1 and 2 days of incubation, the culture fluid was withdrawn and assayed for interferon activity. As shown in Fig. 1, a considerable amount of interferon was detected in the culture fluid at 38”,

FIG. 1. Effects of temperature shift-up from 32 to 38” on spontaneous interferon production. Symbols: +O, at 38”; O-- 0, at 32”; H----m, shiftup from 32” ( t ) to 38”.

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FIG. 2. Effects on interferon production of temperature shift-down at various hours of incubation. L-HVJ cells grown at 35” were incubated at 38” for 24 hr, and then the cells were superinfected with NDV. After a 1-hr adsorption period at 4”, these cells were further incubated at 38”, and at various times of incubation, these cells were transferred to 32”. At 20 hr after the beginning of the second incubation, the culture fluid was assayed for interferon activity. Symbols: -, at 38”; - - -, at 32”; ( J 1, shift-down 38 to 32”.

that temperature-dependent interferon synthesis occurs during the first 6 hr after viral infection. Effects of Actinomycin D, Puromycin, and Cytochalasin B on Interferon Production In order to determine which step of the interferon-production process in L-HVJ cells is suppressed at 32”, the effects of actinomycin D and puromycin on interferon production were studied in cells in which there was a temperature shift-up from 32 to 38” (spontaneous interferon production). L-HVJ cells, preincubated at 32” for 24 hr, were treated with actinomycin D (10, 1 pg/ml) or puromycin (100, 10 pg/ml) for 30 min at 32”, and the cells were then transferred to 38” without washing. At 24 hr after the temperature shift-up, the culture fluid was collected, dialyzed against MEM to remove these inhibitors, and then assayed for interferon. As shown in Table 3, the spontaneous interferon production con-

ET

AL.

sequent upon the temperature shift-up was suppressed by these inhibitors. This experimental result shows that the suppressive step of the interferon production process in L-HVJ cells at 32” occurs before messenger RNA synthesis. Cytochalasin B inhibits interferon production by mouse spleen cells cocultivated with viral-carrier cell (unpublished data). Therefore, the effects of cytochalasin B on the spontaneous interferon production after temperature shift-up to 38” were examined. Treatment of L-HVJ cells with cytochalasin B resulted in suppression of the interferon yield, as determined in culture fluid collected 24 hr after shift-up to 38”(Table 4). Furthermore, interferon production by L-HVJ cells superinfected with NDV was also suppressed by cytochalsin B, but that by L cells was not influenced by cytochalasin B treatment (Table 5). This finding indicates that L-HVJ cells are more sensitive to cytochalasin B treatment than L cells. Characteristics of L-HVJ at 38 or 32”

Cells Incubated

The L-HVJ cells were grown at 35” for 2 days. After confluent monolayers had formed, the cells in maintenance medium TABLE

3

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN SFQNTANEOUS INTERFERON PRODUCTIOEP Treatmentb Actinomycin Actinomycin Puromycin Puromycin None

Dose D D

(pg/ml) 10 1 100 10

Interferon ter

ON ti-

<4 <4 <4 <4 36

” Interferon production in the cells induced by temperature shift-up from 32 to 38”. b L-HVJ cells, preincubated at 32” for 24 hr, were treated with actinomycin D or puromycin for 30 min at 32”, and the cells were then transferred to 38” without washing. At 24 hr after the temperature shift-up, the culture fluid was obtained, dialyzed against MEM to remove these inhibitors, and then assayed for interferon. r Interferon titers were assayed by the plaquereduction method using L cells and VSV. Titers of interferon were expressed as the reciprocals of dilutions causing 50% plaque-count reduction.

INTERFERON TABLE EFFECTS

PRODUCTION

AND

4

OF CYTOCHALASIN B ON SWNTANEOUS INTERFERON PRODUCTION"

Dose (pg/ml)

Interferon

titer” <3 30 90 90

100 10 1 0

R L-HVJ cells, preincubated at 32” for 24 hr, were treated with cytochalasin B for 30 min at 32”, and the cells were then transferred to 38” without washing. At 24 hr after temperature shift-up, the culture fluid was collected, dialyzed against MEM to remove the drug, and then assayed for interferon. b Interferon titers were assayed by the plaquereduction method using L cells and VSV. Titers of interferon were expressed as the reciprocals of the dilutions causing 50% plaque-count reduction. TABLE

5

EFFECTS OF CYTOCHALASIN B ON INTERFERON PRODUCTION STIMULATED WITH NDV’ Cell L-HVJ

L Cell

Cell

Dose

(@g/ml) 100 10 0 100 10 0

1nterff;m

ti-

180 720 1100 1030 1140 1110

” The cells, preincubated at 38” for 24 hr, were treated with cytochalasin B for 30 min at 38”, the culture fluid was removed, and the cells were infected with NDV (m.o.i. about 501. After a 1-hr adsorption period, the virus inoculum was removed, the cells were washed three times with MEM, and then 3 ml of maintenance medium containing cytochalasin B were added. After 20 hr of incubation at 38”, the culture fluid was collected, dialyzed against MEM to remove the drug, and then assayed for interferon. li Interferon titers were assayed by the plaquereduction method using L cells and VSV. Titers of interferon were expressed as the reciprocals of the dilutions causing 50% plaque-count reduction.

were incubated for 24 hr at 38 or 32”. The characteristics of these cells were investigated. The results are shown in Table 6. The presence of viral antigens in the cytoplasm of the cells was tested by fluorescent antibody staining. Nucleocapsid protein antigen was present in the cytoplasm of the cells incubated at both 38 or 32”. Envelope protein antigen was not de-

VIRUS-INFECTED

467

CELLS

tectable in the cells incubated at 38”, whereas it was present in the cytoplasm of the cells at 32”. Cells incubated at 38 or 32 showed neither hemadsorption nor cell-associated hemagglutination. Synthesis of actinomycin D-resistant RNA could not be detected in the cells at 38”. The rate of protein and RNA synthesis in L-HVJ cells incubated at 32” was about 70% of that in the cells at 38”. These findings may indicate that L-HVJ cells are endowed with interferon-producing capacity at the expense of synthesis of envelope protein antigen and virus-specific RNA indicated by actinomycin D-resistant incorporation. Effects of L-Glutamine Deprivation Interferon Production

on

The previous study (Ito et al., 1974) showed that L-glutamine is essential for replication of HVJ, and that omission of LTABLE CHARACTERISTICS

6

OF L-HVJ CELLS INCUBATED 38 OR 32” Temperature 38”

Nucleocapsid antigen” (% antigen positive cells) Envelope protein antigen” (% antigen positive cells) Hemadsorption Cell-associated HA” Actinomycin D-resistant RNA’ RNA synthesis’ Protein synthesis’

AT

32”

>99

>99


>99



I’ For fluorescent antibody staining of intracytoplasmic envelope protein and nucleocapsid antigen, cells grown on coverslips were used after fixation by acetone for 20 min at room temperature. b For measurement of cell-associated hemagglutinin, the cells in one petri dish (40-mm diameter), detached by EDTA treatment, were suspended in 2.0 ml of PBS and sonicated for 30 sec. I’ Measured by radioactivity incorporated into macromolecules. Added to culture media were 5 pg/ ml of actinomycin D, 0.5 &i/ml of [Wlleucine, and 1 &i/ml of lDHluridine. Rates of RNA or protein synthesis at 38” were taken as 100%. Actinomycin Dresistant RNA synthesis in L-HVJ cells = RNA synthesis in L-HVJ cells in the presence of actinomycin D-RNA synthesis in control L cells in the presence of actinomycin D.

468

IT0

ET AL.

HVJ cells) were incubated at 32”, the interferon-producing capacity of the cells was suppressed. Interferon-producing capacity of L-HVJ cells was restored by the temperature shift-up to 38”. A similar phenomenon had been reported in L cells persistently infected with tick-borne encephalitis virus by Stancek (1965). Wagner and Huang (1966) reported that NDV induced interferon production in Krebs-2 carcinoma cells but a marked reduction in interferon production took place when the cells were coinfected with VSV. Their experimental data suggest that the inhibition of interferon production is due to suppression of cellular RNA synthesis caused by the coinfecting virus. However, this mechanism seemsunlikely in our system, because L-HVJ cells incubated at 32” could synthesize protein and RNA at about 70% of the rate of 38”, and glutaminestarved L-HVJ cells incubated at 32” could produce a considerable amount of interferon, although the rate of RNA synthesis was decreased by glutamine-starvation (It0 et al., 1974). DISCUSSION Another possibility, that this suppresThe present study shows that when L sive state of interferon production may be cells persistently infected with HVJ CL- mediated by an inhibitory factor produced by viral-carrier cells, was considered since some evidence for the production of such an inhibitor [blocker (Isaacs et al., 1966) and interferon depressor (Kate et al., 1969)l in some virus-host systems has been reported. However, the suppression of interferon production was brought about immediately after the shift-down to 32” and we failed to demonstrate a suppressive effect of either culture fluid or cell homogenate of L-HVJ cells incubated at 32” on interferon production (unpublished data). Therefore, the suppressive state of inter0 feron production in our system could not be considered to be due to some factor(s) FIG. 3. Effects of L-glutamine deprivation on inblocking the interferon production. terferon production. After confluent monolayers of Synthesis of envelope protein antigen L-HVJ cells had formed, the cells were washed with was not detected in the L-HVJ cells incuglutamine-deprived MEM, and then incubated in bated at 38” which could produce interglutamine-deprived MEM at 32” for a further 12 hr. Lferon. Moreover, glutamine-starved These glutamine-starved cells were incubated in HVJ cells did produce interferon even at glutamine-deprived MEM at 32”, and 1 or 2 days 32”. GGlutamine is essential for replication after incubation, interferon in the culture fluid was of HVJ, and omission of Lglutamine from assayed. Symbols: O-O, glutamine-deprived culture fluid resulted in a marked inhibiMEM, O-O, normal MEM. glutamine from culture medium resulted in a markedly inhibitory effect of the release of infectious virus and synthesis of envelope protein, although synthesis of virus-specific RNA and nucleocapsid antigen was readily detected. An experiment was performed to test whether the omission of L-glutamine from culture medium would bring about induction of interferon production in the L-HVJ cells at 32”. After confluent monolayers of L-HVJ cells had formed, the cells were washed with glutamine-deprived MEM, and then incubated in glutamine deprived MEM at 32” for further 12 hr. These glutaminestarved cells were incubated in glutaminedeprived MEM at 32”, and 1 or 2 days after incubation, interferon in the culture fluid was assayed. As shown in Fig. 3, L-HVJ cells at 32” produced a considerable amount of interferon when incubated in glutamine-deprived medium. However, glutamine deprivation did not influence NDV-induced interferon production in LHVJ cells (data not shown).

INTERFERON

- PODUCTION

tory effect of synthesis of envelope protein. Futhermore, there is another carrier culture of HVJ-infected L cells in our laboratory in which the envelope protein antigen is detectable at 38”. These cells incubated at 38” can produce neither interferon induced by temperature shift-up to 38” nor NDV-induced interferon (unpublished data). Therefore, it is inferred that the suppression of interferon production observed in L-HVJ cells may involve a process of envelope protein synthesis. Any relationship between the suppression of interferon production and synthesis of viral envelope protein is not yet known, but a common feature was found in several points in regard to process of interferon synthesis and of synthesis of viral envelope protein. Little information is available on the protein metabolism in HVJinfected cells. With regard to NDV, another paramyxovirus, Nagai et al. (personal communication) recently reported that the envelope proteins of NDV are synthesized and processed to the membrane system. Interferon synthesis is also associated to membrane system. The formation of envelope protein in several enveloped virus was shown to be selectively inhibited by %deoxy-n-glucose or n-glucosamine (Kaluza et al., 1972; Klenk et al., 1972). Most available evidence suggests that this inhibition of virus multiplication is due to selective interference with normal glycosylation of viral envelope glycoproteins. Either of these two inhibitors seems to suppress the production of biologically active interferon in the same manner (Havell et al., 1975). Therefore, it is quite likely that competition may play an important role on the suppression of interferon production in L-HVJ cells. A further study is now under way to clarify this point. It is not clear why glutamine-starved LHVJ cells at 32” could not produce NDVinduced interferon. A similar result was obtained when L-HVJ cells, preincubated at 32” for 24 hr, were infected with NDV within 12 hr after temperature shift-up to 38”. The cells could produce only a small amount of interferon (unpublished data). These results may suggest that the suppressive state of NDV-induced interferon

AND

VIRUS-INFECTED

CELLS

469

production in L-HVJ cells was not desuppressed at once after arrest of envelope protein synthesis and was influenced by the presence of envelope protein antigen. Which step of interferon production process is suppressed? It is neither the step of interferon-inducer adsorption (ex. NDV) nor that of interferon release. Since either actinomycin D or puromycin inhibits interferon production induced by temperature shift-up to 38”, the suppressive step should occur before m:-ssenger RNA and protein synthesis. Any interferon suppressive step might be related with the cellular site of action of cytochalasin B, for cytochalasin B prevented L-HVJ cells from producing interferon upon the shift-up to 38”. Cytochalasin B is known to depress active transport of some monosaccharides across the plasma membrane and to alter the function of the microfilaments of cortical cytoplasma, although the primary cellular site of its action has not been unequivocally demonstrated. Further investigation is required to clarify this point. ACKNOWLEDGMENTS We wish the thank Mrs. Yamaguchi, Mrs Iwata, and Miss Hattori for able technical assistance. This research was supported by a grant from the Ministry of Education, Tokyo, Japan. REFERENCES HAVELL,

E. A., VILCEK, J., FALCOFF, E., and BERB. (1975). Suppression of human interferon production by inhibitors of glycosylation. Virology 63, 475-483. HECHT, T. T., and SUMMERS, D. F. (1974). Newcastle disease virus infection of L cells. J. Viral. 14, 162169. HERMODSSON, S. (1963). Inhibition of interferon by an infection with parainfluenza virus type B(PIV3). Virology 20, 333-343. ISAACS, A., ROTEM, Z., and FANTES, K. H. (1966). An inhibitor of the production of interferon (“blocker”). Virology 29, 248-254. ITO, Y., KIMURA, Y., NAGATA, I., and KUNII, A. (1974). Effects of L-glutamine deprivation of growth of HVJ (Sendai Virus) in BHK cells. J. Virol. 13, 557-566. KALUZA, G., SCHOLTISSEK, C., and ROTT, R. (1972). Inhibition of the multiplication of enveloped RNA-viruses by glucosamine and 2-deoxy-n-glucase. J. Gen. Virol. 14, 251-259. MAN,

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KATO, M., EGGERS, H. J., OHTA, F., and KOBAYASHI, T. (1969). Depressors of interferon synthesis: Further studies on the production, action and properties of the so-called enhancer. J. Gen. Viral. 5, 195-203. KLENK, H. D., SCHOLTISSEK, C., and ROTT, R. (1972). Inhibition of glycoprotein of influenza virus by Dglucosamine and 2-deoxy-n-glucose. Virology 49, 723-734. MAENO, K., YOSHII, S., NAGATA, I., and MATSUMOTO, T. (1966). Growth of Newcastle disease virus in a HVJ carrier culture of HeLa cells. Virology 29, 255-263. NAGATA, I., KIMURA, Y., ITO, Y., and TANAKA, T. (1972). Temperature-sensitive phenomenon of

ET

AL. viral maturation observed in BHK cells persistently infected with HVJ. Virology 49, 453-461. STANCEK, D. (1965). The role of interferon in tickborne encephalitis virus-infected L-cells. III. The effects of temperature on the production of virus and interferon by L cells during acute and persistent infection. Acta Viral. 9, 298-307. TOBA, M., and MATUMOTO, M. (1971). Mechanism of enhancement of Newcastle disease virus growth in culture cells by co-infecting hog cholera virus. Arch. Ges. Virusforsch. 34, 310-322. WAGNER, R. R., and HUANG, A. S. (1966). Inhibition of RNA and interferon synthesis in Krebs-2 cells infected with vesicular stomatitis virus. Virology 28, l-10.