SUMMARY. The ability of wild-type (wt) Sindbis virus and six temperature-sensitive (ts) mutants to establish persistent infection in mouse L cells and a Line of ...
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J. gen. Virol. (1981), 54, 57-65 Printed in GreatBritain
Establishment
o f Persistent Infection in Mouse C e l l s b y Sindbis Virus and
its Temperature-sensitive Mutants By P. N O E L
B A R R E T T - ~ AND G R E G O R Y
J. A T K I N S *
Department o f Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland (Accepted 15 December 1980)
SUMMARY The ability of wild-type (wt) Sindbis virus and six temperature-sensitive (ts) mutants to establish persistent infection in mouse L cells and a Lineof mouse embryo (ME) cells was determined. The wt established persistent infection in both ME cells and L cells at 39 °C. At 30 °C the wt established persistent infection in L cells but not ME cells, which did not recover from the initial infection. For the ts mutants, both cell lines survived the initial infection at 39 °C (the restrictive temperature) but the virus was eventually eliminated. At 30 °C (the permissive temperature) in L cells all mutants established persistent infection. In ME cells at 30 °C, R N A - mutants (unable to synthesize virus-specified RNA at 39 °C) established persistent infection whereas the cells did not recover from infection with RNA + mutants (able to synthesize virus-specified RNA at 39 °C). The wt virus was less cytopathic in L cells than in BHK or ME cells. Interferon was produced by both L a n d ME cells at 30 °C and 39 °C, but its activity could not be detected in either cell line at 30 °C. It is proposed that establishment of persistent infection is dependent on reduced cytopathogenicity in the early stage of infection, and that further evolution of the virus then occurs to a less cytopathic form. Elimination of the virus at 39 °C is probably due to the action of interferon. INTRODUCTION Many factors have been implicated in the establishment and maintenance of persistent infection by RNA viruses: these include the production of temperature-sensitive (ts) mutants, defective interfering (DI) particles and interferon (Preble & Youngner, 1975; Rima &Martin, 1976; Holland & Levine, 1978; Pringle, 1979). For alphaviruses, interferon has been implicated in the establishment of persistent infection by Inglot et al. (1973) and by Meinkoth & Kennedy (1980). The role of DI particles has been studied by Weiss et al. (1980) ha BHK cells, a line which does not produce interferon. We have previously studied the establishment of persistent infection in BHK cells by ts mutants of Sindbis virus (Atkins, 19.79) and shown that persistent infection may be established by ts mutants showing low reversion or interference with the multiplication of the wild-type (wt). In this study we have extended this to two mouse cell lines capable of producing interferon. As in our previous study, we have concentrated on the conditions necessary to establish persistent infection rather than following the evolution of the virus over a large number of cell passages. This is because we wish eventually to extend this work to the whole animal, where establishment probably involves infection of cells of limited division potential at low multiplicity of infection (rmo.i.). We have shown that infection by Sindbis virus may lead to the death of the cell, establishment of persistent infection or elimination of the virus, and we have defined the conditions under ~"Present address: Institut ffir Virologicund Immunobiologieder Universit~itWfirzburg~Versbacher Strasse 7, 8700 Wfirzburg,West Germany. 0022-1317/81/0000-4447 $02.00 © 1981 SGM
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which these may occur. We also propose a mechanism for the establishment of persistent infection by this virus in mammalian cells. METHODS
Virus. All ts mutants are derivatives of the AR339 strain of Sindbis virus and have been described by Atkins et aL (1974a). The permissive temperature for the ts mutants is 30 °C, the restrictive temperature 39 °C. The mutants N2 and F294 accummulate protein precursors on the non-structural cleavage pathway (Brzeski & Kennedy, 1978; G. J. Atkins, unpublished data) on shift from 30 to 39 °C. A82 is a presumed double mutant, accumulating precursors on both the structural and non-structural pathways (G. J. Atkins, unpublished data), whereas the lesion in F104 is unknown. N2, F294, A82 and F104 are all R N A mutants (unable to synthesize virus-specified RNA at 39 °C) whereas A93 and H18 are RNA + mutants (able to synthesize virus-specified RNA at 39 °C). A93 has a cleavage defect on the structural protein cleavage pathway (Brzeski et al., 1978), whereas H18 is maturation defective (G. J. Atkins, unpublished data). Cells. The growth of BHK cells and the method of plaque assay have been described previously (Atkins et al., 1974b). Mouse L cells were obtained from the Department of Biological Sciences, University of Warwick, Coventry, U.K. and were grown in medium 199 plus 10% calf serum (Gibco-Biocult). They were split 1/3 at 3- to 4-day intervals at 39 °C and at 10-day intervals at 30 °C. ME cells are a line of mouse embryo cells isolated in our laboratory from Balb/c mice. They were grown in Dulbecco's minimal essential medium plus 10% foetal bovine serum (Gibco-Biocult), and were routinely split 1/3 at 7-day intervals at 39 °C and at 14-day intervals at 30 °C; they are fibroblastic in morphology. To facilitate their growth from low cell density, ME cells were infected at passages 55 to 60 from isolation. Establishment of persistent infection. Cells were seeded into 32 oz screw-capped glass bottles and grown until nearly confluent. They were then infected at a multiplicity of infection (m.o.i.) of 1 p.f.u./cell in 1 ml medium and the virus allowed to adsorb for 1 h. Thirty ml medium were added, and the ceils incubated at the appropriate temperature. Medium was changed when the cells showed cytopathic effect (c.p.e.), or every 3 to 4 days if they did not. When the monolayers were confluent the medium was changed and 24 h later this was removed and stored at - 6 0 °C for subsequent plaque assay, The cells were then split 1/3 into fresh bottles and frozen in liquid nitrogen after six passages. Fluorescent antibody staining. Cells were grown to confluence on revival from storage in liquid nitrogen, then split 1/3 into fresh bottles. When confluent, one of these cultures was used for fluorescent antibody staining, another for electron microscopy (see below). Four 90 mm diam. plastic dishes containing sterile glass microscope slides were set up from each confluent culture. The microscope slides were marked with a circle of 0.5 cm diam. to facilitate staining. Each dish received 10 ml cell suspension and the cells on each slide were fixed the following day after incubation overnight at 30 or 39 °C. The slides were washed three times with phosphate-buffered saline (PBS), then soaked in acetone for 5 min. They were then air dried an d stored at - 2 0 °C. For staining, the slides were warmed to room temperature and 50/~1 rabbit anti-Sindbis serum, diluted 1/30 in PBS, added to the marked area of each slide for 30 min at 37 °C. The slides were washed twice in PBS and once in distilled water for 10 min with agitation, then 50/tl fluorescein-conjugated sheep anti-rabbit immonoglobulin (Wellcome), diluted 1/20 in PBS, added to the marked area of each slide. The slides were again incubated at 37 °C, washed with PBS and water and coverslips placed over the stained area on FA mounting fluid (Difco). They were then viewed in a Reichert u.v. microscope. To prepare rabbit anti-Sindbis serum, a 9-month-old rabbit was injected four times subcutaneously at 2-week intervals with Sindbis virus purified by banding on a sucrose
Persistent infection by Sindbis virus
59
gradient. Two weeks after the final injection, the rabbit was bled and the serum stored in aliquots at - 2 0 °C. Electron microscopy. Confluent monolayers were washed three times with PBS, fixed in 3 % glutaraldehyde for 1 h, then scraped from the bottle and pelleted. The pellet was washed three times with PBS and treated with 3 % osmium tetroxide for 1.5 h. The cells were then dehydrated in ascending concentrations of ethanol and embedded in Araldite. Thin (70 nm) sections were cut, stained with uranyl acetate and lead citrate, and viewed in a Hitachi HU-12A electron microscope. To estiinate the proportion of cells showing virus multiplication, 100 cells were counted in random fields. Inhibition ofmacromolecular synthesis. Cells were seeded into 5 cm diam. plastic dishes at a concentration of 2 x 105 cells/ml in 3 ml growth medium. The following day, when the cells were still subconfluent, the monolayers were drained and virus was added to give an m.o.i, of 5 p.f.u./cell. After adsorption for 1 h at 39 °C the monolayers were drained and 2 ml warm (39 °C) growth medium added. For fiaeasurement of virus-specified RNA synthesis, 5/~g/ml actinomycin D (Sigma) was incorporated in the medium; for measurement of protein and DNA synthesis, no actinomycin D was present. At 6 h after infection, the monolayers were drained and washed twice with 4 mt PBS. Two ml medium containing the appropriate radioisotope were added and the cells incubated at 39 °C for a further 2 h. The monolayers were then drained, the medium frozen at - 6 0 °C for plaque assay and the cells washed twice with 4 ml PBS and dissolved in 2 ml 1% sodium lauryl sulphate. This solution was removed from the dishes, 2 ml 10% trichloroacetic acid (TCA) added, and the sample left on ice for 30 rain. The precipitate was collected on glass fibre discs by vacuum filtration, then washed twice with 5% TCA and twice with ethanol. The discs were dried overnight at 37 °C, placed in scintillation vials, scintillation fluid added and radioactivity counted in a scintillation counter. For measurement of RNA synthesis, 1 /~Ci/ml 3H-uridine (40 Ci/mmol) was incorporated into the medium during the pulse; for measurement of protein synthesis and DNA synthesis, 1 ¢tCi/ml 3H-leucine (48 Ci/mmol) and 1 /.tCi/ml 3H-thymidine (46 Ci/mmol) were incorporated respectively. All results quoted are the average of triplicate determinations. Induction and action of interferon. Confluent cell monolayers in 75 cm 2 plastic flasks were infected with virus to give an m.o.i, of 1 p.f.u./cell in 1 ml virus suspension. After adsorption for 1 h the inoculum was removed and 10 ml growth medium added. The cells were incubated at the appropriate temperature for 24 h, the medium was then removed and I M-HC1 added to pH 2. The medium was stored at 4 °C for 48 h, then 1 M-NaOH added to pH 6.7. This sample was stored at - 2 0 °C until an interferon assay could be performed. The interferon assay was the inhibition of nucleic acid synthesis (INAS) method described previously by Atkins et al. (1974a) except that L or ME cells rather than chick embryo cells were used. Cells were challenged with Semliki Forest virus (SFV) 24 h after addition of interferon, and the interferon titre measured in units of INASs0. RESULTS
Establishment of persistent infection in ME cells Initially, duplicate cultures were infected with each mutant or the wt at 39 and 30 °C. At 39 °C, infectious virus was eliminated from the culture supernatants by passage 6. Duplicate cultures gave similar results, and data for one culture for each mutant and the wt are shown in Table 1. The wt differed from the ts mutants in that it caused a c.p.e, in the culture by 24 h after infection. However, patches of cells survived this c.p.e, and grew out to form a confluent monolayer; the cells could then be passaged further, but showed sporadic phases of c.p.e, and recovery, which coincided with the production of infectious virus. The mutant-infected cells showed no c.p.e, at any stage.
60
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Virus initiating infection
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L
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Properties of virus produced by persistently infected cultures at passage 6
T a b l e 1.
Cell line
N.
RNA phenotype
Temperature of incubation (°C)
wt
+
F 104
--
F294
--
A82
--
N2
--
A93
+
HI8
+
wt
+
F104
--
F294
--
A82
--
N2
--
A93
+
H18
+
39 30 39 30 39 30 39 30 39 30 39 30 39 30 39 30 39 30 39 30 39 30 39 30 39 30 39 30
Titre of virus produced (p.f.u./ml)* < 1~: c.p.e.(1)§ e(3) 3 x 103 e(2) 9 × 103 e(2) c.p.e.(4) e(3) 2 x 103 e(2) c.p.e.(1) e(2) c.p.e.(1) 4 x 102 7 x 104 e(1) 3 x 103 e(2) 103 e(2) 6 x 103 6 2 x 10a e(3) 2 × 10 4 e(3) 104
e.o.p.'~ (39/30 °C)
% plaques < 1 m m in diam.
< 10 -a 6 × 10-2 0.1 0.5 0.3 < 10 -3 2 x 10 -3 3 x 10 -3 < 10-1 < 10 -3 3 x 10 -3 10-2
100(2)11 100(1) 0 100 51(3) 100(3) 100(5) 0 0 0 0 68(6)
* As assayed at 30 °C; e, infectious virus eliminated from supernatant, with passage number at which this occurred in parentheses. "~ e.o.p., Efficiency of plating. ~: At passages before the 6th a gradual reduction in plaque size was observed (see Results); infectious virus was detected at passages after the 6th. § The culture underwent a cytopathic effect from which it did not recover, the passage number at which this occurred in parentheses. IIThe passage number at which a proportion of small plaques greater than 10% was observed is indicated in parentheses.
At 30 °C neither the wt nor RNA + mutants could establish persistence, but caused a c.p.e. f r o m w h i c h t h e cells d i d n o t r e c o v e r . I n i t i a l l y , s o m e R N A 30 °C
in some
cultures, whereas
infected with each RNAand
F104
in two. No
others
mutants established persistence at
did not. Therefore,
a t o t a l o f five c u l t u r e s w e r e
m u t a n t . N 2 e s t a b l i s h e d p e r s i s t e n c e in all five c u l t u r e s , F 2 9 4 i n t h r e e cultures
passages. Data for representative
infected with A82
could be maintained
longer than
four
c u l t u r e s w h e r e p e r s i s t e n c e w a s e s t a b l i s h e d a r e p r e s e n t e d in
T a b l e 1. For many
cultures at 30 °C small-plaque variants were produced
eventually replaced the large-plaque
v i r u s in t h e s u p e r n a t a n t .
at later passages
which
These small plaques were less
t h a n 1 m m in d i a m . , w h e r e m o r e t h a n 9 5 % o f p l a q u e s p r o d u c e d
by the wt virus were more
t h a n 2 m m in d i a m . F e w i n t e r m e d i a t e - s i z e p l a q u e s w e r e o b s e r v e d i n a s s a y s o f t h e s u p e r n a t a n t fluids from these cultures. ME cells persistently infected with wt virus at 39 °C also produced s m a l l - p l a q u e v a r i a n t s a t l a t e r p a s s a g e s , b u t t h e r e w a s a g r a d u a l r e d u c t i o n in p l a q u e s i z e w i t h passage number.
Persistent infection by Sindbis virus
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Table 2. Fluorescent antibody staining and electron microscopy of persistently infected
cultures at passage 8
Cell line ME
Virus wt F294 H18 Uninfected
L
wt F294 H18 Uninfected
Temperature of incubation (°C) 39 39 30 39 39 30 39 30 39 30 39 30 39 30
Degree of fluorescence* + -+ ---+ + -+ -+ ---
% cells showing virus multiplication in EM~" 2 0 16 0 0 0 2 18 0 23 0 42 0 0
* +, Bright fluorescence; --, no fluorescence. I" A total of 100 cells were counted in random fields.
Establishment of persistent infection in L cells All mutants and the wt established persistent infection in duplicate cultures at both 30 and 39 °C. Duplicate cultures gave similar results and d a t a for one culture for each m u t a n t and the wt are shown in Table 1. A t 39 °C, infectious virus was eliminated from the cultures by passage 6 for all ts mutants except N2. N o ts m u t a n t caused c.p.e, on initial infection, and the wt caused only slight c.p.e, from which the culture quickly recovered. The wt-infected cultures showed sporadic production of low titres o f infectious virus at later passages. A t 30 °C, most cultures showed slight c.p.e, on initial infection, but no c.p.e, was evident at subsequent passages. All cultures continued to produce infectious virus in the supernatant up to passage 6. A s with M E cells, small-plaque variants were often found in the cell supernatants at 30 °C, and were also produced b y the wt at 39 °C after elimination of the large-plaque form.
Fluorescent antibody staining Persistently infected cultures established with the mutants F 2 9 4 and H18, and with the wt, were examined by fluorescent antibody staining. Both cells infected with the wt for 24 h at 39 ° C and control uninfected cells were tested. All persistently infected cultures incubated at 30 ° C , and those established with the wt at 39 °C, showed bright fluorescence in the m a j o r i t y o f cells. F o r cultures established with H18 and F 2 9 4 at 39 °C, fluorescence could not be detected (Table 2).
Electron microscopy Cells tested b y fluorescent antibody staining were also examined by electron m i c r o s c o p y (EM) for the presence o f intermediates in virus multiplication, as described by G r i m l e y et al. (1972). The results are shown in Table 2. The p r o p o r t i o n of cells showing signs o f virus multiplication correlated with the production o f infectious virus by the cultures as m e a s u r e d in Table 1.
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Table 3. Yield of persistently infected cultures on superinfection Cell line ME
Mutant wt F294 H18 Uninfected
L
wt F294 H18 Uninfected
Temperature (°C)
Yield on superinfection (p.f.u./106 cells)*
39 39 30 39 39 30 39 30 39 30 39 30 39 30
1.6 2.1 2.2 3.6 2.4 1.2 1.9 2.6 8.2 2-8 5.8 2.4 5-6 4-5
× × x x x x x x x × x x x x
105 106 105 106 106 106 106 106 107 106 107 106 107 107
* Collected 2 to 24 h p.i. and assayed at 39 °C.
Temperature shift experiments L and ME cells infected at 39 °C with the mutants F294 and H18 (passages 7 to 8 p.i.) were shifted to 30 °C for 24 h, the cells washed three times with PBS and fresh medium added for a further 24 h. No virus could be detected in this medium by plaque assay at 30 °C, either for cells shifted to 30 °C or for parallel cultures maintained at 39 °C. Also, L cells infected with the mutants F294 and H18 at 30 °C and ME cells infected with the mutant F294 (passages 7 to 8 p.i.) were shifted in a similar manner to 39 °C. No virus could be detected in the supernatant fluids of cells shifted to 39 °C, although virus was easily detectable in parallel cultures maintained at 30 °C. Thus, we conclude that cultures which had ceased to produce infectious virus at 39 °C could not be induced to do so by shift to 30 °C and that cultures shifted from 30 to 39 °C ceased to produce virus, although they continued to do so if left at 30 °C.
Superinfection experiments L and ME cells infected at 39 °C by the mutants H18 and F294, and the Wt (passages 7 to 8 p.i.) were superinfected with the wt virus at 39 °C and the yield plaque assayed at 39 °C. As shown in Table 3, cells infected with the mutants were as sensitive to subsequent infection by wt virus as uninfected ceils, although wt-infected cells gave a reduced yield after superinfection. Similarly, L cells infected with the mutants H18 and F294, and the wt, and ME cells infected with the mutant F294 were superinfected with the wt at 30 °C and the yield plaque assayed at 39 °C. As shown in Table 3, the yield was substantially reduced on superinfection of these persistently infected cells compared to uninfected cells. Thus we conclude that cells infected at 39 °C with ts mutants were sensitive to wt superinfection, whereas those infected at 30 °C showed a degree of superinfection immunity.
Inhibition of macromolecular synthesis Initial observations suggested that the wt virus had greater cytopathogenicity in ME cells than in L cells. Also, previous studies in our laboratory had shown that the wt virus is highly cytopathic in B H K cells (Atkins, 1977) and is unable to establish persistent infection in these cells (Atkins, 1979). We therefore attempted to quantify these results by measuring inhibition of cellular protein and D N A synthesis, and the production of infectious particles and virus-specified R N A in the three cell lines at 39 °C. As shown in Table 4, cellular macromolecular synthesis was inhibited to approximately the same extent in BHK and ME
Persistent infection by Sindbis virus
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Table 4. Inhibition of maeromolecular synthesis in ME, L and B H K cells infected with
Sindbis virus
Cell line
DNA synthesis* (% control)
Protein synthesis* (% control)
Viral R N A synthesis~ (ct/min)
Infectious virus production~ (p.f.u./ml)
c.p.e. §
ME L BHK
39 92 56
46 106 53
634 434 2431
9 x 105 8 x 104 2 x 106
++ + ++
* Expressed as a percentage of the TCA-precipitable ct/min obtained for uninfected controls (50 000 to 70 000 ct/min for 3H-thymidine; 1000 to 1500 ct/min for 3H-leucine). "~Measured in the presence of 5 /tg/ml actinomycin D; 150 to 250 ct/min have been subtracted for the uninfected control. $ Measured 6 to 8 h after infection. § Observed 24 h after infection: + +, strong c.p.e.; +, c.p.e, just detectable.
Table 5. Interferon induction and action at 39 and 30 ° C Cell line* L
Induction temperature (°C)
Assay temperature (°C)
Interferon titre (INASs0)
30
30 39 30 39 30 39 30 39
Undetectable
39 ME
30 39
160
Undetectable 162 Undetectable 7 Undetectable 16
* Interferon was induced and assayed in the same cell line.
cells, but inhibition was not detected in L cells, and this correlated with the production of infectious virus and virus-induced c.p.e. Thus we conclude that the wt is less cytopathic in L cells than in ME or B H K cells.
Induction and action of interferon As shown in Table 5, both ME and L cells produced interferon at 39 and 30 °C, although ME ceils produced less interferon or were less sensitive to its action than L cells. However, although interferon induced an antiviral state in both cell lines at 39 °C, it was inactive at 30 °C. We also examined supernatant fluids from L cells persistently infected with the mutants H18 and F294, and ME cells persistently infected with F294 at 30 °C (passages 8 to 15), for the presence of interferon. Interferon titres of 5 to 100 units could be detected in all instances. DISCUSSION
This study has shown that the establishment of persistent infection by Sindbis virus depends both on the genotype of the virus and the cell. L cells and ME cells showed differing responses to mutant and wt infections. The establishment of persistent infection in ME cells was clearly dependent on the R N A phenotype of the virus at permissive temperature. It is possible that ts mutants are somewhat defective at permissive as well as restrictive temperature (Brzeski et al., 1978; Brzeski & Kennedy, 1978; G. J. Atkins, unpublished data). This may be the reason for the differing abilities of R N A + and R N A - mutants to establish
64
p. N. BARRETT AND G. J. ATKINS
persistent infectio,as at 30 °C in ME cells, since RNA + mutants may be more cytopathic than R N A - mutants, as is the case for BHK cells at 39 °C (Atkins 1976, 1977). In L cells at 30 ° C all mutants and the wt were able to establish persistent infection. The probable reason for this is that Sindbis virus is inherently less cytopathic in L cells than in ME or BHK cells, and so infected cells survive to enable persistent infection to be established. At 39 °C, virus was eliminated from both L and ME cells and so persistent infection was not established. Elimination of virus was shown by the failure of the cells to produce infectious virus even after shift to 30 °C, the absence of virus antigen in the cells, the lack of superinfection immunity and the failure to detect intermediates in virus multiplication by electron microscopy. Since both cell lines produce and are sensitive to interferon at 39 °C, we believe that this is the probable reason for virus elimination. Further evidence for this is that infectious virus production by ceUs persistently infected at 30 °C ceases when the cultures are shifted to 39 °C. The lower efficiency of interferon action at 30 °C results in persistent infection rather than virus elimination. Although some R N A - mutants of Sindbis virus are defective in interferon induction at 39 °C, at least in chick cells (Atkins et al., 1974 a; Marcus & Fuller, 1979) it is probable that the mutation results in a delay in interferon production, and that interferon is eventually produced by the processes of leak and reversion (Atkins & Lancashire, 1976). We have confirmed that interferon production by the R N A - mutants F104 and F294 at 39 °C in L cells is undetectable at 24 h p.i. but that small amounts of interferon (5 to 10 units) are produced by 48 h. The mechanism of maintenance of persistent infection in L and ME cells at 30 °C is not clear. We have so far been unable to detect DI particle RNA in these cells at 30 °C by sucrose gradient centrifugation, although total RNA synthesis is low (103 to 104 ct/min could be detected in 42S and 26S RNA peaks). Therefore, it is possible that small quantities of DI RNA are synthesized but not detected using sucrose gradients. However, Meinkoth & Kennedy (1980), in their analysis of L cells persistently infected with SFV, also failed to detect DI RNA. It is possible that interferon produced by persistently infected cultures has a minimum effect at 30 °C, sufficient to maintain a persistent infection but not to eliminate the virus. Inglot et al. (1973) in their study of persistent infection of similar cells to those used in the present study, showed that virus isolates which differed in plaque morphology also differed in their ability to establish persistent infection. However, since the basis of the difference in plaque morphology was unknown, no conclusions could be drawn concerning the mechanism of establishment of persistent infection. Both persistently infected cell lines periodically produced small amounts of interferon, and the addition of anti-interferon globulin stimulated virus production and destruction of the cultures. Thus, interferon was involved in the maintenance of the infection and may have mediated its establishment. For SFV, Meinkoth & Kennedy (1980) have shown that interferon may be involved in both the establishment and maintenance of persistent infection in L cells. Our results show that interferon was probably involved in the elimination of Sindbis virus ts mutants from L and ME ceils at 39 °C, but that the wt established a persistent infection. Possibly the reduced rate of multiplication of the ts mutants resulted in reduced ability to combat the interferon-induced antiviral state. Our results show tlaat virus produced by most persistently infected ME and L cell cultures gave reduced plaque size. For many of them the total virus population gave plaques less than 1 mm in diam. after six passages. Schw6bel & Ahl (1972), Atkins (1979) and Weiss et al. (1980) also described the production of virus with much reduced plaque size from BHK cells persistently infected with Sindbis virus. Weiss et al. (1980) reported that this virus is less cytopathic in BHK cells than wt Sindbis virus, and we have also made this observation (G. J. Atkins, unpublished data).
Persistent infection by Sindbis virus
65
We believe that our data (this study and Atkins, 1979), as well as those of others (Schqeobel & Ahl, 1972; Inglot et al., 1973; Weiss et al. 1980; Meinkoth & Kennedy, 1980), suggest that persistent infection by alphaviruses of mammalian cells is an equilibrium state between virus multiplication and factors restricting this process such as DI particle and interferon production. The essential prerequisite to the establishment of persistent infection is survival of the infected cells; this may be achieved through the action of DI particles, interferon, non-cytocidal virus mutants or inherent resistance of the cell to the virus. If restriction is too severe, the virus is eliminated but an intermediate degree of restriction allows a rate of virus multiplication compatible with cell survival. This initial stage may be unstable, but survival of infected cells leads to selection pressure on the virus to evolve to a less cytopathic state, as cells infected by cytopathic virus would be killed and the virus eventually eliminated. This less-cytopathic state may involve the generation of DI particles, small plaque variants, ts mutants, the continuous induction of interferon or a combination of these factors. W e t h a n k M r s L y n d a W i n s t o n for p e r f o r m i n g t h e electron m i c r o s c o p y a n d for excellent t e c h n i c a l
assistance. T h i s w o r k w a s s u p p o r t e d b y t h e M e d i c a l R e s e a r c h C o u n c i l o f I r e l a n d a n d the Irish C a n c e r Society. REFERENCES ATKINS, G. J. (1976). The effect of infection with Sindbis virus and its temperature-sensitive mutants on cellular protein and D N A synthesis. Virology 71, 593-597. ATKrNS, G. J. (1977). Cytopathogenicity of temperature-sensitive mutants of Sindbis virus. FEMS Microbiology Letters 2, 51-55. ArKINS, G. J. (1979). Establishmerit of persistent infection in BHK-21 cells by temperature-sensitive mutants of Sindbis virus. Journal of General Virology 45, 201-207. ATKINS, G. J. & LANCASHIRE, C. L. (1976). The induction of interferon by temperature-sensitive mutants of Sindbis virus: its relationship to double-stranded R N A synthesis and cytopathic effect. Journal of General Virology 30, 157-165. ATKINS, G. J., JOHNSTON, M. D., WESTMACOTT,L. M. & BURKE, D. C. (1974a). Induction of interferon in chick cells by temperature-sensitive mutants of Sindbis virus. Journal of General Virology 25, 381-390. ATKINS, G. J., SAMUELS, S. & KENNEDY, S. I. T. (1974b). Isolation and preliminary characterization of temperature-sensitive mutants of Sindbis virus strain AR339. Journal of General Virology 25, 371-380. B~ZESrd, a. ~ KENNEDY, S. I. T. (1978). Synthesis of alphavirus specified R N A . Journal of Virology 25, 630-640. BRZESKI, H., CLEGG, J. C. S., ATKINS, G. J. & KENNEDY, S. I. T. (1978). Regulation of the synthesis o f Sindbis virus specified R N A : role of the virion core protein. Journal of General Virology 38, 461-470. GRIMLEY, P. M., LEVIN, J. O., BEREZESKY, I. K. & FRIEDMAN, R. M. (1972). Specific membranous structures associated with the replication of group A arboviruses. Journal of Virology 10, 492-503. HOLLAND, J. J. & LEVINE, A. (1978). Mechanisms of virus persistence. Cell 14, 447-452. rNGLOT, A. D., ALBnq, M. & CHUDZIO, T. (1973). Persistent infection of mouse cells with Sindbis virus: role of virulence of strains, auto-interfering particles and interferon. Journal of General Virology 20, 105-110. MARCUS, P. I. & FULLER, F. J. (1979). Interferon induction by viruses. II. Sindbis virus: interferon induction requires one-quarter of the genome-genes G and A. Journal of General Virology 44, 169-178. MEIr~ZOTH, J. & IO~ZNEDY, S. I. T. (1980). Semliki Forest virus persistence in mouse L929 cells. Virology 100, 141-155. PREBLE, O. T. & YOtmGNER, S. S. (1975). Temperature-sensitive viruses and the etiology of chronic and inapparent infections. Journal of Infectious Diseases 131, 467-473. PRINOLE, C. R. (1979). Virus evolution during persistent infection. Nature, London 280, 16. RIMA, B. & MARTrN, S. J. (1976). Persistent infection of tissue-culture cells by R N A viruses. Medical Microbiology. and Immunology 162, 89-118. SCHW6BEL, S. & AHL, R. (1972). Persistence of Sindbis virus in BHK-21 cell cultures. Archives of Virology 38, 1-10. WEISS, B., ROSENTHAL, R. & SCHLESINGER, S. (1980). Establishment and maintenance of persistent infection by Sindbis virus in B H K cells. Journal of Virology 33, 463-474.
(Received 30 September 1980)
