Mutual Exclusion Occurs in a Chlorella

J. gen. Virol. (1989), 70, 1829-1836. Printed in Great Britain
1829
Key words: mutual exclusion/Chlorella viruses/endonucleases
Mutual Exclusion Occurs in a Chlorella-like Green Alga Inoculated with
Two Viruses
By T H O M A S E. C H A S E , ' ~ J E N N I F E R
A. NELSON,
D W I G H T E. B U R B A N K
AND J A M E S L. V A N E T T E N *
Department o f Plant Pathology, University o f Nebraska, Lincoln, Nebraska 68583-0722, U.S.A.
(Accepted 22 March 1989)
SUMMARY
Progeny viruses resulting from dual inoculations with different and near-isogenic
viruses of a Chlorella-like green alga were distinguished by immunoblotting. Plaques
arising from single cells inoculated with two viruses usually contained only one of the
viruses. Thus the viruses mutually exclude one another. In some combinations the ratio
of viruses (as infective centres) recovered differed significantly from the input ratio.
INTRODUCTION
Thirty large (150 to 190 nm in diameter), polyhedral, dsDNA-containing, plaque-forming
viruses which replicate in a unicellular, eukaryotic Chlorella-like green alga have been partially
characterized (for review see Van Etten et al., 1986a, 1987, 1988). These viruses can be
distinguished by differences in plaque size, antigenic specificity, D N A restriction fragment
patterns and the nature and abundance of methylated bases in their genomic D N A s (Schuster et
al., 1986). Each of the virus D N A s contains 5-methylcytosine (5mC); the concentration of 5mC
varies from 0.1 ~ to 47"5~o of the total cytosine residues. In addition, 18 of the 30 virus D N A s
also contain N6-methyladenine (6mA); the concentration of 6 m A varies from 1.45 ~o to 37 ~ of
the'total adenine residues. At least some of these viruses code for D N A methyltransferases and
D N A site-specific (restriction) endonucleases (Xia & Van Etten, 1986; Xia et al., 1986a, b,
1987a, b, 1988; N a r v a et al., 1987).
This report describes dual inoculations of Chlorella N C 6 4 A with genetically distinct viruses as
well as nearly isogenic viruses. These experiments were conducted for two reasons: first, to
determine whether the algal host can support simultaneous replication of two different viruses
and secondly, to determine whether one function of the site-specific endonucleases is to exclude
other viruses. The results indicate that usually only one virus replicates in an individual cell
inoculated with two distinct viruses, i.e. the viruses exhibit mutual exclusion. However, the sitespecific endonucleases are probably not solely, if at all, responsible for exclusion.
METHODS
Culture conditions. The growth of the host Chlorella strain NC64A on MBBM medium, the production of the
viruses (listed in Table 1), and the procedures used to measure virus adsorption and virus growth have been
described (Van Etten et al., 1983a, b; Schuster et al., 1986).
In the double infection experiments 10 ml of actively growing Chlorella cells (1.6 x 107 to 1-8 × 107 cells/ml)
were inoculated simultaneously with two viruses at an m.o.i, of 10 to 35 (values for specific experiments are listed
in the tables) and incubated for 1 h. Cells were separated from unattached virus by centrifugation (3000 g for
5 min), washed several times in MBBM, resuspended in MBBM at their original concentration and then titrated
for infective centres as previously described (Van Etten et al., 1983a, b). At the m.o.i, used at least 98 ~ of the cells
should be infected with both viruses within 30 rain assuming infection follows a Poisson distribution; when cells
w e r e inoculated at this m.o.i, with each virus alone essentially all the cells were infected.
t Present address: Pacific Southwest Forest and Range Experiment Station, 1960 Addison Street, P.O. Box 245,
Berkeley, California 94701, U.S.A.
0000-8896 © 1989 SGM
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T. E. CHASE AND OTHERS
T a b l e 1. Properties o f the C h t o r e l l a N C 6 4 A viruses used in this study
Virus
Class*
Replication
time (h)t
NE-8D
NYb-I
CA-4B
NY-2C
NC- 1D
PBCV- 1
EPA1
IL-3A
SC- 1B
NC- 1A
NC- 1B
NY-2B
1
1
1
2
2
3
3
4
5
6
7
9
4-9
4-9
4-9
4-9
4-9
4-9
4-9
44
8-15
4-9
4-9
9-18
Reacts with
antiserum to
Site-specific
r
~------------~ endonuclease
PBCV-1
NY-2C
produced
Yes
Yes
Yes
No
No
Yes
No
Yes
Yes
Yes
Yes
No
No
No
No
Yes
Yes
No
No
No
No
No
No
Yes
Sensitivity of virus
DNA to site-specific
endonuclease
r
*
CviAI
CviBI
CviJI
CviAI
CviAI
CviJI
CviBI
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
No
* The criteria used to classify the viruses are in Schuster et al. (1986).
t The first number is when progeny viruses are first released and the second number when release is complete.
Chlorella cells were also inoculated with one virus before inoculation with a second virus. In these experiments
the first virus was incubated with the cells for 30 min, unattached virus was removed, and then the second virus
was added. After removal of the unattached second virus 30 rain later, cells were washed and titrated for infective
centres as described above.
The effect on burst size of inoculating the host with two viruses was determined by allowing the viruses (m.o.i. of
20 for cells inoculated with one virus or 10 each for cells inoculated with two viruses) to adsorb to the host
(1.7 × 107 cells/ml) for 1 h. The inoculated cells were pelleted by centrifugation, suspended in 10 ml of MBBM,
incubated for 2 days and titrated. The burst size was calculated by dividing the total p.f.u, by the number of cells.
Irnmunoblotting. Viruses in the infective centres (plaques) were identified using the Promega immunoblotting
system. Duplicate plaque lifts on nitrocellulose filters were made after incubating the titration plates for 3 to 4 days
and then chilling the plates at 4 °C for several hours. The filters were incubated for 30 min in TBST buffer (1%
bovine serum albumin, 10 mM-Tris-HC1, 150 mM-NaCI, 0.05 % Tween 20, pH 8.0) to reduce non-specific binding
to the filters. Individual filters were then incubated for 30 min with primary antiserum, usually raised against virus
PBCV-I or NY-2C, at a dilution of 1:750 in TBST buffer. The filters were rinsed in TBST buffer (three times,
10 rain each) and then incubated in the secondary antiserum (goat anti-rabbit IgG-alkaline phosphatase
conjugate, diluted 1:7500 in TBST buffer) for 30 min. Filters were rinsed in TBST buffer (three times, 10 rain
each) and incubated in a reaction mixture containing 40 mM-nitro blue tetrazolium, 40 mM-5-bromo-4-chloro-3indolyl phosphate in alkaline phosphatase buffer (100 mM-Tris-HCl pH 9-5, 100 mM-NaCI, 5 mM-MgC12). Filters
were incubated until the plaques were clearly visible (purple colour) and the reaction was terminated by placing
the filters in stop buffer (20 mM-Tris-HCl pH 8-0, 5 mM-EDTA). One-hundred to 400 plaques were counted in a
typical experiment.
Analysis of individual infective centres. Plaques arising from infective centres were removed with sterile
toothpicks and transferred to 200 ~tl of 50 mM-Tris-HC1 pH 7.5. A portion of this virus suspension was titrated,
plaques were lifted and virus composition was determined by immunoblotting. Fifty ml of actively growing
Chlorella NC64A cells was also inoculated with a second portion of the virus suspension. After overnight
incubation, virus was pelleted from the lysate by centrifugation (Van Etten et al., 1983b) and suspended in 1 ml of
50 mM-Tris-HCl pH 7.5, 10 mM-MgC12 containing 55 p.g DNase I. After incubation at 22 °C for I h to digest
contaminating host DNA, 18 ~tl of 0.5 M-EDTA was added. The virus was disrupted by incubating at 60 °C for 1 h
with 350 ~tg of autodigested proteinase K (37 °C, 1 h) and 90 pl of sodium N-lauroylsarcosine. Protein was
removed by several extractions with phenol and chloroform : isoamyl alcohol (25 : 1 v/v). The aqueous phase was
adjusted to 0.2 M-sodium acetate and DNA was precipitated with ethanol. The DNA was washed several times
with 70~ ethanol, suspended in 10mM-Tris-HC1, 1 mM-EDTA, pH8, and treated with BamHI or EcoRI
restriction endonucleases as recommended by the suppliers.
Other procedures. Attachment rates of the viruses were determined as described previously (Van Etten et al.,
1983 b). Spontaneous serotype mutants of PBCV- 1 were isolated by treating I x 109 p.f.u, of PBCV- 1 wth PBCV- 1
antiserum (1:100 dilution) for 30 min, removing agglutinated virus by centrifugation (3000 g for 5 rain) and then
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Mutual exclusion of Chlorella viruses
1831
Fig. 1. Immunoblotting of duplicate plaque lifts of a titration plate containing a mixture of PBCV-1
and NY-2C viruses. The lift on the left was treated with PBCV-1 antiserum and the one on the right
with NY-2C antiserum. Plaques that produced a dark colour with PBCV-1 antiserum had a light colour
with NY-2C antiserum and vice versa.
plating the supernatant fraction. Under these conditions about 1 x 103 plaques were recovered; viruses purified
from these plaques were insensitive to PBCV-1 antiserum. Polyclonal antisera to PBCV-1, NY-2C and EPA 1 were
raised against purified viruses as described previously (Van Etten et al., 1982).
Most experiments were repeated two or more times and representative results are reported.
RESULTS
Immunoblotting procedure
Immunoblots of duplicate plaque lifts from a titration plate containing a mixture of PBCV-1
and NY-2C viruses are shown in Fig. 1. The blot pictured on the left was reacted with PBCV-1
antiserum and the one on the right with NY-2C antiserum. All plaques were detected on both
filters but plaques that produced a dark colour with PBCV-1 antiserum had a lighter colour with
NY-2C antiserum and vice versa. To confirm plaque identity, virus D N A was isolated from 20
plaques identified either as PBCV-1 or NY-2C and digested with BamHI. In every instance the
D N A restriction pattern confirmed the identity. Thus the immunoblotting technqiue clearly
distinguished the two viruses.
Progeny from cells inoculated with PBCV-1 and NY-2C
Viruses PBCV-1 and NY-2C were chosen for the first dual inoculation experiment because
they adsorb to and replicate at similar rates in Chlorella NC64A. Nearly every infective centre
plaque resulting after inoculation with PBCV-1 and NY-2C (m.o.i. of 12 for each virus) was
unambiguously identified as either PBCV-1 (121 plaques) or NY-2C (70 plaques) by
immunoblotting. Two of the 193 infective centres examined a p p e a r e d to contain a mixture of
the two viruses because they were intermediate in colour intensity with both antisera.
To check the accuracy of the initial scoring of these plaques, the virus compositions of 36
infective centres (35 plaques identified as either PBCV-1 or NY-2C and one mixed plaque) were
analysed further by titrating and immunoblotting and by examining D N A restriction patterns.
Thirty-two of 36 plaques were identified correctly in the initial screening (Table 2). Four of the
35 infective centres initially classified as containing only one virus contained small amounts (1 to
1 2 ~ ) of the other virus. The plaque identified as ' m i x e d ' contained both NY-2C ( 2 3 ~ ) and
PBCV-1 (77 ~). Thus the immunoblotting procedure correctly identified the virus composition
of the plaques 89 9/ooof the time.
W e conclude that a cell inoculated with both PBCV-1 and NY-2C usually produces progeny of
one but not both viruses, i.e. most of the time the viruses mutually exclude one another.
Furthermore, PBCV-1 has a slightly better chance of replicating in a given cell than NY-2C if
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T. E. C H A S E A N D O T H E R S
T a b l e 2. Scoring of plaques, titrated single plaque isolates and DNA restriction patterns from
infective centres resulting from a dual inoculation with PBCV-1 and NY-2C*
Scoring of
plaque
Viruses in the plaques
c
A
•
PBCV-1 (~)
NY-2C (~)
NY-2C
PBCV-1
PBCV-1
PBCV-1
Mixed
1.5
89.7
88-3
98-3
77-0
98.5
10.3
11.6
1.7
23.0
DNA restriction
profile
NY-2C
Mixed
Mixed
PBCV-1
Mixed
* Thirty-six infective centres were picked from cells simultaneously inoculated with NY-2C and PBCV-I.
Viruses from each of these plaques were titrated and scored by immunoblotting. The restriction patterns from the
DNAs were also examined. Of the 36 plaques examined 15 contained only PBCV-1 and 16 contained only NY-2C
and were scored correctly by immunoblotting. The compositions of the other five plaques are shown in the table.
T a b l e 3. Progeny (infective centres) from Chlorella NC64A cells simultaneously inoculated with
two viruses
Viruses
A+B
Input
ratio
PBCV-1 + NY-2C
PBCV-1 + NY-2C
PBCV-1 + NC-1D
NYb-lt + NC-1D
NE-8D + NC-1D
CA-4B + NC-1D
IL-3At + NY-2C
IL-3At + NY-2C
SC-1B + NY-2C
NC-1A + NY-2C
NC-1B + NY-2C
NC-1B + NY-2C
SC-1B t + NY-2B
1: 1
1:3
1: 1
1:1
1:1
1: 1
1: 1
1:3
1:2
1:1
3:2
2 :1
1: 1
M.o.i.
Outcome
(A : B : M)
proportion*
Percentage
mixed
24
18
20
27
20
26
16
27
33
11
16
35
12
121:70:2
55:94:4
61:39 : 1
220:56:17
81:209:l
123:90 : 1
247: 36 : 3
310:69:4
146:131:4
69:28:2
53:41:0
105 : 87:0
57 : 15 : 1
1-0
2.6
1.0
5.8
0-3
0.5
1.0
1-0
1.4
2.0
0
0
1.4
* Numbers refer to plaques from infective centres scored as containing virus A, virus B or both viruses (M).
t The underlined viruses dominated (> 3:1) in the dual inoculation.
the m.o.i, are identical. C h a n g i n g the m.o.i, ratio o f P B C V - 1 to N Y - 2 C to 1:3 increased the
fraction of N Y - 2 C infective centres (Table 3, line 2).
Cells inoculated with other virus combinations
T h e infective centres f r o m dual inoculations w i t h n i n e o t h e r virus c o m b i n a t i o n s were
analysed (Table 3). W i t h the e x c e p t i o n o f viruses SC-1B a n d N Y - 2 B all of the viruses adsorbed
to cells and replicated in t h e m at about the s a m e rate as PBCV-1 (Table 1). T h e s e e x p e r i m e n t s
led to several conclusions. First, all virus c o m b i n a t i o n s s h o w e d mutual exclusion. T h e highest
percentage o f plaques c o n t a i n i n g virus mixtures was 6 ~ (NYb-1 and N C - 1 D , T a b l e 3, line 4);
m o r e typically 1 to 2 ~ o f the plaques c o n t a i n e d b o t h viruses. Secondly, the ratio of infective
centres often differed f r o m the m.o.i, ratio of the inoculating viruses but usually by no m o r e t h a n
two- to three-fold. T h e most notable exceptions i n v o l v e d viruses I L - 3 A and N Y - 2 C where I L - 3 A
d o m i n a t e d (approx. 7 : 1 ; T a b l e 3, line 7), NYb-1 and N C - I D where NYb-1 d o m i n a t e d (approx.
4 : 1 ; T a b l e 3, line 4), and SC-1B and N Y - 2 B w h e r e SC-1B d o m i n a t e d (approx. 4 • 1 ; T a b l e 3, line
13). A l t e r i n g the m.o.i, ratio in favour o f N Y - 2 C in the I L - 3 A and N Y - 2 C e x p e r i m e n t slightly
affected the infective centres ratio but I L - 3 A still d o m i n a t e d (Table 3, line 8). Thirdly, a faster
replicating virus did not necessarily p r e d o m i n a t e o v e r a slower replicating virus. F o r example,
N Y - 2 C replicates a b o u t twice as fast as SC-1B and yet the ratio of infective centres after
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Mutual exclusion o f Chlorella viruses
1
2
3
4
5
6
7
8
9
10
11
12
1833
13 14
23.1 - -
9"4 J
6.7--
4.4--
2"3~
2.0--
0.56
Fig. 2. A comparison of PBCV-1 DNA (even-numbered lanes) and a serotype mutant of PBCV-1
(EPA1) DNA (odd-numbered lanes) after treatment with PstI (lanes 1, 2), HindlII (lanes 3, 4), BamHI
(lanes 5, 6), EcoRI (lanes 7, 8), Sinai (lanes 9, 10), MboI (lanes 11, 12) and DpnI (lanes 13, 14). EPA1
DNA lacks one BamHI fragment (arrowhead, lane 6) and one EcoRI fragment (arrowhead, lane 8).
Lambda DNA HindlII fragments were used as size markers.
inoculation with these two viruses was about 1 : 1 (Table 3, line 9) even though the NY-2C m.o.i.
was twice that of SC-1B in this particular experiment.
Cells inoculated with near-isogenic viruses
The preceding experiments used viruses which not only contained different concentrations of
methylated bases in their D N A s but which had, necessarily, different serotypes. To determine
whether closely related viruses exclude one another, a stable spontaneous mutant of PBCV-1
(named EPA1) that did not react with PBCV-1 antiserum was isolated. Except for the loss of a
single PBCV-1 BamHI fragment (arrowhead in lane 6, Fig. 2) and EcoRI fragment (arrowhead
in lane 8, Fig. 2), D N A from EPA1 was indistinguishable from that of PBCV-1 as determined by
restriction patterns and sensitivity to seven restriction endonucleases (Fig. 2). EPA1 adsorbed to
and replicated in host cells at the same rate as PBCV-1, and most importantly antiserum to
EPA1 did not react with PBCV-1 (data not shown).
Progeny from dual inoculations with EPA1 and PBCV-1 serotypes are reported in Table 4.
Mutual exclusion occurred in all experiments including dual inoculations with EPA1 and
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T.
E. C H A S E
AND
OTHERS
T a b l e 4. Progeny (infective centres) from Chlorella NC64A cells simultaneously inoculated with
a serotype mutant ofPBCV-1 (EPA1) and a second virus
Virus
A+B
Input
ratio
M.o.i.
PBCV-1 + EPA1
PBCV-1 + EPA1
PBCV-1 + EPA1
NE-8D + EPA1
IL-3At + EPA1
NC-1A + EPA1
1:1
1:3
5:2
1:1
1:1
1:2
20
17
43
18
19
32
Outcome
(A : B : M)
proportion
57:69 : 1
78:136:6
85:20 : 1
75:100:0
129:5:0
67:96:6
* Numbers refer to plaques from infective centres scored as containing virus A, virus B or both viruses (M).
t The underlined virus dominated (> 3:1) in the dual inoculation.
T a b l e 5. The burst size o f C h l o r e l l a NC64A inoculated with one or two viruses
Virus
Burst size*
A
r
A,
•
A
B
PBCV-1
PBCV-1
NYb-1
NE-8D
CA-4B
IL-3A
SC-1B
NC-1A
NC-1B
SC-1B
NY-2C
NC-1D
NC-1D
NC-1D
NC-1D
NY-2C
NY-2C
NY-2C
NY-2C
NY-2B
rA
227
227
336
318
89
250
174
406
295
218
B
A+o
164
292
292
292
292
164
164
164
164
93
176
241
88
94
111
318
79
160
155
84
* A and B refer to the first and second viruses, respectively, listed in columns 1 and 2.
T a b l e 6. Progeny (infective centres) from Chlorella NC64A cells inoculated with one virus
30 min before inoculation with a second virus*
First virus
Second virus
PBCV-1
NY-2C
Simultaneous
NY-2C
PBCV-1
Outcome
(PBCV- 1 : NY-2C)
681 : 138
167:680
124 : 63
* The m.o.i, was 10 for each virus.
PBCV-1 (Table 4, lines 1 to 3). R e c o v e r y ratios were usually related to the m.o.i, ratios. O n e
e x c e p t i o n was the d o m i n a n c e o f I L - 3 A (Table 4, line 5). T h e virus identities in the infective
centres resulting f r o m inoculation with E P A 1 and PBCV-1 a n d E P A 1 and N C - 1 A were
confirmed by titrating and i m m u n o b l o t t i n g virus p r o g e n y f r o m these plaques.
Effect of dual inoculations on burst size
T h e burst sizes f r o m cells inoculated w i t h one or two viruses were also determined. As s h o w n
in T a b l e 5 the burst size was reduced slightly for s o m e c o m b i n a t i o n s but, in general, dual
inoculations did not affect burst size.
Exclusion is probably an early event
T o d e t e r m i n e w h e t h e r m u t u a l exclusion o c c u r r e d early in the virus replication cycle, P B C V - I
was i n c u b a t e d w i t h cells for 30 m i n before inoculation w i t h N Y - 2 C and vice versa. T h e virus
a d d e d first d o m i n a t e d in these situations (Table 6). T o d e t e r m i n e w h e t h e r infection by one virus
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Mutual exclusion of Chlorella viruses
1835
prevented attachment of a second virus, algae were infected with PBCV-1 (m.o.i. of 10) and the
cells were collected at 1 h after infection. The cells were resuspended in MBBM and tested for
their ability to adsorb PBCV-1 and NY-2C. Both viruses attached equally well to uninfected and
infected cells (data not shown). We conclude from these experiments that exclusion is probably
an early event. However, it is not at the level of virus attachment.
DISCUSSION
Plaque lifts can be combined with immunoblotting to identify Chlorella NC64A virus
serotypes rapidly in plaques although not if a plaque contains a mixture of two viruses. Plaques
containing 9 0 ~ of one virus and 10~ of another will probably be identified as containing only
one virus, i.e. be misidentified. Plaques containing two viruses at a ratio of 3 : 1 will be identified
as a mixture.
The immunoblotting procedure was used to study the progeny resulting from Chlorella cells
inoculated with two viruses. Approximately 9 0 ~ of the time only one virus replicated in a cell
inoculated with two viruses, i.e. mutual exclusion occurred. The estimation that both viruses
replicate in 10~ of the cells may be artificially high. Vegetative cells of Chlorella NC64A
increase in size and typically produce four progeny cells (called autospores) inside the mother
cell. If different viruses infect different autospores in a single mother cell, the result would be an
infective centre containing both viruses. If this occurs, the infecting virus would have to
penetrate two walls (the mother cell wall and the autospore wall).
The results allow us to make some general comments about Chlorella NC64A cells inoculated
with two viruses. (i) Infection of the alga by one virus does not prevent attachment of a second
virus. (ii) Cells inoculated with one virus 30 min before inoculation with a second virus will
preferentially replicate the first virus. Thus the mechanism responsible for mutual exclusion
probably occurs within the first 30 to 45 min after infection. (iii) A faster growing virus does not
necessarily predominate in a dual inoculation since SC-1B competed very well with NY-2C
(Table 3, line 9) even though it replicated considerably slower than NY-2C. (iv) Some viruses
dominate in certain combinations. For example, IL-3A dominates NY-2C and PBCV-1
(EPA1). (v) It is unlikely that the virus-encoded site-specific endonucleases play a major role in
mutual exclusion. If they did, certain viruses should dominate in particular combinations. For
example, viruses PBCV-I and NC-1A produce the site-specific endonucleases CviAI and
CviBI, respectively (Xia et al., 1986a, b). In vitro, CviBI digests PBCV-1 D N A but CviAI does
not digest N C - I A DNA. Consequently if the site-specific endonucleases are involved in
exclusion, inoculation with both NC-1A and PBCV-1 (EPA1) should yield predominantly NC1A. This did not occur (Table 4, line 6). It is possible, however, that PBCV-1 and N C - I A code
for additional site-specific endonucleases that have not been detected. Finding that exclusion
also occurs in ceils inoculated with the isogenic viruses PBCV- 1 and EPA 1, which have the same
site-specific endonuclease, also indicates that these enzymes are not involved in exclusion.
Mutual exclusion was first described by Delbr~ick (1945) for T1 and T7 bacteriophages. When
cells were inoculated with both T1 and T7, 3 3 ~ of the cells yielded only T1, whereas the
remaining 6 6 ~ yielded only T7. Cells producing both viruses were not recovered. Further
experiments with closely related coliphage in the T-even series (reviewed in Doermann, 1983)
led to the general conclusion that unrelated phages usually exclude or partially exclude, whereas
closely related phages (e.g. those differing by only a few mutational steps) are compatible. The
present results demonstrate that mutual exclusion also occurs in a lower eukaryotic host-virus
system. Mutual exclusion occurred between viruses which differed from each other in several
ways, including D N A restriction patterns and levels of D N A methylation. In this respect the
results are similar to those obtained for coliphage. A surprising result was the mutual exclusion
between PBCV-1 and its mutant derivative EPA1. It will be interesting to determine how
mutual exclusion occurs between two viruses which are essentially identical except for
mutations in genes encoding capsid antigenic determinants. These data also suggest that genetic
recombination between Chlorella NC64A viruses is relatively rare although Tessman (1985)
recovered wild-type recombinants from temperature-sensitive mutants of PBCV-1 but at low (1
to 2 ~ ) frequencies. This low frequency of recombinants could result from mutual exclusion.
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1836
T. E. C H A S E A N D O T H E R S
We thank K e n N a r v a for help with the immunoblotting, Ellen Ball for providing the virus antisera, and Les
Lane, Myron Brakke and Irwin T e s s m a n for helpful discussions and critically reading the manuscript. This work
was supported in part by Public Health Service G r a n t GM32441 from the National Institutes of General Medical
Sciences and grant DE-ACO2-82ERI2086 from the Department of Energy. This manuscript has been assigned
Journal Series No. 8787, Agricultural Research Division, University of Nebraska.
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(Received 30 January 1989)
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