VIROLOGY
Murine
84, 222-226 (19’78)
Leukemia
BRUCE
Virus Infectious Centers are Dependent of Virus Production by Infected Cells
CHESEBRO,
KATHY WEHRLY, KENNETH AND KEVIN CHESEBRO’
on the Rate
WATSON,
U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840, and Department of Chemistry, University of Montana, Missoula, Montana 59812 Accepted September 16,1977 A group of in vitro-maintained mouse leukemia cell lines has been used to analyze a murine leukemia virus S+L- infectious center assay. The rate of virus production by leukemia cell lines was determined by two methods and was compared to the observed incidence of infectious centers for each cell line in an effort to define the exact nature of an infectious center. The results show that detection of virus-producing cells in the infectious center assay is dependent on the rate of virus production by individual cells. Although 100% of high-virus-producer cells give positive infectious center plaques, only 0.01-l% of low virus producers are detectable. The results roughly fit the prediction that a virus producer cell is scored as an infectious center if it secretes 1 PFU of virus within 40 hr after seeding onto the target cell culture.
passage over a long period of time and should be relatively homogeneous cell populations. One of the low IC lines (D2N) was cloned to see if high- and low-virus-producing subpopulations could be isolated. Nine separate clones of the line D2N were test.ed. All clones had an incidence of infectious centers (IC/106 cells) similar to that of the uncloned population. No nonproducer clones were obtained. These observations appeared to exclude the idea that heterogeneity of virus production among the cells of the D2N line was responsible for the low percentage of cells giving infectious center plaques. Since cells of all clones released plaque-forming virus, all cells in the original population should have been capable of virus production. The low percentage of infectious centers observed in the cloned and uncloned lines appeared to have resulted from a general characteristic of virus production kinetics of all cells in the population. We have attempted to relate the observed value of IC/106 cells to the level of virus production of the entire culture. Our
Infectious center (10 assays have been valuable in many virus systems to evaluate the number of virus-infected cells in various experimental situations. The two commonly used plaque assays for murine leukemia viruses (MuLV), the XC assay and the S+L- assay, have been used to identify infectious centers by plating live infected cells instead of free virus in the infection step of the assay (1,2). We have attempted to define the exact nature of the infectious centers detected in the mouse S+L- plaque assay using a number of mouse leukemia cell lines. Several leukemia lines known to be virus producers had extremely low titers in the IC assay (2.3-3.8 log IC/106 cells -0.01 to 1.0% positive) (Table 1). This observation suggested that in these lines there might be a large proportion of cells which did not produce plaque-forming virus. This was a surprising finding since these leukemia lines have been maintained in laboratory ’ Present address: School of Medicine, University of California at Los Angeles, Los Angeles, California. 222 0042-6822/78/0841-0222$02.00/O Copyright All rights
0 1978 by Academic F’ress, Inc. of reproduction in any form reserved.
SHORT
TABLE CORRELATION
Cell line”
AA41 DlB (early)’ Al3 EL4 (G+) DlB (lateIfT2 BCl D2N EL4 (G-J
OF RATE
OF
VIRUS
PRODUCTION
Log IWO6 cell@
6.12 5.63 5.69 5.04 3.84 3.61 3.72 3.75 2.31
223
COMMUNICATIONS 1
WITH INCIDENCE CELL LINES
Log PFU/lOG cells’
r 0.13 k 0.28 i 0.21 2 0.30 + 0.57 c 0.32 +- 0.32 i 0.27 t- 0.41
6.22 5.52 4.71 4.20 3.44 2.55 1.79 1.67 0.84
OF INFECTIOUS
CENTERS
IN LEUKEMIA
Log PFLJ produced/lo6
cells/40 hr
Calculated by Calculated by cell washing equilibrium method’ methodd -~__ __-__ 6.42 7.21 5 0.47 5.62 6.51 r 0.23 5.51 5.70 t 0.32 nt” k 0.18 5.19 nt 4.43 -c 0.44 nt 3.54 f 0.87 2.78 nt ? 0.13 nt 2.66 + 0.37 nt 1.83 t 0.53 ____ previously (3). AA41 is a new FV-B-induced line
a Origin and maintenance of cell lines were described derived from a (BIO.A x A)F, leukemic mouse spleen. b Values observed in infectious center assay on D56 (mouse) S+L- cells (2). Various numbers of washed cells to be assayed were added directly to dishes containing 4 ml of medium. Exposure of cells to be tested to 2000 R of X-irradiation to prevent cell division, or to treatment with trypsin to inactivate exposed virus (41, did not affect the IC titer observed. c PFU/lOG cells in tissue culture supernatant = (PFU/ml of supernatant)/(cells/ml of suspension) x lo”, as assayed on D56 (mouse) S+L- cells (2,3). d Calculated from equation (3), assuming T% = 2 hr. ’ Calculated from virus concentration achieved 3 to 4 hr after washing cells and resuspending in fresh medium. ‘Early passages (up to 30th) of the DlB line produced more virus than late passages (later than 50th). Therefore, these sublines were considered separately in this report, g nt = not tested.
assumption was that each infectious center plaque observed might simply represent the release of a single viral PFU by a plated cell and the successful infection of a S+L- assay target cell by this PFU. Because of the critical timing involved in successful detection of PFU in this assay, this initial target cell infection event would probably have to occur within some restricted time period following plating of virus or cells in order to have time to produce a visible plaque on the fifth day, when plaques are counted (Eq. 1). The initial problem was to determine the rate of production of viral PFU for each cell line and to see how this figure compared with the observed IWO6 cells. In preliminary experiments, it was found that regardless of the cell density of a given culture the number of viral PFU/ml in the cell-free supernatant had a constant relationship to the cell concentration. Thus for each individual cell line (PFU/
mlY(cells/ml) or PFU/106 cells was a constant. Virus production by the cells and inactivation of cell-free virus appeared to be in equilibrium. Thus virus produced/hr equaled virus inactivatedlhr. By the delinition of half-life (Tf/2) the amount of virus inactivated in one half-life is equal to one-half the cell-free virus concentration (Eq. 2). Since the virus/cell ratio (PFU/106 cells) remained constant for each cell line, the effect of cell concentration could be neglected by using PFU/106 cells instead of PFU/ml as virus concentration (Eq. 3). (1) ICYlO6 cells = (PFU produced/lo6 cells/hr) (t) (E 1. E = efficiency of infection of target cell by virus produced by a plated cell in the IC assay; t = time (hr) after seeding virus-producing cells into IC assay during which target cell infection yields a countable plaque read on day 5. (2) PFU produced/hr = PFU inactivated/hr = l/2 PFU present/(Tl/z).
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(3) PFU produced/lo6 cells/hr = PFU inactivated/lo6 cells/hr = l/z (PFU/106 cells)/(TVz) T1/2 = half-life of cell-free virus under tissue culture conditions. PFU/ lo6 cells = (cell-free PFU/ml)/(cells/ml) (106) = cell-free PFU present in tissue culture supernatant per lo6 cells present in the culture suspension. Combining equations (1) and (3) yields equations (4) and (5) which could be evaluated experimentally. (4) IC/106 cells = l/2 (PFU/106 cells) (t)
-A13 =.DlB(l.t.) bD2N q.E14(G+) nEL4(G-)
7 i
ec 1 ~.DlB(e.rly) @.A A41
(E)/(TVz)
(5) Log PFU/106 cells = log IC/106 cells + log [(TVz)/Vz (t) (El]. Thus when log PFU/106 cells is plotted against log ICYlO cells (Fig. l), equation (5) would predict a straight line with slope = 1 and x-intercept = log [l/2 (t) (E)I(T112)] if t, E, and TV2 are constant for all the cell lines. We have determined the T1/2 for the viruses produced by several of our cell lines, and all appear to be in the range of 1.5 to 2.5 hr. The time (t) as defined in equation (1) was estimated to be approximately 30 to 40 hr by seeding 20 to 40 cells (= 20 to 40 IC) of a high producer line in the S+L- dishes at various times after the usual infection time in this assay. Although a sharp cutoff point was difficult to determine precisely, good plaques were observed when cells to be assayed were seeded up to 30 to 40 hr after the usual time of seeding (data not shown). The data comparing IC/106 cells with PFU/106 cells for several cell lines are given in Table 1 and Fig. 1. The general trend of the data obtained from different cell lines supports the idea that the incidence of infectious centers observed is related to the rate of virus production by a cell line. For lines A13, DlB (early), and AA41 the rate of virus production was also determined in a different manner. Cell-free virus was washed out of an exponentially growing cell culture, and cells were recultured in fresh medium at the original concentration. Samples of culture supernatant were examined for the rate of reappearance of virus. Attainment of equilibrium status of virus production (i.e., constant PFU/106 cells) was achieved by 8 to 12 hr (data not shown). However, during
. 0
1
2
3 4 LOG IC/106CELLS
$
b
7
FIG. 1. Observed incidence of infectious centers (ICYlO cells) was plotted against the cell-free virus concentration (expressed as PFU/106 cells present in the suspension culture before centrifugation). The line drawn is the theoretical line expected from equation (4) (in text). The intercept would equal 1 ifE = 1, t = 40 hr, and T1/z = 2 hr; these appear to be the approximate values observed for t and T%.
the initial time prior to equilibrium, virus concentration increased at a very rapid rate; and the amount of virus inactivated was small, since the actual virus concentration was low.* Therefore the rate of virus production (PFU produced/lo6 cells/ hr) could be approximated by determining the virus concentration 3 to 4 hr after washing. The results are shown in Table 1. The rate of virus production determined 2 The rate of virus inactivation (duldt) is directly proportional to the cell-free virus concentration (VI. Thus dvldt = AV, where A = 0.693/T%. Following the washing of the cultured cells, V is a function of time (t) before equilibrium between virus production and inactivation is attained. Therefore, the amount of virus inactivated in the first 4 hr (V = AJb V dt) can be calculated by plotting virus concentration vs time and measuring the area under the curve from t = 0 to 4 hr. For example, with the Al3 cell line, virus inactivation during the initial 4 hr was 1.8 x lo3 PFU/lOB cells/4 hr. However, net virus production was 3.2 x 10’ PFU/108 cells/4 hr. Therefore, we have chosen for simplicity to neglect this small amount of total virus production which was lost due to inactivation in the first 4 hr.
SHORT
225
COMMUNICATIONS
by this method is in fair agreement with that calculated by the equilibrium data also shown in Table 1. Both methods appear to give values in the range of the observed values of lC/106 cells for each line tested. These studies indicate that infectious center assays for MuLV are strongly dependent on the rate of virus production by the cells being assayed. For a cell population producing large amounts of plaqueforming virus the IC assay appears to detect all infected cells. However, for populations producing lower amounts of virus only a small fraction of virus-producing cells are detected. These findings make interpretation of IC assay data from heterogeneous cell populations difficult. For example, one cannot distinguish a high incidence of low producers from a low incidence of high producers. Furthermore, in a mixed population a 1% incidence of high producers could give more lC/106 cells (i.e., 10”) than a 99% incidence of low producers, such as the EL4 (G-I or D2N lines (2 x lo’ and 5.6 x lo3 lC/106, respectively). This type of phenomenon has not been troublesome with IC assays in other virus systems. However, MuLV have several properties which might lead to these results. First, MuLV are released from producer cells by membrane budding in a continuous fashion; no lytic burst of large numbers of viruses occurs. Second, after being released from the cell MuLV are very labile (Tl/z = 2 hr). Third, the effciency of infection by cell-free virus is low. Therefore, rate of virus production might be a critical limiting factor in determining whether or not a virus-producing cell actually makes enough virus to score a plaque in the IC assay. These findings provide a possible explanation for the “refractoriness” and/or “lack of productive viral expression” noted by Pincus et al. (4) in Fv-l-resistant cells which were doubly hit by virus but did not score as virus producers. If the rate of initial virus production was low in Fv-l-resistant cells even when doubly infected, then some of these cells might not be scored as infectious centers. Average virus yield per infected cell has been shown to be a function
of multiplicity of infection (5). This finding, as well as the observation of DuranTroise et al. (6) that all Fv-l-resistant cells can be productively infected if the multiplicity of infection is high enough, support the interpretation that the apparent refractoriness seen in these systems might be related to the failure to score low virus producer cells in the IC assay. In our attempts to correlate lC/lO” cells with rates of virus production (Fig. 1, Table l), it is clear that a number of oversimplifications have been made which could account for the failure of some cell populations to fit the expected theoretical curve more precisely. The calculation of virus production based on equilibrium between virus production and inactivation for each cell line assumed a constant T'iz (2 hr) for all viruses. In addition, we have assumed an equal efficiency of infection for all the viruses in both IC and free virus plaque assays. There could be differences among the viruses, which could lead to variations in the relation of ICs to free virus production. As we have demonstrated, a number of our leukemia cell lines produce only small amounts of ecotropic S’L- plaque-forming virus. However, some of these same lines (T2, D2N, and BCl) release large amounts of reverse transcriptase (data not shown). Several alternatives could explain these findings: (i) production of xenotropic MuLV (7, 8); (ii) production of MCF “recombinant” viruses of Hartley et al. (9 ); (iii) production of defective [noninfectious or non-plaque-forming] MuLV (IO). We are presently testing growth of these viruses on SC-l cells and mink lung cells to examine some of these possibilities. ACKNOWLEDGMENTS This work was supported in part by NIH Grant No. CA 16315 and by an American Cancer Society Faculty Research Award to Kenneth Watson. The authors wish to thank Dr. T. Pincus for helpful discussions, and Mrs. Helen Blahnik for preparation of the manuscript. REFERENCES 1. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W., Virology ‘I2, 1136-1139 (19701. 2. BASSIN, R. H., TUTTLE, N., and FISCHINGER, P.
226
SHORT
COMMUNICATIONS
J., Nature (London) 229, 564-566 (1971). 3. CHESEBRO, B., WEHRLY, K., CHESEBRO, K., and PORTIS, J., J. Zmmunol. 117, 1267-1274 (1976). 4. PINCUS, T., HARTLEY, J. W., and ROWE, W. P., ViroZogy 65, 333-342 (1975). 5. O’DONNELL, P. F., DEITCH, C. J., and PINCUS, T., Virology 73, 23-35 (1976). 6. DURAN-TROISE, G., BASSIN, R. H., REIN, A., and GERWIN, B. I., Cell 10, 479-488 (1977).
7. LEVY, J. A., Science 182, 1X11-1153 (1973). 8. TODARO, G. J., ARNSTEIN, P., PARES, W. P., LENNETTE, E. H., and HUEBNER, R. J., Proc. Nat. Acad. Sci. USA 70, 859-862 (1973). 9. HARTLEY, J. W., WOLFORD, N. K., OLD, L. J., and ROWE, W. P., Proc. Nat. Acad. Sci. USA 74, 789-792 (1977). 10. HOPKINS, N., and JOLICOEUR, P., J. Viral. 16, 991-999 (1975).
Murine
84, 222-226 (19’78)
Leukemia
BRUCE
Virus Infectious Centers are Dependent of Virus Production by Infected Cells
CHESEBRO,
KATHY WEHRLY, KENNETH AND KEVIN CHESEBRO’
on the Rate
WATSON,
U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840, and Department of Chemistry, University of Montana, Missoula, Montana 59812 Accepted September 16,1977 A group of in vitro-maintained mouse leukemia cell lines has been used to analyze a murine leukemia virus S+L- infectious center assay. The rate of virus production by leukemia cell lines was determined by two methods and was compared to the observed incidence of infectious centers for each cell line in an effort to define the exact nature of an infectious center. The results show that detection of virus-producing cells in the infectious center assay is dependent on the rate of virus production by individual cells. Although 100% of high-virus-producer cells give positive infectious center plaques, only 0.01-l% of low virus producers are detectable. The results roughly fit the prediction that a virus producer cell is scored as an infectious center if it secretes 1 PFU of virus within 40 hr after seeding onto the target cell culture.
passage over a long period of time and should be relatively homogeneous cell populations. One of the low IC lines (D2N) was cloned to see if high- and low-virus-producing subpopulations could be isolated. Nine separate clones of the line D2N were test.ed. All clones had an incidence of infectious centers (IC/106 cells) similar to that of the uncloned population. No nonproducer clones were obtained. These observations appeared to exclude the idea that heterogeneity of virus production among the cells of the D2N line was responsible for the low percentage of cells giving infectious center plaques. Since cells of all clones released plaque-forming virus, all cells in the original population should have been capable of virus production. The low percentage of infectious centers observed in the cloned and uncloned lines appeared to have resulted from a general characteristic of virus production kinetics of all cells in the population. We have attempted to relate the observed value of IC/106 cells to the level of virus production of the entire culture. Our
Infectious center (10 assays have been valuable in many virus systems to evaluate the number of virus-infected cells in various experimental situations. The two commonly used plaque assays for murine leukemia viruses (MuLV), the XC assay and the S+L- assay, have been used to identify infectious centers by plating live infected cells instead of free virus in the infection step of the assay (1,2). We have attempted to define the exact nature of the infectious centers detected in the mouse S+L- plaque assay using a number of mouse leukemia cell lines. Several leukemia lines known to be virus producers had extremely low titers in the IC assay (2.3-3.8 log IC/106 cells -0.01 to 1.0% positive) (Table 1). This observation suggested that in these lines there might be a large proportion of cells which did not produce plaque-forming virus. This was a surprising finding since these leukemia lines have been maintained in laboratory ’ Present address: School of Medicine, University of California at Los Angeles, Los Angeles, California. 222 0042-6822/78/0841-0222$02.00/O Copyright All rights
0 1978 by Academic F’ress, Inc. of reproduction in any form reserved.
SHORT
TABLE CORRELATION
Cell line”
AA41 DlB (early)’ Al3 EL4 (G+) DlB (lateIfT2 BCl D2N EL4 (G-J
OF RATE
OF
VIRUS
PRODUCTION
Log IWO6 cell@
6.12 5.63 5.69 5.04 3.84 3.61 3.72 3.75 2.31
223
COMMUNICATIONS 1
WITH INCIDENCE CELL LINES
Log PFU/lOG cells’
r 0.13 k 0.28 i 0.21 2 0.30 + 0.57 c 0.32 +- 0.32 i 0.27 t- 0.41
6.22 5.52 4.71 4.20 3.44 2.55 1.79 1.67 0.84
OF INFECTIOUS
CENTERS
IN LEUKEMIA
Log PFLJ produced/lo6
cells/40 hr
Calculated by Calculated by cell washing equilibrium method’ methodd -~__ __-__ 6.42 7.21 5 0.47 5.62 6.51 r 0.23 5.51 5.70 t 0.32 nt” k 0.18 5.19 nt 4.43 -c 0.44 nt 3.54 f 0.87 2.78 nt ? 0.13 nt 2.66 + 0.37 nt 1.83 t 0.53 ____ previously (3). AA41 is a new FV-B-induced line
a Origin and maintenance of cell lines were described derived from a (BIO.A x A)F, leukemic mouse spleen. b Values observed in infectious center assay on D56 (mouse) S+L- cells (2). Various numbers of washed cells to be assayed were added directly to dishes containing 4 ml of medium. Exposure of cells to be tested to 2000 R of X-irradiation to prevent cell division, or to treatment with trypsin to inactivate exposed virus (41, did not affect the IC titer observed. c PFU/lOG cells in tissue culture supernatant = (PFU/ml of supernatant)/(cells/ml of suspension) x lo”, as assayed on D56 (mouse) S+L- cells (2,3). d Calculated from equation (3), assuming T% = 2 hr. ’ Calculated from virus concentration achieved 3 to 4 hr after washing cells and resuspending in fresh medium. ‘Early passages (up to 30th) of the DlB line produced more virus than late passages (later than 50th). Therefore, these sublines were considered separately in this report, g nt = not tested.
assumption was that each infectious center plaque observed might simply represent the release of a single viral PFU by a plated cell and the successful infection of a S+L- assay target cell by this PFU. Because of the critical timing involved in successful detection of PFU in this assay, this initial target cell infection event would probably have to occur within some restricted time period following plating of virus or cells in order to have time to produce a visible plaque on the fifth day, when plaques are counted (Eq. 1). The initial problem was to determine the rate of production of viral PFU for each cell line and to see how this figure compared with the observed IWO6 cells. In preliminary experiments, it was found that regardless of the cell density of a given culture the number of viral PFU/ml in the cell-free supernatant had a constant relationship to the cell concentration. Thus for each individual cell line (PFU/
mlY(cells/ml) or PFU/106 cells was a constant. Virus production by the cells and inactivation of cell-free virus appeared to be in equilibrium. Thus virus produced/hr equaled virus inactivatedlhr. By the delinition of half-life (Tf/2) the amount of virus inactivated in one half-life is equal to one-half the cell-free virus concentration (Eq. 2). Since the virus/cell ratio (PFU/106 cells) remained constant for each cell line, the effect of cell concentration could be neglected by using PFU/106 cells instead of PFU/ml as virus concentration (Eq. 3). (1) ICYlO6 cells = (PFU produced/lo6 cells/hr) (t) (E 1. E = efficiency of infection of target cell by virus produced by a plated cell in the IC assay; t = time (hr) after seeding virus-producing cells into IC assay during which target cell infection yields a countable plaque read on day 5. (2) PFU produced/hr = PFU inactivated/hr = l/2 PFU present/(Tl/z).
224
SHORT
COMMUNICATIONS
(3) PFU produced/lo6 cells/hr = PFU inactivated/lo6 cells/hr = l/z (PFU/106 cells)/(TVz) T1/2 = half-life of cell-free virus under tissue culture conditions. PFU/ lo6 cells = (cell-free PFU/ml)/(cells/ml) (106) = cell-free PFU present in tissue culture supernatant per lo6 cells present in the culture suspension. Combining equations (1) and (3) yields equations (4) and (5) which could be evaluated experimentally. (4) IC/106 cells = l/2 (PFU/106 cells) (t)
-A13 =.DlB(l.t.) bD2N q.E14(G+) nEL4(G-)
7 i
ec 1 ~.DlB(e.rly) @.A A41
(E)/(TVz)
(5) Log PFU/106 cells = log IC/106 cells + log [(TVz)/Vz (t) (El]. Thus when log PFU/106 cells is plotted against log ICYlO cells (Fig. l), equation (5) would predict a straight line with slope = 1 and x-intercept = log [l/2 (t) (E)I(T112)] if t, E, and TV2 are constant for all the cell lines. We have determined the T1/2 for the viruses produced by several of our cell lines, and all appear to be in the range of 1.5 to 2.5 hr. The time (t) as defined in equation (1) was estimated to be approximately 30 to 40 hr by seeding 20 to 40 cells (= 20 to 40 IC) of a high producer line in the S+L- dishes at various times after the usual infection time in this assay. Although a sharp cutoff point was difficult to determine precisely, good plaques were observed when cells to be assayed were seeded up to 30 to 40 hr after the usual time of seeding (data not shown). The data comparing IC/106 cells with PFU/106 cells for several cell lines are given in Table 1 and Fig. 1. The general trend of the data obtained from different cell lines supports the idea that the incidence of infectious centers observed is related to the rate of virus production by a cell line. For lines A13, DlB (early), and AA41 the rate of virus production was also determined in a different manner. Cell-free virus was washed out of an exponentially growing cell culture, and cells were recultured in fresh medium at the original concentration. Samples of culture supernatant were examined for the rate of reappearance of virus. Attainment of equilibrium status of virus production (i.e., constant PFU/106 cells) was achieved by 8 to 12 hr (data not shown). However, during
. 0
1
2
3 4 LOG IC/106CELLS
$
b
7
FIG. 1. Observed incidence of infectious centers (ICYlO cells) was plotted against the cell-free virus concentration (expressed as PFU/106 cells present in the suspension culture before centrifugation). The line drawn is the theoretical line expected from equation (4) (in text). The intercept would equal 1 ifE = 1, t = 40 hr, and T1/z = 2 hr; these appear to be the approximate values observed for t and T%.
the initial time prior to equilibrium, virus concentration increased at a very rapid rate; and the amount of virus inactivated was small, since the actual virus concentration was low.* Therefore the rate of virus production (PFU produced/lo6 cells/ hr) could be approximated by determining the virus concentration 3 to 4 hr after washing. The results are shown in Table 1. The rate of virus production determined 2 The rate of virus inactivation (duldt) is directly proportional to the cell-free virus concentration (VI. Thus dvldt = AV, where A = 0.693/T%. Following the washing of the cultured cells, V is a function of time (t) before equilibrium between virus production and inactivation is attained. Therefore, the amount of virus inactivated in the first 4 hr (V = AJb V dt) can be calculated by plotting virus concentration vs time and measuring the area under the curve from t = 0 to 4 hr. For example, with the Al3 cell line, virus inactivation during the initial 4 hr was 1.8 x lo3 PFU/lOB cells/4 hr. However, net virus production was 3.2 x 10’ PFU/108 cells/4 hr. Therefore, we have chosen for simplicity to neglect this small amount of total virus production which was lost due to inactivation in the first 4 hr.
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225
COMMUNICATIONS
by this method is in fair agreement with that calculated by the equilibrium data also shown in Table 1. Both methods appear to give values in the range of the observed values of lC/106 cells for each line tested. These studies indicate that infectious center assays for MuLV are strongly dependent on the rate of virus production by the cells being assayed. For a cell population producing large amounts of plaqueforming virus the IC assay appears to detect all infected cells. However, for populations producing lower amounts of virus only a small fraction of virus-producing cells are detected. These findings make interpretation of IC assay data from heterogeneous cell populations difficult. For example, one cannot distinguish a high incidence of low producers from a low incidence of high producers. Furthermore, in a mixed population a 1% incidence of high producers could give more lC/106 cells (i.e., 10”) than a 99% incidence of low producers, such as the EL4 (G-I or D2N lines (2 x lo’ and 5.6 x lo3 lC/106, respectively). This type of phenomenon has not been troublesome with IC assays in other virus systems. However, MuLV have several properties which might lead to these results. First, MuLV are released from producer cells by membrane budding in a continuous fashion; no lytic burst of large numbers of viruses occurs. Second, after being released from the cell MuLV are very labile (Tl/z = 2 hr). Third, the effciency of infection by cell-free virus is low. Therefore, rate of virus production might be a critical limiting factor in determining whether or not a virus-producing cell actually makes enough virus to score a plaque in the IC assay. These findings provide a possible explanation for the “refractoriness” and/or “lack of productive viral expression” noted by Pincus et al. (4) in Fv-l-resistant cells which were doubly hit by virus but did not score as virus producers. If the rate of initial virus production was low in Fv-l-resistant cells even when doubly infected, then some of these cells might not be scored as infectious centers. Average virus yield per infected cell has been shown to be a function
of multiplicity of infection (5). This finding, as well as the observation of DuranTroise et al. (6) that all Fv-l-resistant cells can be productively infected if the multiplicity of infection is high enough, support the interpretation that the apparent refractoriness seen in these systems might be related to the failure to score low virus producer cells in the IC assay. In our attempts to correlate lC/lO” cells with rates of virus production (Fig. 1, Table l), it is clear that a number of oversimplifications have been made which could account for the failure of some cell populations to fit the expected theoretical curve more precisely. The calculation of virus production based on equilibrium between virus production and inactivation for each cell line assumed a constant T'iz (2 hr) for all viruses. In addition, we have assumed an equal efficiency of infection for all the viruses in both IC and free virus plaque assays. There could be differences among the viruses, which could lead to variations in the relation of ICs to free virus production. As we have demonstrated, a number of our leukemia cell lines produce only small amounts of ecotropic S’L- plaque-forming virus. However, some of these same lines (T2, D2N, and BCl) release large amounts of reverse transcriptase (data not shown). Several alternatives could explain these findings: (i) production of xenotropic MuLV (7, 8); (ii) production of MCF “recombinant” viruses of Hartley et al. (9 ); (iii) production of defective [noninfectious or non-plaque-forming] MuLV (IO). We are presently testing growth of these viruses on SC-l cells and mink lung cells to examine some of these possibilities. ACKNOWLEDGMENTS This work was supported in part by NIH Grant No. CA 16315 and by an American Cancer Society Faculty Research Award to Kenneth Watson. The authors wish to thank Dr. T. Pincus for helpful discussions, and Mrs. Helen Blahnik for preparation of the manuscript. REFERENCES 1. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W., Virology ‘I2, 1136-1139 (19701. 2. BASSIN, R. H., TUTTLE, N., and FISCHINGER, P.
226
SHORT
COMMUNICATIONS
J., Nature (London) 229, 564-566 (1971). 3. CHESEBRO, B., WEHRLY, K., CHESEBRO, K., and PORTIS, J., J. Zmmunol. 117, 1267-1274 (1976). 4. PINCUS, T., HARTLEY, J. W., and ROWE, W. P., ViroZogy 65, 333-342 (1975). 5. O’DONNELL, P. F., DEITCH, C. J., and PINCUS, T., Virology 73, 23-35 (1976). 6. DURAN-TROISE, G., BASSIN, R. H., REIN, A., and GERWIN, B. I., Cell 10, 479-488 (1977).
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