Cell, Vol. 15, 597406,
October
1978, Copyright
0 1978 by MIT
The tdCE and hrCE Phenotypes: Host Range Mutants of Vesicular Stomatitis Virus in Which Polymerase Function Is Affected C. Ft. Pringle M.R.C. Virology Unit Institute of Virology Church Street Glasgow, Scotland Gil
5JR
Summary Conditional host range mutants of VSV New Jersey (designated fdCE mutants), which multiplied at 31” and 39% in BHK-21 cells but only at 31” C in chick embryo (CE) cells, were Isolated at a higher frequency than conventional fs mutants after 5fluorouracil mutagenesis. Three types of fdCE mutant could be distinguished by their degree of temperature-sensitivity in other avian cells. Nonconditional host range mutants (hrCE), which failed to multiply In CE cells at both 31” and 39”C, were isolated rarely. The h&E mutants also failed to produce plaques on MDBK cells. Temperature-shift experiments showed that the host restriction operated early in the viral growth cycle. Nevertheless, pseudotypes of Chandipura virus with envelopes supplied by tdCE or h&E mutants were not restricted in CE cells, indicating that restriction did not occur at the cell surface. This was consistent with the observation of the in vitro temperature-sensitivity of the virlon polymerase of two of the three types of tdCE mutant and the hypothesis that the host range phenotype was determined by host factors which Interacted with the virion polymerase (SzilBgyi and Pringle, 1975). Comparison of tdCE and h&E mutants in other cultured cells did not reveal any association of conditional temperature-sensitivity with species of origin, degree of transformation, time in culture, chronic infection with cytoplasmic (RS virus) or nuclear (ALV) RNA viruses, or morphological type. The tdCE mutants, however, tended to be temperature-sensitive in the embryonic ceils of some species. The differentiation of pluripotent murine embryonal carcinoma cells to embryoid bodies was accompanied by a decrease in restrictiveness. These results suggest that several host factors may interact with the VSV polymerase, and that the absence of these factors at certain stages of differentiation may have a protective effect. introduction The expression of the genome of negative-stranded RNA viruses is dependent upon the normal functioning of the RNA-dependent RNA polymerase of the virion. In the rhabdoviruses and paramyxovi-
ruses, subgenomic sequences which function as monocistronic messages are transcribed directly from a linear nonsegmented genome, whereas replication occurs by synthesis of a complete positivestranded template of the genome. The fine details and control of polymerase activity are unknown, but recently mutants of the rhabdovirus, vesicular stomatitis virus (VSV), have been isolated which indicate that host factor(s) may be involved (SzilBgyi and Pringle, 1975). The origin, prevalence and characteristics of these mutants, which have been designated temperature-dependent chick embryo @ICE) mutants, will be described in greater detail and compared with the properties of nonconditional host range (hr) mutants of VSV and conventional temperature-sensitive (ts) mutants. Three phenotypically distinct classes of conditional mutants of VSV have been described in which the in vitro activity of the virion polymerase is affected : -Thermolabile temperature-sensitive (ts) mutants in which polymerase activity was irreversibly inactivated at the restrictive temperature of 39°C and in which the ts phenotype could be attributed to the thermolability of the virion polymerase (SzilBgyi and Pringle, 1972). Dissociation and reconstitution experiments have shown that the L polypeptide was the site of the mutational lesion in the case of mutant ts G114 of complementation group I of the Indiana serotype of VSV (Hunt and Wagner, 1974). -Nonthermolabile ts mutants belonging to the same complementation group where in vitro polymerase activity was not diminished at 39°C. Presumably mutation affected the polymerase in such a way that it produced defective transcripts (SzilBgyi and Pringle, 1972). -A reversible inhibition of in vitro polymerase activity at 39°C was exhibited by some t&E mutants of VSV New Jersey (SzilBgyi and Pringle, 1975) [and also ts Bl of complementation group B (J. F. Szilagyi and C. R. Pringle, unpublished data)]. In the case of mutant t&E 3, the molecular site of the mutational lesion was also shown to be the L polypeptide by dissociation and reconstitution experiments (SzilBgyi, Pringle and Macpherson, 1977). The rdCE mutants are conditionally ts mutants which multiply normally in BHK-21 clone 13 cells at either 31” or 39X, but only at 31°C in chick embryo (CE) cells. This phenotype is expressed irrespective of the cells in which the virus has been grown, and therefore the host range component of the phenotype implies that a factor in the cell environment (absent in CE cells and in the in vitro reaction mixture) is involved in the functioning of the VSV polymerase, maintaining it in an active conformation at 39°C (SzilBgyi and Pringle, 1975).
Cell 598
The data presented here indicate that more than one host factor may be involved, since three types of tdCE mutant have been distinguished according to their ability to multiply in other avian cells. The t&E mutants showed a tendency to be ts in the embryonic cells of some species, and the differentiation of pluripotent murine embryonal carcinoma stem ccl Is (Martin, 1975) was accompanied by a decrease in their temperature-dependent, restrictiveness. Thus the hypothetical host factors which maintain the viral polymerase in a functional configuration at 39°C may not normally be expressed until the host cell has undergone a degree of differentiation.
Results Frequency of Isolation The t&E mutants were isolated from wild-type stocks of both the Indiana and New Jersey serotypes of VSV after mutagenization by growth in the presence of 50-200 kg/ml 5fluorouracil (5Fu). The mutagenized virus was plated on mixed cultures of BHK-21 and CE cells (in proportions 3:1, 1 :l and 1:3, respectively) in an attempt to obtain host-restricted mutants preferentially by cloning virus from small or hazy plaques. The virus was also plated on monocultures of BHK-21 and CE cells. The frequency and phenotype of the mutants isolated from each type of culture were similar, however, and the figures have been combined to provide the data presented in Tables 1 and 2. Clones isolated in this way were screened for plaque-forming ability on BHK-21 and CE cells at 31°C to detect host-restricted (hr) mutants. At the same time, the isolates were screened at 39°C to detect conventional ts mutants and to monitor the efficacy of mutagenization. The phenotypes detectable in this screening procedure are indicated in Table 1. Conventional ts mutants and tdCE mutants were isolated from VSV Indiana, and ts, tdCE and regular host range mutants restricted in CE cells at 31” and 39°C (hrCE) were isolated from VSV New Jersey. It is significant that no mutants conditionally (tdBHK) or totally (hrBHK) restricted on BHK21 cells were obtained from either mixed or monocultures of BHK-21 and CE cells. The t&E mutants were isolated frequently, but the hrCE mutants appeared rarely and only from the VSV New Jersey wild-type; tdCE mutants were more frequent than fs mutants in VSV New Jersey but not VSV Indiana. Table 2 illustrates that the yield of tdCE mutants increased with increasing concentrations of mutagen, suggesting that the t&E mutants arose by independent mutational events and that they were not all clonally related. Table 3 indicates the efficiency of plating (Log,,
pfu at 31”C-Log,, pfu at 39°C) of two hrCE and four t&E mutants on permissive (BHK-Pl), semi-permissive (BS-C-1) and restrictive (CE) cells. One of the mutants (t&E 3) was slightly ts on permissive cells, and the wild-type virus was slightly restricted on CE cells.
Discrimination
of Different fdCE Mutants
Only the VSV New Jersey mutants have been studied in detail because of their greater abundance and the existence of the hrCE phenotype. The individual t&E mutants failed to complement one another or conventional ts mutants of the RNAnegative groups, A, B, E and F (Pringle, Duncan and Stevenson, 1971). Weak but significant complementation was only observed with mutants representing the RNA-positive groups C and D in semipermissive cells (Table 4). Nevertheless, the tdCE mutants are not identical, as illustrated in Figure 1 where the efficiency of plating of three mutants in four different avian cells is compared. Mutant tdCE 5 was temperature-sensitive in CE cells at 39X, but not in the other three avian cells [quail embryo (QE), duck embryo (DE), turkey embryo (TE)], whereas tdCE 3 was fs in all three. Mutant t&E 2 was intermediate, being ts in CE and DE cells. The activity of the virion polymerase of mutants t&E 3 and t&E 5 was reversibly temperaturesensitive at 39°C in vitro, whereas mutant t&E 2 was indistinguishable from wild-type (Szilagyi and Pringle, 1975, and unpublished data).
Effect of Temperature
Shift on Yield
Figures 2 and 3 illustrate the growth of mutant tdCE 2 (in vitro polymerase-positive) and mutant tdCE 4 (in vitro polymerase-negative) in CE cells at 31” and 39%. Temperature-shift experiments showed that in both cases the temperature-dependent restriction operated early in the viral growth cycle. The shift-up curves in Figures 2 and 3 show that the block was partially circumvented as early as 2 hr after infection. In the same experiments, the virus yield was virtually constant following shift-down at hourly intervals up to 8 hr (data not shown), indicating that the virus was not appreciably inactivated at 39°C and that the cells remained susceptible. Similar results were obtained in duplicate experiments.
Host Restriction in Vivo Is Not Determined Viral Envelope
by the
Pseudotype particles containing the core of Chandipura virus (CHV), an unrelated rhabdovirus, and the envelope of mutants tdCE 2, tdCE 3, tdCE 5, h&E 1 and hrCE 2 were obtained by mixed infection of CE cells. After cell lysis, the fluid from these cultures was clarified by low speed centrifugation and mixed with anti-CHV serum. The CHV(VSV)
VSV Host 599
Table Plaque
Range
Mutants
1. Phenotype Forming
and Frequency
of Isolation
of Mutants
Ability”
Mutant Designation
VSV Indiana Number
VSV New Jersey Number
BHKi31”
BHKl39”
CE13l”
CE139”
ts+ ts
+
+
+
+
+
-
+
21
0.14
6
0.03
tdCE
+
+
+
7
0.05
17
0.09
tdBHK
+
-
+
+
0
5.0
-
11
tdCE
5
0.76
0.14
11
1.16
0.08
8
>6.0
-
12
0.32
0.13
12
0.82
0.12
11
0.18
13
ts+ (x) = The
mean (Log,,, pfu at 3l”C-Log,, (S.E.) = Standard error. (n) = Number of separate determinations. (N.P.) = No plaques at either temperature.
Table 4. Complementation Conventional ts Mutants Cells Complementation
Indices in Mixed Infections and fdCE 3 in Semi-Permissive
3
between ES-C-1
Index”
VSV New Jersey
tdCE
1.44
pfu at 39°C).
ts Mutants
ts Al
ts Bl
ts Cl
ts Dl
ts El
ts Fl
0.15
1 .o
11.7
7.2
0.28
0.01
B Complementation indices were calculated according to Pringle (1970). and the test was carried out as described in Experimental Procedures. The significant values are in italics. Figure 1. The Efficiency of Plating of Three fdCE Wild-Type Virus in Four Avian Cell Types at 39°C
Mutants
and
infection did not markedly affect the characteristic restrictiveness of any of these cells. These results suggest that the presumptive host factor(s) did not turn over rapidly. The hrCE phenotype showed some variability on CE monolayers prepared from different batches of eggs, but the tdCE mutants behaved consistently on monolayers from all batches of eggs, whether primary, secondary or high passage cultures.
Mutants tdCE 2, tdCE 3 and tdCE 5 and wild-type (ts+) virus were assayed on monolayers of hamster (CER), chick embryo (CE), Japanese quail embryo (QE). Pekin duck embryo (DE) and turkey embryo (TE) cells. Plaques were counted after 48 hr of incubation at 3l”and 39”C, and the mean values obtained from 5experiments were plotted as histograms. The data on the in vitro polymerase activity of these mutants are shown at the bottom of the figure, and are taken from Sziltigyi and Pringle (1975) and unpublished results.
Loss of Restriction upon Differentiation of Pluripotent Murine Embryonal Carcinoma Cells Mutant t&E 3 was restricted on both murine pluripotent embryonal carcinoma stem cells (PSA4TG12) and the mitomycin C-treated ST0 mouse embryo cells used to provide a feeder layer. If these stem cells were allowed to differentiate to embtyoid bodies (EB) in the absence of feeder cells, a decrease in restrictiveness was observed. Figure 5 shows that mutant hrCE 1 multiplied in both PSA4TG12 stem cell cultures and embryoid body cultures at 31” and 39°C. The yields were comparable to those obtained with wild-type virus.
It was observed consistently, however, that the yield of mutant t&E 3 from EB cultures at 39% (0- - -0) was approximately 100 fold greater than that from stem cell cultures (O- - -0). Examination of cell lines derived from embryoid bodies (EB lines) showed that the decrease in restrictiveness could be attributed to the appearance of more permissive cell types as a result of differentiation. Figure 6 shows that wild-type virus exhibited the same efficiency of plating (that is, LogI,, pfu at 31”CLog,,, pfu at 39°C) in PSA4TG12 stem cells, ST0 feeder cells, embryoid body cultures and all 8 embryoid body-derived (EB) lines. The tempera-
VSV Host 601
Range
Mutants
3-3-- 4 .2 :
‘8
3s”
-
. 20 24 30 12 “OURS lFTER AOSORPTION
Figure 2. The Growth of Mutant f&E 2 in CE Cells at 31” and 39°C. and the Yield Obtained from fdCE P-Infected Cells after Shift-Up from 31” to 39°C at Various Times up to 30 hr PostInfection Monolayers of CE cells were infected as described and replicate cultures were harvested by rapid freeze-thawing at the times indicated. Total infectivity was assayed by titration on BS-C-1 cells and the mean values were plotted. The 31” + 39% shift-up curve was obtained by transfer of pairs of cultures from 31”-39°C at the times indicated, and incubation was continued up to 36 hr post-infection before freeze-thawing. Growth at 31°C (O---O); growth at 39°C (o--O); 31” + 39°C shift-up (W - -m).
ture-sensitivity of mutant tdCE 3, however, was reduced markedly in embryoid body cultures and in 3 (EB 28110, EB 22120, EB 37110) of the 8 embryoid body-derived lines. In one instance, 5 clones from one of these EB-derived cultures (EB 28/10) were available, and each individual clone displayed the same degree of restrictiveness as the parental culture (data not shown). 3 of the remaining 5 EB lines were similar to the PSA4TG12 stem cell parent in their restrictiveness for mutant tdCE 3, and 2 (EB 37/16, EB 37/17) were intermediate. These results indicate that the presumptive host factors were not expressed until the embryonal carcinoma cells had undergone a degree of differentiation. The situation was complicated by the observation that cultures of embryoid bodies, unlike the parental stem cells, survived infection even by wild-type virus, and persistent infection ensued. The relationship of this phenomenon to differentiation is under investigation.
Discussion VSV multiplies well in almost all types of cultured animal cells. Nonetheless, a number of host range effects have been described, and host range mu-
Figure 3. The Growth of Mutant 39°C and the Yield Obtained after Times up to 30 hr Post-Infection Legend (0-O);
as Figure 2. Growth 31” -f 39°C shift-up
tdCE 3 in CE Cells 31°C --t 39°C Shift-Up
at 31°C (O--O); (W- - -m).
growth
at 31” and at Various at 39°C
tants are a source of cryptic variation which remains unrecognized in the absence of appropriate discriminatory cells. It is well established that VSV (and other viruses) do not multiply in primary lymphocytes unless activated by antigens or mitogens. Nowakowskilet al. (1973), however, reported that VSV Indiana was restricted in Raji cells, but not in Wil-2 cells, another similar human lymphoblastoid cell line. Thacore and Youngner (1975) reported that VSV Indiana was restricted in rabbit cornea1 cells but not in rabbit kidney cells, and recently Levinson et al. (1978) have described a stock of VSV Indiana which was restricted in duck embryo cells but not in chick, quail or pheasant embryo cells. Finally, Simpson and Obijeski (1974) have described induced mutants of VSV Indiana which were simultaneously temperature-sensitive in CE cells and restricted in human cells, although they concluded that the ts and host range phenotypes were determined by independent mutations. The t&E and hrCE mutants described here are also distinctive host range phenotypes. The tdCE mutants have been isolated from stocks of VSV Indiana and VSV New Jersey, and also from Chandipura virus (D. A. Gadkari and C. R. Pringle, unpublished data). The results presented in Figure 1 show that at least three distinct types of t&E mutant have been isolated, and consequently that the prevalence of these mutants is not entirely due to reisolation of sister mutants. Temperature-sensitive host range mutants of herpes simplex type 2 have also been isolated (Koment and Rapp, 1975;
Cell
602
Table
5. Lack of Restriction
of CHV(VSV)
Pseudotypes
on CE Cells at 39°C B pfu on CE Cells at 39°C (x 10-q by:
Experiment
Virus lnoculum
1
2
tdCE 2
Plaque Type -
(CO.001)
(
October
1978, Copyright
0 1978 by MIT
The tdCE and hrCE Phenotypes: Host Range Mutants of Vesicular Stomatitis Virus in Which Polymerase Function Is Affected C. Ft. Pringle M.R.C. Virology Unit Institute of Virology Church Street Glasgow, Scotland Gil
5JR
Summary Conditional host range mutants of VSV New Jersey (designated fdCE mutants), which multiplied at 31” and 39% in BHK-21 cells but only at 31” C in chick embryo (CE) cells, were Isolated at a higher frequency than conventional fs mutants after 5fluorouracil mutagenesis. Three types of fdCE mutant could be distinguished by their degree of temperature-sensitivity in other avian cells. Nonconditional host range mutants (hrCE), which failed to multiply In CE cells at both 31” and 39”C, were isolated rarely. The h&E mutants also failed to produce plaques on MDBK cells. Temperature-shift experiments showed that the host restriction operated early in the viral growth cycle. Nevertheless, pseudotypes of Chandipura virus with envelopes supplied by tdCE or h&E mutants were not restricted in CE cells, indicating that restriction did not occur at the cell surface. This was consistent with the observation of the in vitro temperature-sensitivity of the virlon polymerase of two of the three types of tdCE mutant and the hypothesis that the host range phenotype was determined by host factors which Interacted with the virion polymerase (SzilBgyi and Pringle, 1975). Comparison of tdCE and h&E mutants in other cultured cells did not reveal any association of conditional temperature-sensitivity with species of origin, degree of transformation, time in culture, chronic infection with cytoplasmic (RS virus) or nuclear (ALV) RNA viruses, or morphological type. The tdCE mutants, however, tended to be temperature-sensitive in the embryonic ceils of some species. The differentiation of pluripotent murine embryonal carcinoma cells to embryoid bodies was accompanied by a decrease in restrictiveness. These results suggest that several host factors may interact with the VSV polymerase, and that the absence of these factors at certain stages of differentiation may have a protective effect. introduction The expression of the genome of negative-stranded RNA viruses is dependent upon the normal functioning of the RNA-dependent RNA polymerase of the virion. In the rhabdoviruses and paramyxovi-
ruses, subgenomic sequences which function as monocistronic messages are transcribed directly from a linear nonsegmented genome, whereas replication occurs by synthesis of a complete positivestranded template of the genome. The fine details and control of polymerase activity are unknown, but recently mutants of the rhabdovirus, vesicular stomatitis virus (VSV), have been isolated which indicate that host factor(s) may be involved (SzilBgyi and Pringle, 1975). The origin, prevalence and characteristics of these mutants, which have been designated temperature-dependent chick embryo @ICE) mutants, will be described in greater detail and compared with the properties of nonconditional host range (hr) mutants of VSV and conventional temperature-sensitive (ts) mutants. Three phenotypically distinct classes of conditional mutants of VSV have been described in which the in vitro activity of the virion polymerase is affected : -Thermolabile temperature-sensitive (ts) mutants in which polymerase activity was irreversibly inactivated at the restrictive temperature of 39°C and in which the ts phenotype could be attributed to the thermolability of the virion polymerase (SzilBgyi and Pringle, 1972). Dissociation and reconstitution experiments have shown that the L polypeptide was the site of the mutational lesion in the case of mutant ts G114 of complementation group I of the Indiana serotype of VSV (Hunt and Wagner, 1974). -Nonthermolabile ts mutants belonging to the same complementation group where in vitro polymerase activity was not diminished at 39°C. Presumably mutation affected the polymerase in such a way that it produced defective transcripts (SzilBgyi and Pringle, 1972). -A reversible inhibition of in vitro polymerase activity at 39°C was exhibited by some t&E mutants of VSV New Jersey (SzilBgyi and Pringle, 1975) [and also ts Bl of complementation group B (J. F. Szilagyi and C. R. Pringle, unpublished data)]. In the case of mutant t&E 3, the molecular site of the mutational lesion was also shown to be the L polypeptide by dissociation and reconstitution experiments (SzilBgyi, Pringle and Macpherson, 1977). The rdCE mutants are conditionally ts mutants which multiply normally in BHK-21 clone 13 cells at either 31” or 39X, but only at 31°C in chick embryo (CE) cells. This phenotype is expressed irrespective of the cells in which the virus has been grown, and therefore the host range component of the phenotype implies that a factor in the cell environment (absent in CE cells and in the in vitro reaction mixture) is involved in the functioning of the VSV polymerase, maintaining it in an active conformation at 39°C (SzilBgyi and Pringle, 1975).
Cell 598
The data presented here indicate that more than one host factor may be involved, since three types of tdCE mutant have been distinguished according to their ability to multiply in other avian cells. The t&E mutants showed a tendency to be ts in the embryonic cells of some species, and the differentiation of pluripotent murine embryonal carcinoma stem ccl Is (Martin, 1975) was accompanied by a decrease in their temperature-dependent, restrictiveness. Thus the hypothetical host factors which maintain the viral polymerase in a functional configuration at 39°C may not normally be expressed until the host cell has undergone a degree of differentiation.
Results Frequency of Isolation The t&E mutants were isolated from wild-type stocks of both the Indiana and New Jersey serotypes of VSV after mutagenization by growth in the presence of 50-200 kg/ml 5fluorouracil (5Fu). The mutagenized virus was plated on mixed cultures of BHK-21 and CE cells (in proportions 3:1, 1 :l and 1:3, respectively) in an attempt to obtain host-restricted mutants preferentially by cloning virus from small or hazy plaques. The virus was also plated on monocultures of BHK-21 and CE cells. The frequency and phenotype of the mutants isolated from each type of culture were similar, however, and the figures have been combined to provide the data presented in Tables 1 and 2. Clones isolated in this way were screened for plaque-forming ability on BHK-21 and CE cells at 31°C to detect host-restricted (hr) mutants. At the same time, the isolates were screened at 39°C to detect conventional ts mutants and to monitor the efficacy of mutagenization. The phenotypes detectable in this screening procedure are indicated in Table 1. Conventional ts mutants and tdCE mutants were isolated from VSV Indiana, and ts, tdCE and regular host range mutants restricted in CE cells at 31” and 39°C (hrCE) were isolated from VSV New Jersey. It is significant that no mutants conditionally (tdBHK) or totally (hrBHK) restricted on BHK21 cells were obtained from either mixed or monocultures of BHK-21 and CE cells. The t&E mutants were isolated frequently, but the hrCE mutants appeared rarely and only from the VSV New Jersey wild-type; tdCE mutants were more frequent than fs mutants in VSV New Jersey but not VSV Indiana. Table 2 illustrates that the yield of tdCE mutants increased with increasing concentrations of mutagen, suggesting that the t&E mutants arose by independent mutational events and that they were not all clonally related. Table 3 indicates the efficiency of plating (Log,,
pfu at 31”C-Log,, pfu at 39°C) of two hrCE and four t&E mutants on permissive (BHK-Pl), semi-permissive (BS-C-1) and restrictive (CE) cells. One of the mutants (t&E 3) was slightly ts on permissive cells, and the wild-type virus was slightly restricted on CE cells.
Discrimination
of Different fdCE Mutants
Only the VSV New Jersey mutants have been studied in detail because of their greater abundance and the existence of the hrCE phenotype. The individual t&E mutants failed to complement one another or conventional ts mutants of the RNAnegative groups, A, B, E and F (Pringle, Duncan and Stevenson, 1971). Weak but significant complementation was only observed with mutants representing the RNA-positive groups C and D in semipermissive cells (Table 4). Nevertheless, the tdCE mutants are not identical, as illustrated in Figure 1 where the efficiency of plating of three mutants in four different avian cells is compared. Mutant tdCE 5 was temperature-sensitive in CE cells at 39X, but not in the other three avian cells [quail embryo (QE), duck embryo (DE), turkey embryo (TE)], whereas tdCE 3 was fs in all three. Mutant t&E 2 was intermediate, being ts in CE and DE cells. The activity of the virion polymerase of mutants t&E 3 and t&E 5 was reversibly temperaturesensitive at 39°C in vitro, whereas mutant t&E 2 was indistinguishable from wild-type (Szilagyi and Pringle, 1975, and unpublished data).
Effect of Temperature
Shift on Yield
Figures 2 and 3 illustrate the growth of mutant tdCE 2 (in vitro polymerase-positive) and mutant tdCE 4 (in vitro polymerase-negative) in CE cells at 31” and 39%. Temperature-shift experiments showed that in both cases the temperature-dependent restriction operated early in the viral growth cycle. The shift-up curves in Figures 2 and 3 show that the block was partially circumvented as early as 2 hr after infection. In the same experiments, the virus yield was virtually constant following shift-down at hourly intervals up to 8 hr (data not shown), indicating that the virus was not appreciably inactivated at 39°C and that the cells remained susceptible. Similar results were obtained in duplicate experiments.
Host Restriction in Vivo Is Not Determined Viral Envelope
by the
Pseudotype particles containing the core of Chandipura virus (CHV), an unrelated rhabdovirus, and the envelope of mutants tdCE 2, tdCE 3, tdCE 5, h&E 1 and hrCE 2 were obtained by mixed infection of CE cells. After cell lysis, the fluid from these cultures was clarified by low speed centrifugation and mixed with anti-CHV serum. The CHV(VSV)
VSV Host 599
Table Plaque
Range
Mutants
1. Phenotype Forming
and Frequency
of Isolation
of Mutants
Ability”
Mutant Designation
VSV Indiana Number
VSV New Jersey Number
BHKi31”
BHKl39”
CE13l”
CE139”
ts+ ts
+
+
+
+
+
-
+
21
0.14
6
0.03
tdCE
+
+
+
7
0.05
17
0.09
tdBHK
+
-
+
+
0
5.0
-
11
tdCE
5
0.76
0.14
11
1.16
0.08
8
>6.0
-
12
0.32
0.13
12
0.82
0.12
11
0.18
13
ts+ (x) = The
mean (Log,,, pfu at 3l”C-Log,, (S.E.) = Standard error. (n) = Number of separate determinations. (N.P.) = No plaques at either temperature.
Table 4. Complementation Conventional ts Mutants Cells Complementation
Indices in Mixed Infections and fdCE 3 in Semi-Permissive
3
between ES-C-1
Index”
VSV New Jersey
tdCE
1.44
pfu at 39°C).
ts Mutants
ts Al
ts Bl
ts Cl
ts Dl
ts El
ts Fl
0.15
1 .o
11.7
7.2
0.28
0.01
B Complementation indices were calculated according to Pringle (1970). and the test was carried out as described in Experimental Procedures. The significant values are in italics. Figure 1. The Efficiency of Plating of Three fdCE Wild-Type Virus in Four Avian Cell Types at 39°C
Mutants
and
infection did not markedly affect the characteristic restrictiveness of any of these cells. These results suggest that the presumptive host factor(s) did not turn over rapidly. The hrCE phenotype showed some variability on CE monolayers prepared from different batches of eggs, but the tdCE mutants behaved consistently on monolayers from all batches of eggs, whether primary, secondary or high passage cultures.
Mutants tdCE 2, tdCE 3 and tdCE 5 and wild-type (ts+) virus were assayed on monolayers of hamster (CER), chick embryo (CE), Japanese quail embryo (QE). Pekin duck embryo (DE) and turkey embryo (TE) cells. Plaques were counted after 48 hr of incubation at 3l”and 39”C, and the mean values obtained from 5experiments were plotted as histograms. The data on the in vitro polymerase activity of these mutants are shown at the bottom of the figure, and are taken from Sziltigyi and Pringle (1975) and unpublished results.
Loss of Restriction upon Differentiation of Pluripotent Murine Embryonal Carcinoma Cells Mutant t&E 3 was restricted on both murine pluripotent embryonal carcinoma stem cells (PSA4TG12) and the mitomycin C-treated ST0 mouse embryo cells used to provide a feeder layer. If these stem cells were allowed to differentiate to embtyoid bodies (EB) in the absence of feeder cells, a decrease in restrictiveness was observed. Figure 5 shows that mutant hrCE 1 multiplied in both PSA4TG12 stem cell cultures and embryoid body cultures at 31” and 39°C. The yields were comparable to those obtained with wild-type virus.
It was observed consistently, however, that the yield of mutant t&E 3 from EB cultures at 39% (0- - -0) was approximately 100 fold greater than that from stem cell cultures (O- - -0). Examination of cell lines derived from embryoid bodies (EB lines) showed that the decrease in restrictiveness could be attributed to the appearance of more permissive cell types as a result of differentiation. Figure 6 shows that wild-type virus exhibited the same efficiency of plating (that is, LogI,, pfu at 31”CLog,,, pfu at 39°C) in PSA4TG12 stem cells, ST0 feeder cells, embryoid body cultures and all 8 embryoid body-derived (EB) lines. The tempera-
VSV Host 601
Range
Mutants
3-3-- 4 .2 :
‘8
3s”
-
. 20 24 30 12 “OURS lFTER AOSORPTION
Figure 2. The Growth of Mutant f&E 2 in CE Cells at 31” and 39°C. and the Yield Obtained from fdCE P-Infected Cells after Shift-Up from 31” to 39°C at Various Times up to 30 hr PostInfection Monolayers of CE cells were infected as described and replicate cultures were harvested by rapid freeze-thawing at the times indicated. Total infectivity was assayed by titration on BS-C-1 cells and the mean values were plotted. The 31” + 39% shift-up curve was obtained by transfer of pairs of cultures from 31”-39°C at the times indicated, and incubation was continued up to 36 hr post-infection before freeze-thawing. Growth at 31°C (O---O); growth at 39°C (o--O); 31” + 39°C shift-up (W - -m).
ture-sensitivity of mutant tdCE 3, however, was reduced markedly in embryoid body cultures and in 3 (EB 28110, EB 22120, EB 37110) of the 8 embryoid body-derived lines. In one instance, 5 clones from one of these EB-derived cultures (EB 28/10) were available, and each individual clone displayed the same degree of restrictiveness as the parental culture (data not shown). 3 of the remaining 5 EB lines were similar to the PSA4TG12 stem cell parent in their restrictiveness for mutant tdCE 3, and 2 (EB 37/16, EB 37/17) were intermediate. These results indicate that the presumptive host factors were not expressed until the embryonal carcinoma cells had undergone a degree of differentiation. The situation was complicated by the observation that cultures of embryoid bodies, unlike the parental stem cells, survived infection even by wild-type virus, and persistent infection ensued. The relationship of this phenomenon to differentiation is under investigation.
Discussion VSV multiplies well in almost all types of cultured animal cells. Nonetheless, a number of host range effects have been described, and host range mu-
Figure 3. The Growth of Mutant 39°C and the Yield Obtained after Times up to 30 hr Post-Infection Legend (0-O);
as Figure 2. Growth 31” -f 39°C shift-up
tdCE 3 in CE Cells 31°C --t 39°C Shift-Up
at 31°C (O--O); (W- - -m).
growth
at 31” and at Various at 39°C
tants are a source of cryptic variation which remains unrecognized in the absence of appropriate discriminatory cells. It is well established that VSV (and other viruses) do not multiply in primary lymphocytes unless activated by antigens or mitogens. Nowakowskilet al. (1973), however, reported that VSV Indiana was restricted in Raji cells, but not in Wil-2 cells, another similar human lymphoblastoid cell line. Thacore and Youngner (1975) reported that VSV Indiana was restricted in rabbit cornea1 cells but not in rabbit kidney cells, and recently Levinson et al. (1978) have described a stock of VSV Indiana which was restricted in duck embryo cells but not in chick, quail or pheasant embryo cells. Finally, Simpson and Obijeski (1974) have described induced mutants of VSV Indiana which were simultaneously temperature-sensitive in CE cells and restricted in human cells, although they concluded that the ts and host range phenotypes were determined by independent mutations. The t&E and hrCE mutants described here are also distinctive host range phenotypes. The tdCE mutants have been isolated from stocks of VSV Indiana and VSV New Jersey, and also from Chandipura virus (D. A. Gadkari and C. R. Pringle, unpublished data). The results presented in Figure 1 show that at least three distinct types of t&E mutant have been isolated, and consequently that the prevalence of these mutants is not entirely due to reisolation of sister mutants. Temperature-sensitive host range mutants of herpes simplex type 2 have also been isolated (Koment and Rapp, 1975;
Cell
602
Table
5. Lack of Restriction
of CHV(VSV)
Pseudotypes
on CE Cells at 39°C B pfu on CE Cells at 39°C (x 10-q by:
Experiment
Virus lnoculum
1
2
tdCE 2
Plaque Type -
(CO.001)
(
