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Mechanism of interferon action Species specificity of interferon and of the interferon-mediated inhibitor of translation from mouse, monkey, and human cells
VIROLOGY
77,
556-565
(1977)
Mechanism Species
Action
Specificity of Interferon and of the Interferon-Mediated Inhibitor Translation from Mouse, Monkey, and Human Cells
CHARLES Section
of Interferon
of Biochemistry
and
E. SAMUEL’
Molecular
AND
DEBORAH
Biology, Department of Biological Santa Barbara, California 93106 Accepted
November
of
A. FARRIS Sciences,
University
of California,
19,1976
The species specificity of interferon and of the interferon-mediated ribosome-associated inhibitor of translation from mouse fibroblast, African green monkey kidney, and human amnion cells was investigated. The following results were obtained: (1) The antiviral activity of mouse tibroblast, monkey kidney, and human leukocyte interferons was maximal on the homologous species of cells. Mouse interferon was not active on heterologous monkey cells, and monkey interferon was not active on mouse cells. Monkey and human interferons were almost equally active on heterologous human and monkey cells, respectively; mouse and human interferons were about 10 and 125 times more active, respectively, on their homologous cell species than on heterologous human and mouse cells. (2) Ribosomal salt-wash fractions prepared from interferon-treated mouse fibroblast, monkey kidney, and human amnion cells inhibited the translation of reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. The corresponding fractions prepared from untreated mouse and human cells either did not affect or stimulated the translation of reovirus mRNA. Ribosomal washes from untreated monkey kidney cells were inhibitory, but not as inhibitory as washes from interferon-treated cells. (3) The concentration of KC1 required to release the interferon-mediated ribosome-associated inhibitor from ribosomes of mouse fibroblast cells was higher than that required for ribosomes from human amnion and monkey kidney cells. (4) The translation of both methylated and unmethylated reovirus mRNA catalyzed by the mouse ascites cell-free system was inhibited by the mouse tibroblast ribosome-associated interferon-mediated factor. These results suggest that the interferon-mediated ribosome-associated antiviral factor(s) is not species specific, even though certain interferons are relatively species specific. INTRODUCTION
and RNA-containing animal viruses is inhibited as a result of interferon treatment appears to be the translation of viral messenger RNA into protein (Ho and Armstrong, 1975). Cell-free extracts prepared from interferon-treated murine cells have been shown to be incapable of catalyzing the translation of encephalomyocarditis RNA (Friedman et al., 19721, mengovirus RNA (Falcoff et al., 19731, reovirus mRNA (Gupta et al., 1974; Samuel and Joklik, 1974), and vaccinia virus mRNA (Samuel and Joklik, 1974). The interferon-mediated inhibitor of viral mRNA translation is ribosome-associated and can be separated from ribosomes by either washing with
Interferons, cellular proteins synthesized in response to viral infection as well as to several nonviral stimuli, mediate the establishment of an antiviral state in a wide variety of animal cells through a mechanism which apparently involves an extracellular interaction between the interferon molecule(s) and the cellular membrane (Finter, 1973; Ho and Armstrong, 1975; Vengris et al., 1975; Revel et al., 1976). In several animal virus-cell systerns, the primary level of gene expression at which the multiplication of both DNA’ To whom
reprint
requests
should
be addressed. 556
Copyright All riahts
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
MECHANISM
OF
INTERFERON
buffers containing concentrated salt (Falcoff et al., 1973; Samuel and Joklik, 1974) or incubation (Gupta et pl., 1973). The specific biochemical step(s) in the process of translation of viral mRNA into viral protein primarily affected by the interferonmediated ribosome-associated inhibitor and directly responsible for the antiviral effect of interferon has not been elucidated. However, recent studies indicate that the interferon-mediated inhibition of viral mRNA translation in vitro is exerted at a step of polypeptide chain biosynthesis which is subsequent to formation of the first peptide bond (Content et al., 1975; Samuel, 1976) and which either directly or indirectly involves the participation of transfer RNA (Colby et al., 1976; Content et al., 1974; Sen et al., 1976) and the sensitivity of protein synthesis to doublestranded RNA (Kerr et al., 1974). Certain interferons display relatively strict species specificity for cells from the homologous species, with less or no antiviral activity in cells from heterologous species (Finter, 1973), whereas other interferons, such as human leukocyte interferon, are active on a wide variety of cells from heterologous species (Finter, 1973; Borecky et al., 1974; Desmyter and Stewart, 1976). However, the characteristic species specificity of the ribosome-associated antiviral factor synthesized in response to interferon treatment is .undefined. The #tudies presented here were therefore undertaken to determine (1) the homologous and heterologous antiviral activity of mouse tibroblast, African green monkey kidney, and human leukocyte interferons on mouse fibroblast, monkey kidney, and human amnion cells, and (2) the effect of the ribosomal salt-wash fractions prepared from ribosomes isolated from untreated and interferon-treated mouse, monkey, and human cells on the translation in vitro of reovirus messenger RNA catalyzed by the Krebs mouse ascites cell-free proteinsynthesizing system prepared from untreated ascites cells. The reasons for choosing this system were primarily the convenient growth characteristics and sensitivity to interferon of the mouse fibroblast (Sanford et al ., 1948; Youngner et al ., 19661,
557
ACTION
African green monkey kidney (Jensen et al., ,1964; Yamamoto et al., 19751, and human amnion (Strander and Cantell, 1966) cells, the ability of the untreated Krebs ascites cell-free protein-synthesizing system to efficiently catalyze the translation of reovirus mRNA into known viral polypeptides (McDowell et al., 1972; Samuel and Joklik, 19761, and the inhibitory action of ribosomal salt washes from interferon-treated mouse cells on viral mRNA translation catalyzed by the mouse ascites cell-free system (Falcoff et al., 1973; Samuel and Joklik, 1974). MATERIALS
AND
METHODS
Materials. L-[4,5-3H,1Leucine, 44 Ci/ from Schwarz/ mmol, was purchased Mann. ‘*C-labeled L-amino acids, S-adenosyl-L-methionine, creatine phosphate, creatine phosphokinase, and dithiothreitol were from Sigma; ATP, GTP, CTP, and UTP were from either Sigma or P. L. Biochemicals; S-adenosyl+homocysteine was from Calbiochem; Eagle’s MEM and Joklik-modified Eagle’s MEM were from Grand Island Biological Co.; fetal calf serum was from Irvine Scientific; and female albino mice (CD-Swiss) were from Hilltop Laboratory. Cells. Clone L929 mouse fibroblast cells, obtained from Dr. W. K. Joklik, Duke University Medical Center, human am-. nion U cells, obtained from Dr. J. Vilcek, New York University Medical Center, and the CV-1 line of African green monkey kidney cells, obtained from Dr. J. A. Carbon, University of California, Santa Barbara, were grown in roller culture in Eagle’s MEM containing 7% fetal calf serum. Krebs ascites tumor cells were maintained in uiuo by injection of 0.15 ml of ascitic fluid into the peritoneal cavity of female albino mice; the cells were passed every 7 to 9 days. Preparation
and
assay
df interferon.
Mouse and monkey interferons induced in confluent aged roller bottle cultures of L,,, and CV-1 cells, respectively, with the Hirts strain of NDV* were prepared as 2 Abbreviations used: DTT, dithiothreitol; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; NDV, Newcastle disease virus; PFU,
558
SAMUEL
AND
previously described (Samuel and Joklik, 1974). Human leukocyte interferon was generously provided by Dr. T. C. Merigan, Stanford University Medical Center. The specific activities of the interferon preparations as determined by a 50% plaque reduction assay on cell monolayers infected with the Indiana strain of VSV (Lai and Joklik, 1973) were: mouse interferon assayed on mouse fibroblast L,,, cells, 3.6 x lo4 units/mg of protein; monkey interferon assayed on monkey kidney CV-1 cells, 6.1 x lo3 units/mg of protein; human interferon assayed on human amnion U cells, 2.1 x lo5 units/mg of protein. One unit of interferon was defined as the reciprocal of the highest dilution that reduced by 50% the number of VSV plaques formed. Protein determination. Protein concentrations were determined by a modification of the phenol reagent method with crystalline bovine serum albumin as the reference standard as described by Rabinowitz and Pricer (1962). Preparation of ribosomal salt-wash fractions. Ribosomal salt-wash fractions
were prepared from untreated and interferon-treated mouse LsZ9 cells, monkey CV-1 cells, and human U cells grown in roller bottles with a growth area of 1330 cm2; 6 to 10 bottles of untreated cells or cells treated for 24 hr with 100 units of interferon/ml were utilized for each preparation. All operations, unless otherwise stated, were carried out at O-4”. Cells were harvested by scraping, washed in phosphate-buffered saline (10 mM phosphate buffer, pH 7.3, containing 137 mM NaCl and 3 n&f KCl), and pelleted at 8OOg for 10 min in an IEC CRU-5000 centrifuge with a 269 rotor. The cell pellet was suspended in 3 packed-cell vol of hypotonic buffer [lo mM HEPES buffer, pH 7.5, containing 15 mM KCl, 1.5 mM Mg(OAc),, and 1 miW D’M’I, allowed to swell for 10 min, and disrupted with about 30 strokes of a tightfitting Dounce homogenizer. The extract plaque-forming units; SAdoHcy, S-adenosyl-L-homocysteine; SAdoMet, S-adenosyl-L-methionine; TCA, tricholoacetic acid; VSV, vesicular stomatitis virus.
FARRIS
was immediately adjusted to a final concentration of 20 mM HEPES buffer, pH 7.5, 5 mit4 Mg(OAc),, 120 mM KCl, and 2 mM DTI’. The homogenate was centrifuged at 5000 g for 5 min, the pellet was discarded, and the supernatant solution was centrifuged at 10,000 g for 20 min. Ribosomes were isolated from the 10,000 g supernatant solution by centrifugation at 150,000 g for 2.5 hr in a DuPont Sorvall OTD-50 ultracentrifuge and T865.1 rotor. The translucent ribosomal pellet was gently suspended in 20 n-&f HEPES buffer, pH 7.5, containing 120 rniW KCl, 5 miW Mg(OAc),, and 1 mM DTI’. The suspension was stirred slowly for 1 hr followed by centrifugation for 2.25 hr at 150,000 g; the resulting supernatant solution was defined as the 0.12 M ribosomal salt wash. The 0.12 M salt-washed ribosomal pellet was suspended, stirred, and centrifuged as before except that the buffer was modified to contain 300 mM KCI; the resulting supernatant solution was defined as the 0.3 M ribosomal salt wash. The 0.3 M saltwashed ribosomal pellet was suspended and stirred for 1 hr as before except that the buffer was modified to contain 800 mM KCl; the suspension was centrifuged overnight at 120,000 g. The resulting supernatant solution, defined as the 0.8 M ribosomal salt wash, as well as the 0.12 and 0.3 M ribosomal salt washes, were all centrifuged for 4 hr at 120,000 g and then dialyzed against 250 vol of 20 n&f HEPES buffer, pH 7.5, containing 50 mit4 KCl, 1 miW Mg(OAc),, 1 mM D’M’, and 10% (v/v) glycerol. The dialysis was carried out overnight with one change of buffer. The fraction recovered by centrifugation of the dialyzed ribosomal washes at 5000 g for 15 min with a DuPont Sorvall RC-5 centrifuge and SS-34 rotor was divided into small aliquots and stored at -90”. Preparation of cell-free protein-synthesis extracts. Preincubated, Sephadex G-25-
treated Krebs II mouse ascites cell-free protein-synthesis extracts were prepared from untreated control cells as previously described (Samuel and Joklik, 1974). Preparation of reovirus mRNA. For the preparation of reovirus mRNA, the Dearing strain of reovirus type 3 was grown in
MECHANISM
OF INTERFERON
mouse L fibroblast cells in suspension culture and purified by the procedure of Smith et al. (1969). Reovirus mRNA was prepared as the in vitro transcription product of the double-stranded RNA-dependent single-stranded RNA polymerase (Skehel and Joklik, 1969); for the preparation of methylated and unmethylated mRNAs, 10 $I!I SAdoMet and 1 n-&I SAdoHcy, respectively, were present in the transcription reaction mixture. Viral mRNA synthesized in vitro for use in cell-free proteinsynthesis experiments was purified from the reaction mixture as previously described (Samuel and Joklik, 1974). Assay ofprotein synthesis in vitro. Protein synthesis in vitro was measured by determining the incorporation of radioactively labeled L-[3H]leucine into hot trichloroacetic acid-insoluble material as described before (Samuel and Joklik, 1974). The standard reaction mixture (50 ~1) contained the following ingredients: 30 mM HEPES, pH 7.5; 96 miI4 KCl; 3 to 4 miI4 Mg(OAc),; 1 n-&I ATP; 0.2 mM GTP; 0.6 m&I CTP; 1 n-&! D’IT; 10 mM creatine phosphate; 0.2 mg/ml of creatine phosphokinase; 19 W-labeled L-amino acids, 50 fl each; 50 &X/ml of r.,-L3Hlleucine; ascites cell-free extract, 0.3 ml/ml; and 75 pglml of reovirus mRNA as indicated. Unless otherwise indicated, 40-~1 aliquots were processed after incubation at 30” for 30 min. Radioactivity was measured with a Beckman LS-230 liquid scintillation spectrometer for 10 min or to give a standard error of counting rate of less than 1.0%. RESULTS
Antiviral activity of interferon on homologous and heterologous cells. The sen-
sitivity of mouse L,,, fibroblast, African green monkey kidney CV-1, and human amnion U cells to homologous and heterologous interferon(s) was assayed by reduction of VSV plaque formation. As shown in Fig. 1, maximal antiviral activity of mouse fibroblast, monkey kidney, and human leukocyte interferon preparations was obtained on the homologous species of cells. However, significant cross-species activity was observed in the case of some
125
A. MOUSE
2 ,251
B. MONKEY
;
C. HUMAN
125 -
559
ACTION
INTERFERON
INTERFERON
INTERFERON
1
INTERFERON
CONCENTRATION
(units
per ml)
FIG. 1. Comparison of the antiviral activity of mouse tibroblast interferon (A), African green monkey kidney interferon (B), and human leukocyte interferon (Cl on mouse fibroblast, African green monkey kidney, and human amnion cells. Confluent monolayers of cells, treated with the indicated concentrations of interferon, were challenged with VSV and the plaques were counted as described under Materials and Methods.
cell:interferon combinations. The crossspecies antiviral activity was comparable between monkey and human interferons and cells; for both monkey and human interferons, the titer determined on the heterologous cell species was about 60 + 10% of the titer determined on the homologous cell species (Table 1). The titer of mouse interferon when assayed on mouse L,,, cells was about lo-fold greater than when assayed on human U cells, whereas the titer of human interferon was about 125fold greater on human U cells than on mouse L,,, cells; no appreciable activity was observed with either mouse interferon
SAMUEL
560 TABLE SPECIFICITY
OF THE
AND
FARRIS
1
ANTIVIRAL
ACTION
801A.
OF
0.!2
M
WASH
INTERFERING
Cell
Interferon specie@ Mouse Mouse Monkey Human
species’
Monkey
Human
7.6 x lo4
<32
<64 2.7 x lo4
6.1 x lo3
6.6 x 103 4.1 x 103
1.9 x 106
3.4 x 106
(1 Interferon titers (units per milliliter) were determined on homologous and heterologous cell species. Titers are expressed as the reciprocal of the dilution of the interferon preparation required to prod&e a 50% reduction in vesicular stomatitis virus plaque formation. b Mouse L,,, tibroblast, African green monkey kidney CV-1, and human leukocyte. r Mouse L,,, tibroblast, African green monkey kidney CV-1, and human amnion U.
--m-o------w___ c~
0 2
C.
80-
M
0.0
---
0
~ -- -c
--__
~
WASH
assayed on monkey cells or monkey interferon assayed on mouse cells (table 1, Fig. 1). Effect of ribosomal treated and translation
homologous and heterologous salt-wash fractions from uninterferon-treated cells on the of viral mRNA. Ribosomal
salt-wash fractions prepared from interferon-treated mouse fibroblast, monkey kidney, and human amnion cells were examined for their ability to affect the translation of reovirus mRNA catalyzed by the mouse ascites tumor cell-free protein-synthesizing system. Ribosomes isolated by centrifugation were washed successively at 4” in buffer solutions containing 0.12, 0.3, and 0.8 M KCl; varying concentrations of each fraction were assayed for their ability to inhibit or stimulate the translation of reovirus mRNA as measured by the incorporation of 13Hlleucine into hot trichloroacetic acid-insoluble material. The results obtained with salt-wash fractions prepared from ribosomes isolated from interferon-treated cells were compared with identical fractions prepared from ribosomes isolated from untreated cells. Mouse cells. As shown in Fig. 2, the 0.3 and 0.8 M ribosomal salt-wash fractions prepared from untreated mouse L,,, fibroblast cells stimulated incorporation of L3Hlleucine in response to reovirus mRNA, whereas the comparable salt-wash fractions prepared from L,,, cells treated with
0
20
RIBOSOMAL
(pg
40
60
SALT-WASH
CONCENTRATION
protein
0.1 ml 1
per
80
FIG. 2. Effect of ribosomal salt-wash fractions from untreated and interferon-treated mouse L,, flbroblast cells on the translation of methylated reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. The standard reaction conditions were as described under Materials and Methods. Ribosomal salt-wash fractions were prepared by washing isolated ribosomes successively with buffers containing (A) 0.12 M KCl; (Bl 0.3 M KCl; (0 0.8 M KC1 as described under Materials and Methods. Wash from untreated cells (C): O---O, endogenous activity; O-O, activity with added reovirus mRNA. Wash from interferon-treated cells (IF): A---A, endogenous activity; A-A, activity with added reovirus mRNA. Small filled circles at zero protein concentration show activity in the absence of added wash. Reaction volume, 50 ~1; a 40-~1 aliquot was processed.
mouse fibroblast interferon inhibited the translation of reovirus mRNA catalyzed by the mouse ascites cell-free system (Figs. 2B and 20. The 0.12 M salt-wash fraction from both interferon-treated and
MECHANISM
6C l- _
A.
0.12
M
OF
INTERFERON
WASH
4c )m b
2C l-
;
c )-
2
6C )- _ B.
B 2
4c , -
8 g
20 I-
0.3
M
WASH
:---,-lloQc--------A
s-
cI
ii6
80 , -.
s J T &!2d
---------^---Q
I
-L
C.
0.8
t
M
L
WASH
1
I
/IF
561
ACTION
feron inhibited the translation of reovirus mRNA catalyzed by the mouse cell-free system; the 0.12 M wash prepared from ribosomes isolated from untreated cells was not inhibitory (Fig. 3A). Although the 0.3 M salt wash from interferon-treated U cells was slightly less active than the corresponding fraction from untreated cells, neither had a significant affect on in vitro translation (Fig. 3B). Both the interferontreated and untreated 0.8 M ribosomal salt washes stimulated incorporation significantly (Fig. 30. Monkey cells. The activity in the mouse ascites cell-free protein-synthesizing system of 0.12, 0.3, and 0.8 M ribosomal salt-
60
r 80 t
40
A.
0.12M
B.
0.3
WASH
20 0
v
IO
RIBOSOMAL
(pg
20 30 40 50 SALT-WASH CONCENTRATION
protein
per 0.1 ml )
FIG. 3. Effect of ribosomal salt-wash fractions from untreated and interferon-treated human amnion U cells on the translation of methylated reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. A, B, C, and symbols are as in Fig. 2.
untreated mouse L,,, cells did not significantly affect the translation of viral mRNA, although the higher concentrations of 0.12 M ribosomal wash derived from interferon-treated cells were slightly more inhibitory than the wash from untreated cells. Ribosomal wash fractions prepared from L929 mouse cells treated with heterologous interferon were not inhibitory.” Human cells. The cross-species activity of ribosomal wash fractions from interferon-treated and untreated human amnion U cells on the translation of reovirus mRNA catalyzed by the mouse ascites system is shown in Fig. 3. The 0.12 M ribosomal salt wash prepared from human U cells treated with human leukocyte inter’ D. A. observations.
Farris
and
C.
E. Samuel,
unpublished
4
80 -
ii
0.
’ ! ‘-7 2l
M
- -----_
“-
C.
WASH
-----‘-‘---B 0.8
M
WASH
60; 40-L -kz=a.a 20 )----mrt-,
1
O-1 0
IO
RIBOSOMAL
(pg
S-Z=------a=-==8 --&TF I 20
30
SALT-WASH
protein
40
CONCENTRATION
per 0.1 ml)
FIG. 4. Effect of ribosomal salt-wash fractions from untreated and interferon-treated African green monkey kidney cells on the translation of methylated reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. A, B, C, and symbols are as in Fig. 2.
562
SAMUEL
AND
wash fractions prepared from interferontreated and untreated African green monkey kidney cells is shown in Fig. 4. Surprisingly, all three salt-wash fractions from both untreated cells and cells treated with monkey kidney interferon inhibited the translation of reovirus mRNA when added to the ascites cell-free system. However, relative to the endogenous level of incorporation, the level of [3H]leucine incorporated in response to reovirus mRNA in the presence of the higher concentrations of 0.12 M ribosomal salt washes was nearly twice as great for the 0.12 M wash prepared from untreated cells as compared to the 0.12 M wash prepared from interferon-treated cells (Fig. 4A). No appreciable difference in activity was observed between untreated and interferon-treated preparations in the case of either 0.3 (Fig. 4B) or 0.8 M (Fig. 4C) washes. Effect of viral mRNA methylation on the activity of ribosomal salt-wash fractions.
The cross-species experiments
0
0
described in
30
FARRIS
Figs. 2, 3, and 4 which established the concentration effect of untreated and interferon-treated ribosomal salt-wash fractions on the translation of reovirus mRNA were all performed with methylated messenger RNA. Figure 5 compares the kinetics of translation of methylated and unmethylated reovirus mRNAs in the absence of added ribosomal salt wash and in the presence of 0.12, 0.3, and 0.8 M ribosomal salt-wash fractions from untreated and interferon-treated mouse fibroblasts. In the absence of added ribosomal salt washes, 75 pglml of methylated reovirus mRNA was about 30% more active than 75 pug/ml of unmethylated mRNA when translated in vitro in the ascites cell-free system in the presence of SAdoHcy (Fig. 5A); comparable results were obtained in the presence of the 0.12 M ribosomal washes from both untreated and interferon-treated cells, neither of which significantly affected the kinetics of translation (Fig. 5B). The level of [3H]leucine incorpo-
45 0 (5 30 45 TIME (min) FIG. 5. Effect of methylation of reovirus mRNA on the activity of ribosomal salt-wash fractions prepared from untreated and from interferon-treated mouse fibroblast cells. The standard reaction mixture, as described under Materials and Methods, contained 75 wg/ml of either methylated (filled symbols) or unmethylated (open symbols) reovirus mRNA, 0.5 n&f SAdoHcy, and the following ribosomal salt-wash fractions: (A) no wash added, (B) 0.12 M ribosomal salt-wash fraction from either untreated (circles, 20 pg of protein/ml) or interferon-treated (triangles, 17 pg of protein/ml) cells; (Cl 0.3 M ribosomal salt-wash fraction from either untreated (circles, 21 pg of protein/ml) or interferon-treated (triangles, 25 wg of protein/ml) cells; (Dl 0.8 M ribosomal salt-wash fraction from either untreated (circles, 21 wg of protein/ml) or interferon-treated (triangles, 22 pg of protein/ml) cells. Reaction volume, 50 ~1; lo-h1 aliquots were processed after incubation for the indicated time.
MECHANISM
OF
INTERFERON
ration in response to the 0.3 and 0.8 M wash fractions from untreated cells (Figs. 5C and 5D) was greater than the level observed in the absence of any added ribosomal salt wash (Fig. 5A); furthermore, the 0.3 and 0.8 M wash fractions from untreated cells stimulated the incorporation in response to methylated reovirus mRNA more than unmethylated reovirus mRNA (Figs. 5C and 5D). The translation of both methylated and unmethylated reovirus mRNA was inhibited by the 0.3 and 0.8 M ribosomal salt-wash fractions prepared from interferon-treated cells (Figs. 5C and 5D). DISCUSSION
The results presented here demonstrate that, while species specificity between certain interferon-cell combinations does occur, the interferon-mediated ribosome-associated antiviral factor does not appear to be species specific. The antiviral activity of the mouse fibroblast, monkey kidney, and human leukocyte interferons utilized in our work was maximal on the homologous species of cells. However, significant heterologous activity was obtained between monkey and human interferons and cells, in accord with the finding of Bucknall(1967). Recent studies indicate the existence of multiple active components of both mouse fibroblast (Knight, 1975; D avey et al., 1976) and human leukocyte (Stewart and Desmyter, 1975; Paucker et aZ., 1975; Chen et aZ., 1976) interferons which can be distinguished by their biochemical and biophysical properties as well as their biological properties. The observations that crude interferon preparations from a variety of cell species are composed of multiple active components may account, in part, for the differential activity expressed in heterologous cells. The translation of reovirus messenger RNA catalyzed by the mouse ascites cellfree protein-synthesizing system prepared from untreated ascites cells was inhibited by fractionated ribosomal salt washes prepared from interferon-treated mouse cells, as reported previously (Falcoff et al., 1973; Samuel and Joklik, 1974). In addition to
ACTION
563
the inhibitory activity in the mouse ascites system of interferon-mediated ribosomeassociated factor(s) isolated from homologous mouse cells, ribosome-associated factor(s) prepared from interferon-treated, but not untreated, human amnion cells inhibited reovirus mRNA translation catalyzed by untreated mouse ascites cells. Ribosomal salt washes from interferontreated African green monkey kidney cells were somewhat more inhibitory than the corresponding washes from untreated monkey cells when tested in the mouse system. Thus, the interferon-mediated ribosome-associated factors from at least three species of cells, mouse fibroblast, monkey kidney, and human amnion, do not display apparent cell type specificity or species specificity when tested in the mouse ascites in vitro protein-synthesizing system. Although species-specific translational factors have been reported in certain eukaryotic systems, the components of the protein biosynthetic machinery in general do not appear to be species specific, but rather are interchangeable among different tissues and cell species (Lodish, 1976). The capacity of ribosomal salt-wash fractions from mouse, monkey, and human cells to affect, that is, either inhibit or stimulate, the translation of viral mRNA depended upon whether the washes were prepared from untreated or interferontreated cells. However, the apparent affinity with which the inhibitory factors present in interferon-treated cells are associated with ribosomes seemed to differ for different cell lines. For example, interferon-mediated factors could be released from ribosomes of human amnion and African green monkey kidney cells with buffer containing 0.12 M KCl, whereas buffer containing 0.3 M KC1 was required to dissociate the inhibitory factor from ribosomes of mouse fibroblast cells. Comparable results in terms of the relative difference in activity between ribosomal saltwash fractions from untreated and interferon-treated cells were obtained with different preparations when tested in the untreated mouse ascites cell-free system. However, the plant cell-free system from
SAMUEL
564
AND FARRIS
wheat, which efficiently catalyzes the translation of reovirus mRNA (Both et al., 1975; Davies and Samuel, 19751, was inhibited by ribosomal salt-wash fractions from both interferon-treated and untreated mammalian cells.3 Although the methylation of reovirus mRNA has been reported to be impaired in cell-free extracts of interferon-treated mouse ascites cells (Sen et al., 1975), both methylated and unmethylated reovirus mRNA translations catalyzed by the untreated murine cell-free system were inhibited by ribosomal washes from interferon-treated murine cells. The interferonmediated ribosome-associated factor(s) from murine cells appears to affect a step of polypeptide chain biosynthesis subsequent to the formation of the first peptide bond in response to viral mRNA in vitro (Content et al., 1975; Samuel, 1976). In addition to the possibility that the factors from mouse, monkey, and human cells might affect the translation of reovirus and most other viral mRNAs in a direct manner, the possibility that they perhaps function in an indirect manner by altering a preexisting component of the protein biosynthetic machinery must also be considered. Purification and characterization of the ribosome-associated interferon-mediated inhibitor(s) of translation4 will hopefully permit the elucidation of its molecular mechanism of action in more defmitive terms. ACKNOWLEDGMENTS This work was supported, in part, by research grants from the American Cancer Society (VC-192) and the National Institute of Allergy and Infectious Diseases (AI-12520). REFERENCES S., and SHATKIN, A. J. (1975). Synthesis of all the gene products of the reovirus genome in vivo and in vitro. Cell 4, 173-180. BORECK~, L., FIJCHSBERGER, N., and HAJNICKA, V. (1974). Electrophoretic profiles and activities of human interferon in heterologous cells. Interuirology 3, 369-377. BUCKNALL, R. A. (1967). “Species-specificity” of inBOTH,
G. W.,
LAVI,
4 D. Eppstein preparation.
and C. E. Samuel,
manuscript
in
terferons: A misnomer? 1022-1023.
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LLondon)
216,
W. J., O’MALLEY, J. A., E., and CARTER, W. A. (1976). Nature of the molecular heterogeneity of human leukocyte interferon. J. Virol. 19, 425-434. COLBY, C., PENHOET, E. E., and SAMUEL, C. E. (1976). A comparison of transfer RNAs from untreated and interferon-treated murine cells. Virology 74, 262-264. CONTENT, J., LEBLEU, B., ZILBERSTEIN, A., BERISSI, H., and REVEL, M. (1974). Mechanism of the interferon-induced block of mRNA translation in mouse L cells: Reversal of the block by transfer RNA. FEBS Lett. 41, 125-130. CONTENT, J., LEBLEU, B., NUDEL, U., ZILBERSTEIN, A., BERISSI, H., and REVEL, M. (1975). Blocks in elongation and initiation of protein synthesis induced by interferon treatment of mouse L cells. Eur. J. Biochem. 54, l-10. DAVEY, M. W., SULKOWSKI, E., and CARTER, W. A. (1976). Purification and characterization of mouse interferon with novel affinity sorbents. J. Virol. 17, 439-445. DAVIES, J. W., and SAMUEL, C. E. (1975). Translation of virus mRNA: Comparison of reovirus and brome mosaic virus single stranded RNAs in a wheat germ cell-free system. Biochem. Biophys. CHEN, J. K., SULKOWSKI,
JANKOWSKI,
Res. Commun. DESMYTER, J.,
65, 788-796.
them. GUPTA, PORI,
Res.
and STEWART, W. E., II. (1976). Molecular modification of interferon: Attainment of human interferon in a conformation active on cat cells but inactive on human cells. Virology 70, 451-458. FALCOFF, E., FALCOFF, R., LEBLEU, B., and REVEL, M. (1973). Correlation between the antiviral effect of interferon treatment and the inhibition of in vitro mRNA translation in noninfected L cells. J. Viral. 12, 421-430. FINTER, N. B. (ed.) (1973). “Interferons and Interferon Inducers. Frontiers of Biology,” Vol. 2. American Elsevier, New York. FRIEDMAN, R. M., METZ, D. H., ESTEBAN, R. M., TOVELL, D. R., BALL, L. A., and KERR, I. M. (19721. Mechanism of interferon action: Inhibition of viral messenger ribonucleic acid translation of L-cell extracts. J. Virol. 10, 1184-1198. GUFTA, S. L., SOPORI, M. L., and LENGYEL, P. (1973). Inhibition of protein synthesis directed by added viral and cellular messenger RNAs in extracts of interferon-treated Ehrlich ascites tumor cells. Location and dominance of the inhibitor(s). BioBiophys.
Commun.
54, 777-763.
S. L., GRAZIADEI, W. D., WEIDELI, H., SoM. L., and LENGYEL, P. (1974). Selective inhibition of viral protein accumulation in interferon-treated cells; nondiscriminate inhibition of the translation of added viral and cellular mes-
MECHANISM
OF
senger RNAs in their extracts. Virology 57,49-63. Ho, M., and ARMSTRONG, J. A. (1975). Interferon. Ann. Rev. Microbial. 29, 131-161. JENSEN, F. C., GIRARDI, A. J., GILDEN, R. V., and KOPROWSKI, H. (1964). Infection of human and simian tissue cultures with Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 52, 53-59. KERR, I. M., BROWN, R. E., and BALL, L. A. (1974). Increased sensitivity of cell-free protein synthesis to double-stranded RNA after interferon treatment. Nature (London) 250, 57-59. KNIGHT, E., JR. (1975). Heterogeneity of purified mouse interferons. J. Biol. Chem. 250,4139-4144. LAI, M.-H. T., and 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. LODISH, H. F. (1976). Translational control of protein synthesis. Ann. Rev. Biochem. 45, 39-72. PAIJCKER, K., DALTON, B. J., OGBURN, C. A., and TBRMK, E. (1975). Multiple active sites on human interferons. Proc. Nat. Acad. Sci. USA 72, 45874591. RABINOWITZ, J. C., and PRICER, W. E., JR. (1962). Formyltetrahydrofolate synthetase. I. Isolation and characterization of the enzyme. J. Biol. Chem. 237, 2898-2902. REVEL, M., BASH, D., and RUDDLE, F. H. (1976). Antibodies to a cell-surface component coded by human chromosome 21 inhibit action of interferon. Nature (London) 260, 139-141. SAMUEL, C. E. (1976). Mechanism of interferon action. Studies on the mechanism of interferon-mediated inhibition of reovirus messenger RNA translation in cell-free protein synthesizing systems from mouse ascites tumor cells. Virology 75, 166-176. SAMUEL, C. E., and JOKLIK, W. K. (1974). A protein synthesizing system from interferon-treated cells that discriminates between cellular and viral messenger RNAs. Virology 58, 476-491. SAMUEL, C. E., and JOKLIK, W. K. (1976). Biosynthesis of reovirus-specified polypeptides. Initiation of reovirus messenger RNA translation in
INTERFERON
ACTION
565
vitro and identification of methionyl-X initiation peptides. Virology 74, 403-413. SANFORD, K. K., EARLE, W. R., and LIKELY, G. D. (1948). The growth in vitro of single isolated tissue cells. J. Nat. Cancer Inst. 9, 229. SEN, G. C., LEBLEU, B., BROWN, G. E., REBELU), M. A., FURUICHI, Y., MORGAN, M., SHATKIN, A. J., and LENGYEL, P. (1975). Inhibition of reovirus messenger RNA methylation in extracts of interferon-treated Ehrlich ascites tumor cells. Biothem. Biophys. Res. Common. 65, 427-434. SEN, G. C., GUPTA, S. L., BROWN, G. E., LEBLEU, B., REBELLO, M. A., and LENGYEL, P. (1976). Interferon treatment of Ehrlich ascites tumor cells: Effects on exogenous mRNA translation and tRNA inactivation in the cell extract. J. Viral. 17, 191-203. SKEHEL, J. J., and JOKLIK, W. K. (1969). Studies on the in vitro transcription of reovirus RNA catalyzed by reovirus cores. Virology 39, 822-831. SMITH, R. E., ZWEERINK, H. J., and JOKLIK, W. K. (1969). Polypeptide components of virions, top component and cores of reovirus type 3. Virology 39, 791-810. STEWART, W. E., II, and DESMYTER, J. (1975). Molecular heterogeneity of human leukocyte interferon: Two populations differing in molecular weights, requirements for renaturation and cross-species antiviral activity. Virology 67, 68-73. STRANDER, H., and CANTELL, K. (1966). Production of interferon by human leukocytes in vitro. Ann. Med. Exp. Fenn. 44, 265-273. VENGRIS, V. E., STOLLAR, B. D., and PITHA, P. M. (1975). Interferon externalization by producing cells before induction of antiviral state. Virology 65, 410-417. YAMAMOTO, K., YAMAGUCHI, N., and ODA, K. (1975). Mechanism of interferon-induced inhibition of early simian virus 40 functions. Virology 68, 58-70. YOUNGNER, J. S., SCOTT, A. W., HALLUM, J. V., and STINEBRING, W. R. (1966). Interferon production by inactivated Newcasle disease virus in cell cultures and in mice. J. Bacterial. 92, 862-868.
77,
556-565
(1977)
Mechanism Species
Action
Specificity of Interferon and of the Interferon-Mediated Inhibitor Translation from Mouse, Monkey, and Human Cells
CHARLES Section
of Interferon
of Biochemistry
and
E. SAMUEL’
Molecular
AND
DEBORAH
Biology, Department of Biological Santa Barbara, California 93106 Accepted
November
of
A. FARRIS Sciences,
University
of California,
19,1976
The species specificity of interferon and of the interferon-mediated ribosome-associated inhibitor of translation from mouse fibroblast, African green monkey kidney, and human amnion cells was investigated. The following results were obtained: (1) The antiviral activity of mouse tibroblast, monkey kidney, and human leukocyte interferons was maximal on the homologous species of cells. Mouse interferon was not active on heterologous monkey cells, and monkey interferon was not active on mouse cells. Monkey and human interferons were almost equally active on heterologous human and monkey cells, respectively; mouse and human interferons were about 10 and 125 times more active, respectively, on their homologous cell species than on heterologous human and mouse cells. (2) Ribosomal salt-wash fractions prepared from interferon-treated mouse fibroblast, monkey kidney, and human amnion cells inhibited the translation of reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. The corresponding fractions prepared from untreated mouse and human cells either did not affect or stimulated the translation of reovirus mRNA. Ribosomal washes from untreated monkey kidney cells were inhibitory, but not as inhibitory as washes from interferon-treated cells. (3) The concentration of KC1 required to release the interferon-mediated ribosome-associated inhibitor from ribosomes of mouse fibroblast cells was higher than that required for ribosomes from human amnion and monkey kidney cells. (4) The translation of both methylated and unmethylated reovirus mRNA catalyzed by the mouse ascites cell-free system was inhibited by the mouse tibroblast ribosome-associated interferon-mediated factor. These results suggest that the interferon-mediated ribosome-associated antiviral factor(s) is not species specific, even though certain interferons are relatively species specific. INTRODUCTION
and RNA-containing animal viruses is inhibited as a result of interferon treatment appears to be the translation of viral messenger RNA into protein (Ho and Armstrong, 1975). Cell-free extracts prepared from interferon-treated murine cells have been shown to be incapable of catalyzing the translation of encephalomyocarditis RNA (Friedman et al., 19721, mengovirus RNA (Falcoff et al., 19731, reovirus mRNA (Gupta et al., 1974; Samuel and Joklik, 1974), and vaccinia virus mRNA (Samuel and Joklik, 1974). The interferon-mediated inhibitor of viral mRNA translation is ribosome-associated and can be separated from ribosomes by either washing with
Interferons, cellular proteins synthesized in response to viral infection as well as to several nonviral stimuli, mediate the establishment of an antiviral state in a wide variety of animal cells through a mechanism which apparently involves an extracellular interaction between the interferon molecule(s) and the cellular membrane (Finter, 1973; Ho and Armstrong, 1975; Vengris et al., 1975; Revel et al., 1976). In several animal virus-cell systerns, the primary level of gene expression at which the multiplication of both DNA’ To whom
reprint
requests
should
be addressed. 556
Copyright All riahts
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
MECHANISM
OF
INTERFERON
buffers containing concentrated salt (Falcoff et al., 1973; Samuel and Joklik, 1974) or incubation (Gupta et pl., 1973). The specific biochemical step(s) in the process of translation of viral mRNA into viral protein primarily affected by the interferonmediated ribosome-associated inhibitor and directly responsible for the antiviral effect of interferon has not been elucidated. However, recent studies indicate that the interferon-mediated inhibition of viral mRNA translation in vitro is exerted at a step of polypeptide chain biosynthesis which is subsequent to formation of the first peptide bond (Content et al., 1975; Samuel, 1976) and which either directly or indirectly involves the participation of transfer RNA (Colby et al., 1976; Content et al., 1974; Sen et al., 1976) and the sensitivity of protein synthesis to doublestranded RNA (Kerr et al., 1974). Certain interferons display relatively strict species specificity for cells from the homologous species, with less or no antiviral activity in cells from heterologous species (Finter, 1973), whereas other interferons, such as human leukocyte interferon, are active on a wide variety of cells from heterologous species (Finter, 1973; Borecky et al., 1974; Desmyter and Stewart, 1976). However, the characteristic species specificity of the ribosome-associated antiviral factor synthesized in response to interferon treatment is .undefined. The #tudies presented here were therefore undertaken to determine (1) the homologous and heterologous antiviral activity of mouse tibroblast, African green monkey kidney, and human leukocyte interferons on mouse fibroblast, monkey kidney, and human amnion cells, and (2) the effect of the ribosomal salt-wash fractions prepared from ribosomes isolated from untreated and interferon-treated mouse, monkey, and human cells on the translation in vitro of reovirus messenger RNA catalyzed by the Krebs mouse ascites cell-free proteinsynthesizing system prepared from untreated ascites cells. The reasons for choosing this system were primarily the convenient growth characteristics and sensitivity to interferon of the mouse fibroblast (Sanford et al ., 1948; Youngner et al ., 19661,
557
ACTION
African green monkey kidney (Jensen et al., ,1964; Yamamoto et al., 19751, and human amnion (Strander and Cantell, 1966) cells, the ability of the untreated Krebs ascites cell-free protein-synthesizing system to efficiently catalyze the translation of reovirus mRNA into known viral polypeptides (McDowell et al., 1972; Samuel and Joklik, 19761, and the inhibitory action of ribosomal salt washes from interferon-treated mouse cells on viral mRNA translation catalyzed by the mouse ascites cell-free system (Falcoff et al., 1973; Samuel and Joklik, 1974). MATERIALS
AND
METHODS
Materials. L-[4,5-3H,1Leucine, 44 Ci/ from Schwarz/ mmol, was purchased Mann. ‘*C-labeled L-amino acids, S-adenosyl-L-methionine, creatine phosphate, creatine phosphokinase, and dithiothreitol were from Sigma; ATP, GTP, CTP, and UTP were from either Sigma or P. L. Biochemicals; S-adenosyl+homocysteine was from Calbiochem; Eagle’s MEM and Joklik-modified Eagle’s MEM were from Grand Island Biological Co.; fetal calf serum was from Irvine Scientific; and female albino mice (CD-Swiss) were from Hilltop Laboratory. Cells. Clone L929 mouse fibroblast cells, obtained from Dr. W. K. Joklik, Duke University Medical Center, human am-. nion U cells, obtained from Dr. J. Vilcek, New York University Medical Center, and the CV-1 line of African green monkey kidney cells, obtained from Dr. J. A. Carbon, University of California, Santa Barbara, were grown in roller culture in Eagle’s MEM containing 7% fetal calf serum. Krebs ascites tumor cells were maintained in uiuo by injection of 0.15 ml of ascitic fluid into the peritoneal cavity of female albino mice; the cells were passed every 7 to 9 days. Preparation
and
assay
df interferon.
Mouse and monkey interferons induced in confluent aged roller bottle cultures of L,,, and CV-1 cells, respectively, with the Hirts strain of NDV* were prepared as 2 Abbreviations used: DTT, dithiothreitol; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; NDV, Newcastle disease virus; PFU,
558
SAMUEL
AND
previously described (Samuel and Joklik, 1974). Human leukocyte interferon was generously provided by Dr. T. C. Merigan, Stanford University Medical Center. The specific activities of the interferon preparations as determined by a 50% plaque reduction assay on cell monolayers infected with the Indiana strain of VSV (Lai and Joklik, 1973) were: mouse interferon assayed on mouse fibroblast L,,, cells, 3.6 x lo4 units/mg of protein; monkey interferon assayed on monkey kidney CV-1 cells, 6.1 x lo3 units/mg of protein; human interferon assayed on human amnion U cells, 2.1 x lo5 units/mg of protein. One unit of interferon was defined as the reciprocal of the highest dilution that reduced by 50% the number of VSV plaques formed. Protein determination. Protein concentrations were determined by a modification of the phenol reagent method with crystalline bovine serum albumin as the reference standard as described by Rabinowitz and Pricer (1962). Preparation of ribosomal salt-wash fractions. Ribosomal salt-wash fractions
were prepared from untreated and interferon-treated mouse LsZ9 cells, monkey CV-1 cells, and human U cells grown in roller bottles with a growth area of 1330 cm2; 6 to 10 bottles of untreated cells or cells treated for 24 hr with 100 units of interferon/ml were utilized for each preparation. All operations, unless otherwise stated, were carried out at O-4”. Cells were harvested by scraping, washed in phosphate-buffered saline (10 mM phosphate buffer, pH 7.3, containing 137 mM NaCl and 3 n&f KCl), and pelleted at 8OOg for 10 min in an IEC CRU-5000 centrifuge with a 269 rotor. The cell pellet was suspended in 3 packed-cell vol of hypotonic buffer [lo mM HEPES buffer, pH 7.5, containing 15 mM KCl, 1.5 mM Mg(OAc),, and 1 miW D’M’I, allowed to swell for 10 min, and disrupted with about 30 strokes of a tightfitting Dounce homogenizer. The extract plaque-forming units; SAdoHcy, S-adenosyl-L-homocysteine; SAdoMet, S-adenosyl-L-methionine; TCA, tricholoacetic acid; VSV, vesicular stomatitis virus.
FARRIS
was immediately adjusted to a final concentration of 20 mM HEPES buffer, pH 7.5, 5 mit4 Mg(OAc),, 120 mM KCl, and 2 mM DTI’. The homogenate was centrifuged at 5000 g for 5 min, the pellet was discarded, and the supernatant solution was centrifuged at 10,000 g for 20 min. Ribosomes were isolated from the 10,000 g supernatant solution by centrifugation at 150,000 g for 2.5 hr in a DuPont Sorvall OTD-50 ultracentrifuge and T865.1 rotor. The translucent ribosomal pellet was gently suspended in 20 n-&f HEPES buffer, pH 7.5, containing 120 rniW KCl, 5 miW Mg(OAc),, and 1 mM DTI’. The suspension was stirred slowly for 1 hr followed by centrifugation for 2.25 hr at 150,000 g; the resulting supernatant solution was defined as the 0.12 M ribosomal salt wash. The 0.12 M salt-washed ribosomal pellet was suspended, stirred, and centrifuged as before except that the buffer was modified to contain 300 mM KCI; the resulting supernatant solution was defined as the 0.3 M ribosomal salt wash. The 0.3 M saltwashed ribosomal pellet was suspended and stirred for 1 hr as before except that the buffer was modified to contain 800 mM KCl; the suspension was centrifuged overnight at 120,000 g. The resulting supernatant solution, defined as the 0.8 M ribosomal salt wash, as well as the 0.12 and 0.3 M ribosomal salt washes, were all centrifuged for 4 hr at 120,000 g and then dialyzed against 250 vol of 20 n&f HEPES buffer, pH 7.5, containing 50 mit4 KCl, 1 miW Mg(OAc),, 1 mM D’M’, and 10% (v/v) glycerol. The dialysis was carried out overnight with one change of buffer. The fraction recovered by centrifugation of the dialyzed ribosomal washes at 5000 g for 15 min with a DuPont Sorvall RC-5 centrifuge and SS-34 rotor was divided into small aliquots and stored at -90”. Preparation of cell-free protein-synthesis extracts. Preincubated, Sephadex G-25-
treated Krebs II mouse ascites cell-free protein-synthesis extracts were prepared from untreated control cells as previously described (Samuel and Joklik, 1974). Preparation of reovirus mRNA. For the preparation of reovirus mRNA, the Dearing strain of reovirus type 3 was grown in
MECHANISM
OF INTERFERON
mouse L fibroblast cells in suspension culture and purified by the procedure of Smith et al. (1969). Reovirus mRNA was prepared as the in vitro transcription product of the double-stranded RNA-dependent single-stranded RNA polymerase (Skehel and Joklik, 1969); for the preparation of methylated and unmethylated mRNAs, 10 $I!I SAdoMet and 1 n-&I SAdoHcy, respectively, were present in the transcription reaction mixture. Viral mRNA synthesized in vitro for use in cell-free proteinsynthesis experiments was purified from the reaction mixture as previously described (Samuel and Joklik, 1974). Assay ofprotein synthesis in vitro. Protein synthesis in vitro was measured by determining the incorporation of radioactively labeled L-[3H]leucine into hot trichloroacetic acid-insoluble material as described before (Samuel and Joklik, 1974). The standard reaction mixture (50 ~1) contained the following ingredients: 30 mM HEPES, pH 7.5; 96 miI4 KCl; 3 to 4 miI4 Mg(OAc),; 1 n-&I ATP; 0.2 mM GTP; 0.6 m&I CTP; 1 n-&! D’IT; 10 mM creatine phosphate; 0.2 mg/ml of creatine phosphokinase; 19 W-labeled L-amino acids, 50 fl each; 50 &X/ml of r.,-L3Hlleucine; ascites cell-free extract, 0.3 ml/ml; and 75 pglml of reovirus mRNA as indicated. Unless otherwise indicated, 40-~1 aliquots were processed after incubation at 30” for 30 min. Radioactivity was measured with a Beckman LS-230 liquid scintillation spectrometer for 10 min or to give a standard error of counting rate of less than 1.0%. RESULTS
Antiviral activity of interferon on homologous and heterologous cells. The sen-
sitivity of mouse L,,, fibroblast, African green monkey kidney CV-1, and human amnion U cells to homologous and heterologous interferon(s) was assayed by reduction of VSV plaque formation. As shown in Fig. 1, maximal antiviral activity of mouse fibroblast, monkey kidney, and human leukocyte interferon preparations was obtained on the homologous species of cells. However, significant cross-species activity was observed in the case of some
125
A. MOUSE
2 ,251
B. MONKEY
;
C. HUMAN
125 -
559
ACTION
INTERFERON
INTERFERON
INTERFERON
1
INTERFERON
CONCENTRATION
(units
per ml)
FIG. 1. Comparison of the antiviral activity of mouse tibroblast interferon (A), African green monkey kidney interferon (B), and human leukocyte interferon (Cl on mouse fibroblast, African green monkey kidney, and human amnion cells. Confluent monolayers of cells, treated with the indicated concentrations of interferon, were challenged with VSV and the plaques were counted as described under Materials and Methods.
cell:interferon combinations. The crossspecies antiviral activity was comparable between monkey and human interferons and cells; for both monkey and human interferons, the titer determined on the heterologous cell species was about 60 + 10% of the titer determined on the homologous cell species (Table 1). The titer of mouse interferon when assayed on mouse L,,, cells was about lo-fold greater than when assayed on human U cells, whereas the titer of human interferon was about 125fold greater on human U cells than on mouse L,,, cells; no appreciable activity was observed with either mouse interferon
SAMUEL
560 TABLE SPECIFICITY
OF THE
AND
FARRIS
1
ANTIVIRAL
ACTION
801A.
OF
0.!2
M
WASH
INTERFERING
Cell
Interferon specie@ Mouse Mouse Monkey Human
species’
Monkey
Human
7.6 x lo4
<32
<64 2.7 x lo4
6.1 x lo3
6.6 x 103 4.1 x 103
1.9 x 106
3.4 x 106
(1 Interferon titers (units per milliliter) were determined on homologous and heterologous cell species. Titers are expressed as the reciprocal of the dilution of the interferon preparation required to prod&e a 50% reduction in vesicular stomatitis virus plaque formation. b Mouse L,,, tibroblast, African green monkey kidney CV-1, and human leukocyte. r Mouse L,,, tibroblast, African green monkey kidney CV-1, and human amnion U.
--m-o------w___ c~
0 2
C.
80-
M
0.0
---
0
~ -- -c
--__
~
WASH
assayed on monkey cells or monkey interferon assayed on mouse cells (table 1, Fig. 1). Effect of ribosomal treated and translation
homologous and heterologous salt-wash fractions from uninterferon-treated cells on the of viral mRNA. Ribosomal
salt-wash fractions prepared from interferon-treated mouse fibroblast, monkey kidney, and human amnion cells were examined for their ability to affect the translation of reovirus mRNA catalyzed by the mouse ascites tumor cell-free protein-synthesizing system. Ribosomes isolated by centrifugation were washed successively at 4” in buffer solutions containing 0.12, 0.3, and 0.8 M KCl; varying concentrations of each fraction were assayed for their ability to inhibit or stimulate the translation of reovirus mRNA as measured by the incorporation of 13Hlleucine into hot trichloroacetic acid-insoluble material. The results obtained with salt-wash fractions prepared from ribosomes isolated from interferon-treated cells were compared with identical fractions prepared from ribosomes isolated from untreated cells. Mouse cells. As shown in Fig. 2, the 0.3 and 0.8 M ribosomal salt-wash fractions prepared from untreated mouse L,,, fibroblast cells stimulated incorporation of L3Hlleucine in response to reovirus mRNA, whereas the comparable salt-wash fractions prepared from L,,, cells treated with
0
20
RIBOSOMAL
(pg
40
60
SALT-WASH
CONCENTRATION
protein
0.1 ml 1
per
80
FIG. 2. Effect of ribosomal salt-wash fractions from untreated and interferon-treated mouse L,, flbroblast cells on the translation of methylated reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. The standard reaction conditions were as described under Materials and Methods. Ribosomal salt-wash fractions were prepared by washing isolated ribosomes successively with buffers containing (A) 0.12 M KCl; (Bl 0.3 M KCl; (0 0.8 M KC1 as described under Materials and Methods. Wash from untreated cells (C): O---O, endogenous activity; O-O, activity with added reovirus mRNA. Wash from interferon-treated cells (IF): A---A, endogenous activity; A-A, activity with added reovirus mRNA. Small filled circles at zero protein concentration show activity in the absence of added wash. Reaction volume, 50 ~1; a 40-~1 aliquot was processed.
mouse fibroblast interferon inhibited the translation of reovirus mRNA catalyzed by the mouse ascites cell-free system (Figs. 2B and 20. The 0.12 M salt-wash fraction from both interferon-treated and
MECHANISM
6C l- _
A.
0.12
M
OF
INTERFERON
WASH
4c )m b
2C l-
;
c )-
2
6C )- _ B.
B 2
4c , -
8 g
20 I-
0.3
M
WASH
:---,-lloQc--------A
s-
cI
ii6
80 , -.
s J T &!2d
---------^---Q
I
-L
C.
0.8
t
M
L
WASH
1
I
/IF
561
ACTION
feron inhibited the translation of reovirus mRNA catalyzed by the mouse cell-free system; the 0.12 M wash prepared from ribosomes isolated from untreated cells was not inhibitory (Fig. 3A). Although the 0.3 M salt wash from interferon-treated U cells was slightly less active than the corresponding fraction from untreated cells, neither had a significant affect on in vitro translation (Fig. 3B). Both the interferontreated and untreated 0.8 M ribosomal salt washes stimulated incorporation significantly (Fig. 30. Monkey cells. The activity in the mouse ascites cell-free protein-synthesizing system of 0.12, 0.3, and 0.8 M ribosomal salt-
60
r 80 t
40
A.
0.12M
B.
0.3
WASH
20 0
v
IO
RIBOSOMAL
(pg
20 30 40 50 SALT-WASH CONCENTRATION
protein
per 0.1 ml )
FIG. 3. Effect of ribosomal salt-wash fractions from untreated and interferon-treated human amnion U cells on the translation of methylated reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. A, B, C, and symbols are as in Fig. 2.
untreated mouse L,,, cells did not significantly affect the translation of viral mRNA, although the higher concentrations of 0.12 M ribosomal wash derived from interferon-treated cells were slightly more inhibitory than the wash from untreated cells. Ribosomal wash fractions prepared from L929 mouse cells treated with heterologous interferon were not inhibitory.” Human cells. The cross-species activity of ribosomal wash fractions from interferon-treated and untreated human amnion U cells on the translation of reovirus mRNA catalyzed by the mouse ascites system is shown in Fig. 3. The 0.12 M ribosomal salt wash prepared from human U cells treated with human leukocyte inter’ D. A. observations.
Farris
and
C.
E. Samuel,
unpublished
4
80 -
ii
0.
’ ! ‘-7 2l
M
- -----_
“-
C.
WASH
-----‘-‘---B 0.8
M
WASH
60; 40-L -kz=a.a 20 )----mrt-,
1
O-1 0
IO
RIBOSOMAL
(pg
S-Z=------a=-==8 --&TF I 20
30
SALT-WASH
protein
40
CONCENTRATION
per 0.1 ml)
FIG. 4. Effect of ribosomal salt-wash fractions from untreated and interferon-treated African green monkey kidney cells on the translation of methylated reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. A, B, C, and symbols are as in Fig. 2.
562
SAMUEL
AND
wash fractions prepared from interferontreated and untreated African green monkey kidney cells is shown in Fig. 4. Surprisingly, all three salt-wash fractions from both untreated cells and cells treated with monkey kidney interferon inhibited the translation of reovirus mRNA when added to the ascites cell-free system. However, relative to the endogenous level of incorporation, the level of [3H]leucine incorporated in response to reovirus mRNA in the presence of the higher concentrations of 0.12 M ribosomal salt washes was nearly twice as great for the 0.12 M wash prepared from untreated cells as compared to the 0.12 M wash prepared from interferon-treated cells (Fig. 4A). No appreciable difference in activity was observed between untreated and interferon-treated preparations in the case of either 0.3 (Fig. 4B) or 0.8 M (Fig. 4C) washes. Effect of viral mRNA methylation on the activity of ribosomal salt-wash fractions.
The cross-species experiments
0
0
described in
30
FARRIS
Figs. 2, 3, and 4 which established the concentration effect of untreated and interferon-treated ribosomal salt-wash fractions on the translation of reovirus mRNA were all performed with methylated messenger RNA. Figure 5 compares the kinetics of translation of methylated and unmethylated reovirus mRNAs in the absence of added ribosomal salt wash and in the presence of 0.12, 0.3, and 0.8 M ribosomal salt-wash fractions from untreated and interferon-treated mouse fibroblasts. In the absence of added ribosomal salt washes, 75 pglml of methylated reovirus mRNA was about 30% more active than 75 pug/ml of unmethylated mRNA when translated in vitro in the ascites cell-free system in the presence of SAdoHcy (Fig. 5A); comparable results were obtained in the presence of the 0.12 M ribosomal washes from both untreated and interferon-treated cells, neither of which significantly affected the kinetics of translation (Fig. 5B). The level of [3H]leucine incorpo-
45 0 (5 30 45 TIME (min) FIG. 5. Effect of methylation of reovirus mRNA on the activity of ribosomal salt-wash fractions prepared from untreated and from interferon-treated mouse fibroblast cells. The standard reaction mixture, as described under Materials and Methods, contained 75 wg/ml of either methylated (filled symbols) or unmethylated (open symbols) reovirus mRNA, 0.5 n&f SAdoHcy, and the following ribosomal salt-wash fractions: (A) no wash added, (B) 0.12 M ribosomal salt-wash fraction from either untreated (circles, 20 pg of protein/ml) or interferon-treated (triangles, 17 pg of protein/ml) cells; (Cl 0.3 M ribosomal salt-wash fraction from either untreated (circles, 21 pg of protein/ml) or interferon-treated (triangles, 25 wg of protein/ml) cells; (Dl 0.8 M ribosomal salt-wash fraction from either untreated (circles, 21 wg of protein/ml) or interferon-treated (triangles, 22 pg of protein/ml) cells. Reaction volume, 50 ~1; lo-h1 aliquots were processed after incubation for the indicated time.
MECHANISM
OF
INTERFERON
ration in response to the 0.3 and 0.8 M wash fractions from untreated cells (Figs. 5C and 5D) was greater than the level observed in the absence of any added ribosomal salt wash (Fig. 5A); furthermore, the 0.3 and 0.8 M wash fractions from untreated cells stimulated the incorporation in response to methylated reovirus mRNA more than unmethylated reovirus mRNA (Figs. 5C and 5D). The translation of both methylated and unmethylated reovirus mRNA was inhibited by the 0.3 and 0.8 M ribosomal salt-wash fractions prepared from interferon-treated cells (Figs. 5C and 5D). DISCUSSION
The results presented here demonstrate that, while species specificity between certain interferon-cell combinations does occur, the interferon-mediated ribosome-associated antiviral factor does not appear to be species specific. The antiviral activity of the mouse fibroblast, monkey kidney, and human leukocyte interferons utilized in our work was maximal on the homologous species of cells. However, significant heterologous activity was obtained between monkey and human interferons and cells, in accord with the finding of Bucknall(1967). Recent studies indicate the existence of multiple active components of both mouse fibroblast (Knight, 1975; D avey et al., 1976) and human leukocyte (Stewart and Desmyter, 1975; Paucker et aZ., 1975; Chen et aZ., 1976) interferons which can be distinguished by their biochemical and biophysical properties as well as their biological properties. The observations that crude interferon preparations from a variety of cell species are composed of multiple active components may account, in part, for the differential activity expressed in heterologous cells. The translation of reovirus messenger RNA catalyzed by the mouse ascites cellfree protein-synthesizing system prepared from untreated ascites cells was inhibited by fractionated ribosomal salt washes prepared from interferon-treated mouse cells, as reported previously (Falcoff et al., 1973; Samuel and Joklik, 1974). In addition to
ACTION
563
the inhibitory activity in the mouse ascites system of interferon-mediated ribosomeassociated factor(s) isolated from homologous mouse cells, ribosome-associated factor(s) prepared from interferon-treated, but not untreated, human amnion cells inhibited reovirus mRNA translation catalyzed by untreated mouse ascites cells. Ribosomal salt washes from interferontreated African green monkey kidney cells were somewhat more inhibitory than the corresponding washes from untreated monkey cells when tested in the mouse system. Thus, the interferon-mediated ribosome-associated factors from at least three species of cells, mouse fibroblast, monkey kidney, and human amnion, do not display apparent cell type specificity or species specificity when tested in the mouse ascites in vitro protein-synthesizing system. Although species-specific translational factors have been reported in certain eukaryotic systems, the components of the protein biosynthetic machinery in general do not appear to be species specific, but rather are interchangeable among different tissues and cell species (Lodish, 1976). The capacity of ribosomal salt-wash fractions from mouse, monkey, and human cells to affect, that is, either inhibit or stimulate, the translation of viral mRNA depended upon whether the washes were prepared from untreated or interferontreated cells. However, the apparent affinity with which the inhibitory factors present in interferon-treated cells are associated with ribosomes seemed to differ for different cell lines. For example, interferon-mediated factors could be released from ribosomes of human amnion and African green monkey kidney cells with buffer containing 0.12 M KCl, whereas buffer containing 0.3 M KC1 was required to dissociate the inhibitory factor from ribosomes of mouse fibroblast cells. Comparable results in terms of the relative difference in activity between ribosomal saltwash fractions from untreated and interferon-treated cells were obtained with different preparations when tested in the untreated mouse ascites cell-free system. However, the plant cell-free system from
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wheat, which efficiently catalyzes the translation of reovirus mRNA (Both et al., 1975; Davies and Samuel, 19751, was inhibited by ribosomal salt-wash fractions from both interferon-treated and untreated mammalian cells.3 Although the methylation of reovirus mRNA has been reported to be impaired in cell-free extracts of interferon-treated mouse ascites cells (Sen et al., 1975), both methylated and unmethylated reovirus mRNA translations catalyzed by the untreated murine cell-free system were inhibited by ribosomal washes from interferon-treated murine cells. The interferonmediated ribosome-associated factor(s) from murine cells appears to affect a step of polypeptide chain biosynthesis subsequent to the formation of the first peptide bond in response to viral mRNA in vitro (Content et al., 1975; Samuel, 1976). In addition to the possibility that the factors from mouse, monkey, and human cells might affect the translation of reovirus and most other viral mRNAs in a direct manner, the possibility that they perhaps function in an indirect manner by altering a preexisting component of the protein biosynthetic machinery must also be considered. Purification and characterization of the ribosome-associated interferon-mediated inhibitor(s) of translation4 will hopefully permit the elucidation of its molecular mechanism of action in more defmitive terms. ACKNOWLEDGMENTS This work was supported, in part, by research grants from the American Cancer Society (VC-192) and the National Institute of Allergy and Infectious Diseases (AI-12520). REFERENCES S., and SHATKIN, A. J. (1975). Synthesis of all the gene products of the reovirus genome in vivo and in vitro. Cell 4, 173-180. BORECK~, L., FIJCHSBERGER, N., and HAJNICKA, V. (1974). Electrophoretic profiles and activities of human interferon in heterologous cells. Interuirology 3, 369-377. BUCKNALL, R. A. (1967). “Species-specificity” of inBOTH,
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