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Inhibition of feline leukemia virus replication by human leukocyte interferon
Antiviral Research, 3 (1983) 115-120 Elsevier
115
Inhibition of feline leukemia virus replication by human leukocyte interferon P a t r i c i a J a m e s o n 1,* a n d M y r o n Essex 2 ~ Department of Microbiology, Medical College of Wisconsin, P.O. Box 26509, Milwaukee, WI 53226, U.S.A. and 2 Department of Microbiology, Harvard University School o f Public Health, Boston, MA 02115, U.S.A. (Received 3 February 1983; accepted 18 February 1983)
The replication of feline leukemia virus (FeLV) is inhibited by treatment of cat cell cultures with crude human leukocyte interferon (HulFN-ct) as evidenced by titration of the infectious progeny. The inhibition can be demonstrated in three different cell lines in which the production of hemagglutinin by encephalomyocarditis (EMC) virus, and plaque formation by vesicular stomatitis virus (VSV) are also inhibited by the HulFN-a. The dose dependency of the inhibition of EMC virus by the H u l F N - a is similar to that obtained with feline interferon in each of the three cell lines. VSV and EMC virus are less than 10 times more sensitive than FeLV to the inhibitory action of H u l F N - a if responses to a single interferon treatment are compared for each of the viruses tested in the most sensitive cell line, FEA. The interferon effect on FeLV is more pronounced when it is added within one day after the inoculation of the cells rather than applied before cell infection. The induction of focus formation by FeLV can also be inhibited by HulFN-~t in cat cells (CCC-81) which contain the murine sarcoma virus genome. feline leukemia virus; human interferon; cat cell lines; FeLV; interferon; leukemia virus
Experimental section Feline l e u k e m i a virus ( F e L V ) is a h o r i z o n t a l l y t r a n s m i t t e d virus o f o u t b r e d cat species which m a y induce a variety o f diseases including several types o f n e o p l a s m s [7]. F o r these reasons the virus provides a useful m o d e l for the study o f inducible malignancies which resemble those o c c u r r i n g in man, a n d for e v a l u a t i o n o f p o t e n t i a l m o d e s o f t h e r a p y , such as interferon a d m i n i s t r a t i o n . One study suggested that this retrovirus might be m u c h less sensitive to the antiviral effects o f interferon than o t h e r types o f viruses [9]. T h e interferon effect on F e L V was d e t e r m i n e d by m e a s u r i n g the r e d u c t i o n in a c c u m u l a t i o n o f virus-specific antigens within infected cells, which m a y not be the most sensitive m e t h o d o f detecting the inhibition o f the viral r e p l i c a t i o n processes by interferon a n d p r e d i c t i n g the possible c o n s e q u e n c e o f interferon t r e a t m e n t on the progress o f the F e L V infectious process in vivo. T h e present w o r k
* To whom correspondence should be addressed. 0166-3542/83/$03.00 © 1983 Elsevier Science Publishers B.V.
116 was initiated to evaluate the effect of interferon on the production of infectious virus after inoculation of cat cells with FeLV.
Sensitivity of feline cells to interferon Three cat cell lines (CCC-81 [ 1], FEA [6], and F L F [2]) were found to be sensitive to the antiviral action of virus-induced [5] feline interferons produced by fibroblastoid (CCC-81) or lymphoblastoid (F422 [8], and FL74 [10]) cell lines and to Sendai virus induced crude human leukocyte interferon (HulFN-ct, from Dr. Cantell) as measured by reduction in yield of EMC virus hemagglutinin (EMC HA-YR) [3]. The dose response patterns for the two species of interferon were similar in a given cell line. FEA and F L F cells were similar to each other in their sensitivity, and were about 10 times more sensitive than the CCC-81 cell line in their response to interferon of either species. HulFN-c~ and samples of interferon from three cat cell lines were titrated in three lines of cat cells using the EMC HA-YR method. HulFN-c~ inhibited virus replication in cat cell cultures about 10 times more than any of the feline interferon samples tested. Three tests of a laboratory reference crude HulFN-et with the FEA cell line using the EMC HA-YR method measured 85 000 U/ml, which is comparable to the sensitivity of the A549 human cell line using the same interferon. Previously the A549 cell line was found to measure 1.8 times the assigned activity of 20 000 reference U/ml for the human leukocyte standard G-023-901-527, National Institutes of Health; this cell line also detected a geometric mean titer of 55 000 U/ml for the laboratory reference HulFN-ct in 84 determinations [4].
Inhibition of FeLV by interferon The sensitivity of FeLV (Subgroup A of feline leukemia virus produced by F422 cells [8]) to inhibition by interferon was tested in the three cat cell lines, along with VSV and EMC viruses (Table 1). A comparison of their responses is made by determining a titer for a single sample of H u l F N - a using inhibition of VSV plaque formation, yield of infectious FeLV or of EMC HA in each of the three different cell lines. Yields of FeLV were determined by assay of focus formation resulting from the rescue of the murine sarcoma virus (MSV) genome from transformed CCC-81 cells [ 1]; titers are expressed as focus-forming units/ml (ffu/ml). EMC virus and VSV were 3 to 50 times more sensitive than FeLV to inhibition by HulFN-ct depending on the cell and interferon treatment tested. A single application of interferon for one day before virus inoculation was used with VSV and EMC viruses; interferon was applied the day before inoculation and again after the attachment period with FeLV. In general, exposure of cultures to interferon was terminated when the medium was removed prior to inoculation with virus or to take samples for determining virus yield. In the FEA and F L F cells, EMC was about 7 to 13 times more sensitive than FeLV, and VSV was about 3 to 8 times more sensitive than FeLV. One or two treatments of CCC-81 cells with H u l F N - a within two days after inoculation with FeLV (day 0) reduced the number of loci of transformed cells detectable two weeks alter virus inoculation to 30 to 60% of the number observed in untreated infected plates (average count of 60 loci/plate).
117 TABLE 1 Comparison of interferon sensitivities of three viruses in three cat cell lines by determining the titer of a single crude human leukocyte interferon sample Cell line
Interferon titer, single observation or geometric mean U/ml ~ (number of titrations) VSV
CCC-81 FLF FEA
4 400 (2) 51 000 (2) 33 500 (2)
FeLV b Day 2
Day 3
EMC
NT 6 800 12 000
650 10 000 6 400
11 000 (10) 60 000 ( 2) 85 000 ( 3)
a
The titer of the interferon is expressed as the reciprocal of the dilution which reduces the yield of EMC virus hemagglutinin or FeLV focus-forming units by 0.5 log, or reduces the number of VSV plaques to 50% relative to that obtained with the untreated control culture. b Virus yields were measured in fluids removed either day 2 or day 3 after inoculation. Interferon was applied to the cells I day before, and immediately after the attachment period for the FeLV. The inhibition o f F e L V replication by interferon t r e a t m e n t was tested in all three cells using a variety o f schedules for the a d d i t i o n o f H u I F N - c t , which was then left in the culture m e d i u m until a d d i t i o n o f virus, or r e m o v a l o f s a m p l e fluid (Table 2). I n s t e a d o f r e p o r t i n g the yield o f F e L V as a measure o f relative sensitivity o f F e L V to the antiviral effect o f interferon, the c o m p a r i s o n o f F e L V response in the different cells u n d e r various c o n d i t i o n s was m a d e by d e t e r m i n i n g titers o f the HuIFN-c~ s a m p l e used for all treatments. N o c o r r e c t i o n factor was used to a c c o u n t for the variable n u m b e r o f interferon a d d i t i o n s m a d e in the various t r e a t m e n t schedules, because the time in the g r o w t h cycle o f the virus in a p a r t i c u l a r cell at which interferon is a d d e d a p p e a r e d to be m o r e i m p o r t a n t than the n u m b e r o f a d d i t i o n s made. T h e r e was little inhibition o f F e L V by the H u I F N - c t in CCC-81; the a p p a r e n t interferon titers ranged from 370 to 800 U / m l , when cells were treated 2 to 4 times with H u I F N - a , beginning one day before i n o c u l a t i o n o f F e L V . Both F E A a n d F L F cells were m o r e sensitive to interferon, a n d a p p a r e n t titers o f the H u I F N - a r a n g e d from 190 to 15 000 U / m l . There was an obvious increase in the antiviral effect, if the a d d i t i o n o f interferon was d e l a y e d until after the i n o c u l a t i o n o f cells with F e L V ; the greatest level o f inhibition o f infectious virus yield o c c u r r e d a b o u t 2 to 3 days after the last a d d i t i o n o f interferon. A p p a r e n t titers o f H u I F N - a r a n g e d from 5 200 to 15 000 U / m l when interferon was a d d e d after viral i n o c u l a t i o n a n d virus yield was m e a s u r e d on d a y 3 after inoculation. The i n h i b i t o r y effect o f two t r e a t m e n t s a p p e a r e d to be slightly greater than a single t r e a t m e n t o f F L F cells. N o striking increase in i n h i b i t o r y effect was o b t a i n e d by a second t r e a t m e n t o f F E A cells either one day before or one day after the a p p l i c a t i o n o f H u I F N - a at the conclusion o f the i n o c u l a t i o n process. The replication o f F e L V in CCC-81 cells is rather slow, with a p l a t e a u o f infectious virus reached a b o u t 3 to 4 days after inoculation o f a very sparse m o n o l a y e r o f r a p i d l y growing cells with a p p r o x i m a t e l y 0.1 flu/cell (Fig. 1). M o r e r a p i d r e p l i c a t i o n o f F e L V
118 TABLE 2 Inhibition of FeLV replication in cat cells by human leukocyte interferon Cell line
CCC-81
FLF
Interferon treatments given on days:
-l
0
+ + + +
+ + +
+1
+ + +
FEA Exp. I
+ + + +
Day 2
+
NT NT NT NT
÷
+ +
2 000 12 000 12 000 2 500 4 400 1 900
+
+ Exp. II
÷ + +
+2
440 6 800 3 600 8 400
+
+
Antiviral effect of interferon (U/ml) ~ measured at the indicated time after FeLV inoculation (day 0) Day 3 650 800 370 560 190 10 000 5 200 9 500
6 6 5 6 15
270 400 700 700 100 000
The titer of interferon is expressed as the reciprocal of the dilution which reduced the yield of FeLV focus-forming units by 0.5 log~0. No adjustment was introduced for the calculation of titers in the groups which received multiple additions of interferon. was o b s e r v e d in the F E A a n d F L F cell cultures, with a p l a t e a u level o f virus yield o b t a i n e d at a b o u t 2 days after i n o c u l a t i o n for b o t h cells. D a t a for F L F cells were similar to results with F E A cells ( d a t a not shown). S i m i l a r a m o u n t s o f virus were m e a s u r e d in culture fluids from a p a r t i c u l a r cell type whether or n o t the cell layers were w a s h e d i m m e d i a t e l y after i n o c u l a t i o n with F e L V ; the yield f r o m F L F cells was a b o u t 0.5 log10 higher t h a n f r o m F E A cells. B o t h o f the cells s u p p o r t i n g m o r e r a p i d viral r e p l i c a t i o n were m o r e sensitive to the antiviral a c t i o n o f HuIFN-c~ m e a s u r e d by F e L V response ( T a b l e 2) as well as by the response o f VSV a n d E M C viruses (Table 1). In view o f these o b s e r v a t i o n s , it is possible that the a p p a r e n t i n t e r f e r o n sensitivity o f F e L V r e p l i c a t i o n in CCC-81 cells might be greater if the virus s a m p l e s were t a k e n as the virus p l a t e a u was a p p r o a c h e d , e.g. on day 4 or 5 (Table 2). F i g u r e 2 shows the d o s e - r e s p o n s e r e l a t i o n s h i p o f interferon i n h i b i t i o n o f F e L V . The change in yield of F e L V (log flu) was r o u g h l y p r o p o r t i o n a l to the m a g n i t u d e of change in the interferon d i l u t i o n in all three cell lines. E n d p o i n t s were i n t e r p o l a t e d at the 0.5 log10 r e d u c t i o n in yield o f F e L V on lines p r o j e c t e d by linear regression using three or m o r e a d j a c e n t points which p r o d u c e d the best c o r r e l a t i o n with linearity (as i n d i c a t e d by the extent o f the line drawn).
CCC-81
119
FEA
z~
o~
3
O
2 14. z~
"o
0
i
~
0
I
i
J
o
after
Day
inoculation
of
cells
with
FeLV
Fig. 1. Replication of FeLV in feline cell lines. Cell monolayers were initiated with 1.5 ml of 2.5 X l0 s cells/ml in a 3-cm well. The next day growth medium was removed and FeLV (4.7 lOgl0 ffu) was inoculated in 0.5 ml of medium containing polybrene (8 pg/ml); after 1 h adsorption, 1.5 ml of medium were added (O) or the inoculum was removed, cells were washed and 2 ml of medium were added ( 0 , A). Samples collected from the unwashed cultures immediately after addition of medium had about 3.6 log ffu/ml; a sample from a washed culture ofCCC-81 cells had 1.9 log ffu/ml). Samples were taken at daily intervals by pooling 0.5 ml aliquots from duplicate cultures; either a 1-ml sample (O, O ) ~ r the entire supernatant fluid was removed (A), and fresh medium was added to replace that removed. W e c o n c l u d e that interferon decreases the yield o f infectious F e L V from infected cat cells. The antiviral activity o f H u I F N - c t is also expressed as a r e d u c t i o n in direct F e L V i n d u c e d focus f o r m a t i o n resulting from the rescue o f the M S V g e n o m e from the
c
I "~ 3.0
. . . . . . . . . . . . . . . . . . . . . .~,. . . .~. . . . . . . . . . . . . . . . . .• .
g2.5 .~_ c~ "~
~
x2. 0
.o_ °~
d o -J
x
o 2.0
Yield
2.5
of
3.0
3.5
FeLV
(log
4.0
ffu/ml)
Fig. 2. Inhibition of FeLV replication by human leukocyte interferon in feline cell lines. Interferon was added to obtain the indicated dilution in the Culture medium one day before and immediately after monolayer inoculation for FEA (O) and CCC-81 ( 0 ) cells; but only after the inoculation of cell monolayers for F L F ( 1 ) and FEA ( a ) cells. Interferon was not removed from the cultures. Virus yields were measured in samples of supernatant fluids collected on day 2 from FEA and F L F cells, and on day 4 from CCC-81 cells. Yields obtained in cultures not treated with interferon are shown at the top. The arrows (--) indicate the endpoint, or interferon titer (U/ml), at 0.5 log below the virus yield in untreated controls.
120 transformed CCC-81 cell line. The fact that this process can be inhibited as late as 1 to 2 days after inoculation of the cell layer suggests that either interferon may inhibit the induction of pseudotype MSV replication and the associated cellular changes may be inhibited to some extent, or that the continued presence of" interferon may inhibit secondary FeLV production which would otherwise induce some loci. But the latter possibility is not supported by observed titration patterns [1]. The antiviral action of H u l F N - ~ on FeLV replication is greatest when it is applied after inoculation of virus; as evidenced in virus production measured 2 to 4 days after inoculation of cells with virus. Although optimal conditions have not been established for determining the relative interferon sensitivity of the three viruses studied, comparisons which can be made of results obtained with single interferon treatments in the FEA cell line suggest that EMC and VSV are less than 10-fold more sensitive than FeLV. Therefore, FeLV resembles most viruses in being subject to control by interferon. The interferon sensitivity observed for FeLV in these experiments appears to be greater than stated in a previous report that VSV is 1000 times as sensitive as FeLV [9]. The different findings may depend partly upon both the cell cultures used, and the methods for detection of virus replication in the two systems. The use of crude human interferon in this study could not account for the relative responses of the three viruses since the effect of the interferon is upon the cell, not upon the virus, and the inhibitory effect on all three viruses was detected in three different cell lines with somewhat different sensitivities to either human or feline interferons.
Acknowledgements This work was supported in part by a grant from the Milwaukee Division, Inc., of the American Cancer Society. We thank Dr. O Jarrett, Glasgow, for the FEA cell line; and Dr. W.D. Hardy for the F L F cell line.
References 1 2 3 4 5
6 7 8 9 10
Fischinger, P.J., Blevins, C.S. and Nomura, S. (1974) J. Virol. 14, 177. Hardy, Jr., W.D., Old, L.H., Hess, P.W., Essex, M. and Cotter, S. (1973) Nature (London) 244, 266. Jameson, P., Dixon, M.A. and Grossberg, S.E.(1977)Proc. Soc. Exp. Biol. Med. 155, 173. Jameson, P. and Grossberg, S.E. (1981) Meth. Enzymol. 78, 357. Jameson, P., Taylor, J,L., Dixon, M., Sedmak, J.J. and Grossberg, S.E. (1980). In: Interferon: Properties and Clinical Uses, Eds.: Khan, A., Hill, N.O. and Dorn, G.L. (Leland Fikes Foundation Press, Dallas) p. 11, 1980. Jarrett, O., Laird, H.M., Hay, D. and Crighton, G.W. (1968) Nature (London) 219,521. Jarrett, W.F.H. (1975) Advances Vet. Sci. Comp. Med. 19, 165. Rickard, C.G., Post, J.E., Noronha, F. and Blair, L.M. (1969) J. Nat. Cancer Inst. 42,987. Rodgers, R., Merigan, T.C., Hardy, Jr., W.D., Old, J.L. and Kassel, R. (1972) Nature New Biol., 237, 270. Theilen, G.H., Kawakami, T.G., Rush, J.D. and Munn, R.J. (1969) Nature (London) 222, 589.
115
Inhibition of feline leukemia virus replication by human leukocyte interferon P a t r i c i a J a m e s o n 1,* a n d M y r o n Essex 2 ~ Department of Microbiology, Medical College of Wisconsin, P.O. Box 26509, Milwaukee, WI 53226, U.S.A. and 2 Department of Microbiology, Harvard University School o f Public Health, Boston, MA 02115, U.S.A. (Received 3 February 1983; accepted 18 February 1983)
The replication of feline leukemia virus (FeLV) is inhibited by treatment of cat cell cultures with crude human leukocyte interferon (HulFN-ct) as evidenced by titration of the infectious progeny. The inhibition can be demonstrated in three different cell lines in which the production of hemagglutinin by encephalomyocarditis (EMC) virus, and plaque formation by vesicular stomatitis virus (VSV) are also inhibited by the HulFN-a. The dose dependency of the inhibition of EMC virus by the H u l F N - a is similar to that obtained with feline interferon in each of the three cell lines. VSV and EMC virus are less than 10 times more sensitive than FeLV to the inhibitory action of H u l F N - a if responses to a single interferon treatment are compared for each of the viruses tested in the most sensitive cell line, FEA. The interferon effect on FeLV is more pronounced when it is added within one day after the inoculation of the cells rather than applied before cell infection. The induction of focus formation by FeLV can also be inhibited by HulFN-~t in cat cells (CCC-81) which contain the murine sarcoma virus genome. feline leukemia virus; human interferon; cat cell lines; FeLV; interferon; leukemia virus
Experimental section Feline l e u k e m i a virus ( F e L V ) is a h o r i z o n t a l l y t r a n s m i t t e d virus o f o u t b r e d cat species which m a y induce a variety o f diseases including several types o f n e o p l a s m s [7]. F o r these reasons the virus provides a useful m o d e l for the study o f inducible malignancies which resemble those o c c u r r i n g in man, a n d for e v a l u a t i o n o f p o t e n t i a l m o d e s o f t h e r a p y , such as interferon a d m i n i s t r a t i o n . One study suggested that this retrovirus might be m u c h less sensitive to the antiviral effects o f interferon than o t h e r types o f viruses [9]. T h e interferon effect on F e L V was d e t e r m i n e d by m e a s u r i n g the r e d u c t i o n in a c c u m u l a t i o n o f virus-specific antigens within infected cells, which m a y not be the most sensitive m e t h o d o f detecting the inhibition o f the viral r e p l i c a t i o n processes by interferon a n d p r e d i c t i n g the possible c o n s e q u e n c e o f interferon t r e a t m e n t on the progress o f the F e L V infectious process in vivo. T h e present w o r k
* To whom correspondence should be addressed. 0166-3542/83/$03.00 © 1983 Elsevier Science Publishers B.V.
116 was initiated to evaluate the effect of interferon on the production of infectious virus after inoculation of cat cells with FeLV.
Sensitivity of feline cells to interferon Three cat cell lines (CCC-81 [ 1], FEA [6], and F L F [2]) were found to be sensitive to the antiviral action of virus-induced [5] feline interferons produced by fibroblastoid (CCC-81) or lymphoblastoid (F422 [8], and FL74 [10]) cell lines and to Sendai virus induced crude human leukocyte interferon (HulFN-ct, from Dr. Cantell) as measured by reduction in yield of EMC virus hemagglutinin (EMC HA-YR) [3]. The dose response patterns for the two species of interferon were similar in a given cell line. FEA and F L F cells were similar to each other in their sensitivity, and were about 10 times more sensitive than the CCC-81 cell line in their response to interferon of either species. HulFN-c~ and samples of interferon from three cat cell lines were titrated in three lines of cat cells using the EMC HA-YR method. HulFN-c~ inhibited virus replication in cat cell cultures about 10 times more than any of the feline interferon samples tested. Three tests of a laboratory reference crude HulFN-et with the FEA cell line using the EMC HA-YR method measured 85 000 U/ml, which is comparable to the sensitivity of the A549 human cell line using the same interferon. Previously the A549 cell line was found to measure 1.8 times the assigned activity of 20 000 reference U/ml for the human leukocyte standard G-023-901-527, National Institutes of Health; this cell line also detected a geometric mean titer of 55 000 U/ml for the laboratory reference HulFN-ct in 84 determinations [4].
Inhibition of FeLV by interferon The sensitivity of FeLV (Subgroup A of feline leukemia virus produced by F422 cells [8]) to inhibition by interferon was tested in the three cat cell lines, along with VSV and EMC viruses (Table 1). A comparison of their responses is made by determining a titer for a single sample of H u l F N - a using inhibition of VSV plaque formation, yield of infectious FeLV or of EMC HA in each of the three different cell lines. Yields of FeLV were determined by assay of focus formation resulting from the rescue of the murine sarcoma virus (MSV) genome from transformed CCC-81 cells [ 1]; titers are expressed as focus-forming units/ml (ffu/ml). EMC virus and VSV were 3 to 50 times more sensitive than FeLV to inhibition by HulFN-ct depending on the cell and interferon treatment tested. A single application of interferon for one day before virus inoculation was used with VSV and EMC viruses; interferon was applied the day before inoculation and again after the attachment period with FeLV. In general, exposure of cultures to interferon was terminated when the medium was removed prior to inoculation with virus or to take samples for determining virus yield. In the FEA and F L F cells, EMC was about 7 to 13 times more sensitive than FeLV, and VSV was about 3 to 8 times more sensitive than FeLV. One or two treatments of CCC-81 cells with H u l F N - a within two days after inoculation with FeLV (day 0) reduced the number of loci of transformed cells detectable two weeks alter virus inoculation to 30 to 60% of the number observed in untreated infected plates (average count of 60 loci/plate).
117 TABLE 1 Comparison of interferon sensitivities of three viruses in three cat cell lines by determining the titer of a single crude human leukocyte interferon sample Cell line
Interferon titer, single observation or geometric mean U/ml ~ (number of titrations) VSV
CCC-81 FLF FEA
4 400 (2) 51 000 (2) 33 500 (2)
FeLV b Day 2
Day 3
EMC
NT 6 800 12 000
650 10 000 6 400
11 000 (10) 60 000 ( 2) 85 000 ( 3)
a
The titer of the interferon is expressed as the reciprocal of the dilution which reduces the yield of EMC virus hemagglutinin or FeLV focus-forming units by 0.5 log, or reduces the number of VSV plaques to 50% relative to that obtained with the untreated control culture. b Virus yields were measured in fluids removed either day 2 or day 3 after inoculation. Interferon was applied to the cells I day before, and immediately after the attachment period for the FeLV. The inhibition o f F e L V replication by interferon t r e a t m e n t was tested in all three cells using a variety o f schedules for the a d d i t i o n o f H u I F N - c t , which was then left in the culture m e d i u m until a d d i t i o n o f virus, or r e m o v a l o f s a m p l e fluid (Table 2). I n s t e a d o f r e p o r t i n g the yield o f F e L V as a measure o f relative sensitivity o f F e L V to the antiviral effect o f interferon, the c o m p a r i s o n o f F e L V response in the different cells u n d e r various c o n d i t i o n s was m a d e by d e t e r m i n i n g titers o f the HuIFN-c~ s a m p l e used for all treatments. N o c o r r e c t i o n factor was used to a c c o u n t for the variable n u m b e r o f interferon a d d i t i o n s m a d e in the various t r e a t m e n t schedules, because the time in the g r o w t h cycle o f the virus in a p a r t i c u l a r cell at which interferon is a d d e d a p p e a r e d to be m o r e i m p o r t a n t than the n u m b e r o f a d d i t i o n s made. T h e r e was little inhibition o f F e L V by the H u I F N - c t in CCC-81; the a p p a r e n t interferon titers ranged from 370 to 800 U / m l , when cells were treated 2 to 4 times with H u I F N - a , beginning one day before i n o c u l a t i o n o f F e L V . Both F E A a n d F L F cells were m o r e sensitive to interferon, a n d a p p a r e n t titers o f the H u I F N - a r a n g e d from 190 to 15 000 U / m l . There was an obvious increase in the antiviral effect, if the a d d i t i o n o f interferon was d e l a y e d until after the i n o c u l a t i o n o f cells with F e L V ; the greatest level o f inhibition o f infectious virus yield o c c u r r e d a b o u t 2 to 3 days after the last a d d i t i o n o f interferon. A p p a r e n t titers o f H u I F N - a r a n g e d from 5 200 to 15 000 U / m l when interferon was a d d e d after viral i n o c u l a t i o n a n d virus yield was m e a s u r e d on d a y 3 after inoculation. The i n h i b i t o r y effect o f two t r e a t m e n t s a p p e a r e d to be slightly greater than a single t r e a t m e n t o f F L F cells. N o striking increase in i n h i b i t o r y effect was o b t a i n e d by a second t r e a t m e n t o f F E A cells either one day before or one day after the a p p l i c a t i o n o f H u I F N - a at the conclusion o f the i n o c u l a t i o n process. The replication o f F e L V in CCC-81 cells is rather slow, with a p l a t e a u o f infectious virus reached a b o u t 3 to 4 days after inoculation o f a very sparse m o n o l a y e r o f r a p i d l y growing cells with a p p r o x i m a t e l y 0.1 flu/cell (Fig. 1). M o r e r a p i d r e p l i c a t i o n o f F e L V
118 TABLE 2 Inhibition of FeLV replication in cat cells by human leukocyte interferon Cell line
CCC-81
FLF
Interferon treatments given on days:
-l
0
+ + + +
+ + +
+1
+ + +
FEA Exp. I
+ + + +
Day 2
+
NT NT NT NT
÷
+ +
2 000 12 000 12 000 2 500 4 400 1 900
+
+ Exp. II
÷ + +
+2
440 6 800 3 600 8 400
+
+
Antiviral effect of interferon (U/ml) ~ measured at the indicated time after FeLV inoculation (day 0) Day 3 650 800 370 560 190 10 000 5 200 9 500
6 6 5 6 15
270 400 700 700 100 000
The titer of interferon is expressed as the reciprocal of the dilution which reduced the yield of FeLV focus-forming units by 0.5 log~0. No adjustment was introduced for the calculation of titers in the groups which received multiple additions of interferon. was o b s e r v e d in the F E A a n d F L F cell cultures, with a p l a t e a u level o f virus yield o b t a i n e d at a b o u t 2 days after i n o c u l a t i o n for b o t h cells. D a t a for F L F cells were similar to results with F E A cells ( d a t a not shown). S i m i l a r a m o u n t s o f virus were m e a s u r e d in culture fluids from a p a r t i c u l a r cell type whether or n o t the cell layers were w a s h e d i m m e d i a t e l y after i n o c u l a t i o n with F e L V ; the yield f r o m F L F cells was a b o u t 0.5 log10 higher t h a n f r o m F E A cells. B o t h o f the cells s u p p o r t i n g m o r e r a p i d viral r e p l i c a t i o n were m o r e sensitive to the antiviral a c t i o n o f HuIFN-c~ m e a s u r e d by F e L V response ( T a b l e 2) as well as by the response o f VSV a n d E M C viruses (Table 1). In view o f these o b s e r v a t i o n s , it is possible that the a p p a r e n t i n t e r f e r o n sensitivity o f F e L V r e p l i c a t i o n in CCC-81 cells might be greater if the virus s a m p l e s were t a k e n as the virus p l a t e a u was a p p r o a c h e d , e.g. on day 4 or 5 (Table 2). F i g u r e 2 shows the d o s e - r e s p o n s e r e l a t i o n s h i p o f interferon i n h i b i t i o n o f F e L V . The change in yield of F e L V (log flu) was r o u g h l y p r o p o r t i o n a l to the m a g n i t u d e of change in the interferon d i l u t i o n in all three cell lines. E n d p o i n t s were i n t e r p o l a t e d at the 0.5 log10 r e d u c t i o n in yield o f F e L V on lines p r o j e c t e d by linear regression using three or m o r e a d j a c e n t points which p r o d u c e d the best c o r r e l a t i o n with linearity (as i n d i c a t e d by the extent o f the line drawn).
CCC-81
119
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i
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Fig. 1. Replication of FeLV in feline cell lines. Cell monolayers were initiated with 1.5 ml of 2.5 X l0 s cells/ml in a 3-cm well. The next day growth medium was removed and FeLV (4.7 lOgl0 ffu) was inoculated in 0.5 ml of medium containing polybrene (8 pg/ml); after 1 h adsorption, 1.5 ml of medium were added (O) or the inoculum was removed, cells were washed and 2 ml of medium were added ( 0 , A). Samples collected from the unwashed cultures immediately after addition of medium had about 3.6 log ffu/ml; a sample from a washed culture ofCCC-81 cells had 1.9 log ffu/ml). Samples were taken at daily intervals by pooling 0.5 ml aliquots from duplicate cultures; either a 1-ml sample (O, O ) ~ r the entire supernatant fluid was removed (A), and fresh medium was added to replace that removed. W e c o n c l u d e that interferon decreases the yield o f infectious F e L V from infected cat cells. The antiviral activity o f H u I F N - c t is also expressed as a r e d u c t i o n in direct F e L V i n d u c e d focus f o r m a t i o n resulting from the rescue o f the M S V g e n o m e from the
c
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. . . . . . . . . . . . . . . . . . . . . .~,. . . .~. . . . . . . . . . . . . . . . . .• .
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~
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3.5
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Fig. 2. Inhibition of FeLV replication by human leukocyte interferon in feline cell lines. Interferon was added to obtain the indicated dilution in the Culture medium one day before and immediately after monolayer inoculation for FEA (O) and CCC-81 ( 0 ) cells; but only after the inoculation of cell monolayers for F L F ( 1 ) and FEA ( a ) cells. Interferon was not removed from the cultures. Virus yields were measured in samples of supernatant fluids collected on day 2 from FEA and F L F cells, and on day 4 from CCC-81 cells. Yields obtained in cultures not treated with interferon are shown at the top. The arrows (--) indicate the endpoint, or interferon titer (U/ml), at 0.5 log below the virus yield in untreated controls.
120 transformed CCC-81 cell line. The fact that this process can be inhibited as late as 1 to 2 days after inoculation of the cell layer suggests that either interferon may inhibit the induction of pseudotype MSV replication and the associated cellular changes may be inhibited to some extent, or that the continued presence of" interferon may inhibit secondary FeLV production which would otherwise induce some loci. But the latter possibility is not supported by observed titration patterns [1]. The antiviral action of H u l F N - ~ on FeLV replication is greatest when it is applied after inoculation of virus; as evidenced in virus production measured 2 to 4 days after inoculation of cells with virus. Although optimal conditions have not been established for determining the relative interferon sensitivity of the three viruses studied, comparisons which can be made of results obtained with single interferon treatments in the FEA cell line suggest that EMC and VSV are less than 10-fold more sensitive than FeLV. Therefore, FeLV resembles most viruses in being subject to control by interferon. The interferon sensitivity observed for FeLV in these experiments appears to be greater than stated in a previous report that VSV is 1000 times as sensitive as FeLV [9]. The different findings may depend partly upon both the cell cultures used, and the methods for detection of virus replication in the two systems. The use of crude human interferon in this study could not account for the relative responses of the three viruses since the effect of the interferon is upon the cell, not upon the virus, and the inhibitory effect on all three viruses was detected in three different cell lines with somewhat different sensitivities to either human or feline interferons.
Acknowledgements This work was supported in part by a grant from the Milwaukee Division, Inc., of the American Cancer Society. We thank Dr. O Jarrett, Glasgow, for the FEA cell line; and Dr. W.D. Hardy for the F L F cell line.
References 1 2 3 4 5
6 7 8 9 10
Fischinger, P.J., Blevins, C.S. and Nomura, S. (1974) J. Virol. 14, 177. Hardy, Jr., W.D., Old, L.H., Hess, P.W., Essex, M. and Cotter, S. (1973) Nature (London) 244, 266. Jameson, P., Dixon, M.A. and Grossberg, S.E.(1977)Proc. Soc. Exp. Biol. Med. 155, 173. Jameson, P. and Grossberg, S.E. (1981) Meth. Enzymol. 78, 357. Jameson, P., Taylor, J,L., Dixon, M., Sedmak, J.J. and Grossberg, S.E. (1980). In: Interferon: Properties and Clinical Uses, Eds.: Khan, A., Hill, N.O. and Dorn, G.L. (Leland Fikes Foundation Press, Dallas) p. 11, 1980. Jarrett, O., Laird, H.M., Hay, D. and Crighton, G.W. (1968) Nature (London) 219,521. Jarrett, W.F.H. (1975) Advances Vet. Sci. Comp. Med. 19, 165. Rickard, C.G., Post, J.E., Noronha, F. and Blair, L.M. (1969) J. Nat. Cancer Inst. 42,987. Rodgers, R., Merigan, T.C., Hardy, Jr., W.D., Old, J.L. and Kassel, R. (1972) Nature New Biol., 237, 270. Theilen, G.H., Kawakami, T.G., Rush, J.D. and Munn, R.J. (1969) Nature (London) 222, 589.