Journal of General Virology (1990), 71, 1775-1783. Printed in Great Britain
1775
A prominent serine-rich region in Vmw175, the major transcriptional
regulator protein of herpes simplex virus type 1, is not essential for virus
growth in tissue culture
T. P a t e r s o n t and R. D. Everett*
M R C Virology Unit, Church Street, Glasgow G l l 5JR, U.K.
Herpes simplex virus type 1 (HSV-1) encodes five
immediate early (IE) genes of which at least three are
involved in the transcriptional regulation of later
classes of viral genes. Perhaps the most important of
these regulatory proteins is Vmw175, a nuclear
phosphoprotein of 1298 predicted amino acid residues.
In the absence of functional Vmwl75 the virus fails to
activate early or late genes or to repress IE gene
expression. All viruses of the sub-family alphaherpesvir~nae encode polypeptides that are closely related to
Vmw175. Mutational studies have shown that regions
of homology within this family of gene regulators are
generally of functional importance. One of the most
striking conserved stretches of amino acid sequence is a
run of serine residues followed by a highly acidic region
in the amino-terminal fifth of the polypeptide. We have
constructed an HSV-1 virus which lacks this serinerich run within Vmw175. Surprisingly, the virus was
viable in tissue culture cells and expressed apparently
normal amounts of viral polypeptides. In plaque assays
it was very slightly temperature-sensitive and, depending on the state of the host cells, could generate plaques
with a syncytial morphology. The mutant protein was
able to bind to DNA in a manner indistinguishable
from that of the wild-type polypeptide. We conclude
that despite its conservation in all of the alphaherpesvirinae so far sequenced, the serine-rich homology is
not important for virus growth in tissue culture.
Introduction
sensitive (ts) mutations which map in Vmw175 result in
an absence of viral growth through failure to activate
early and late promoters at the non-permissive temperature (Preston, 1979). In addition, lack of Vmw175
function results in loss of the autoregulation or repression
of IE gene expression which occurs during normal
infection. Thus Vmw 175 is central to the regulation of all
classes of viral genes and as such has been the focus for
intensive study.
Vmw175 is a representative of a family of regulatory
proteins that are conserved in the subfamily alphaherpesvirinae. It is a phosphorylated nuclear DNAbinding protein with an apparent Mr of 175K in SDSpolyacrylamide gels, and can be poly(ADP-ribosyl)ated
in isolated nuclei (Powell & Purifoy, 1976; Pereira et al.,
1977; Cabral et al., 1980; Hay & Hay, 1980; Preston &
Notarianni, 1983). Phosphorylation of Vmw175 occurs
at both serine and threonine residues (Faber & Wilcox,
1986a) and as pulse-chase experiments have indicated
that phosphate groups can cycle on and off the
polypeptide during infection (Wilcox et al., 1980) it is
possible that phosphorylation plays some role in its
regulatory activities. Vmw175 can be isolated in multimeric (principally dimeric) forms (Metzler & Wilcox,
1985) and has been purified almost to homogeneity
(Kattar-Cooley & Wilcox, 1989).
Herpes simplex virus type 1 (HSV-I) has a linear doublestranded DNA genome of about 150 kb which encodes at
least 70 distinct genes (McGeoch et al., 1988). These
genes can be grouped into three broad classes depending
on their temporal regulation during infection in tissue
culture (for reviews see Wagner, 1985; Everett, 1987).
The five immediate early (IE or 0t) genes can be
transcribed in the absence of de noco viral protein
synthesis by the host RNA polymerase II (Costanzo et
al., 1977). Efficient use of the IE promoters requires
recognition of an IE-specific regulatory sequence by a
host transcription factor (probably NFIII or OTF-I) in
association with a component of the virus particle,
Vmw65 or ct-TIF (Campbell et al., 1984; McKnight et al.,
1987; Preston et al., 1988; O'Hare & Goding, 1988).
Transcription from early promoters requires the expression of IE polypeptides while late gene transcription is
most efficient only after the onset of viral D N A
replication. Of the five IE gene products, that of IE-3
(Vmw175 or ICP4) is perhaps the most crucial for
subsequent viral gene regulation as certain temperaturet Presentaddress:Department of MolecularBiology,Universityof
Edinburgh, Kings Buildings, MayfieldRoad, Edinburgh, U.K.
0000-9390© 1990SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
1776
T. Paterson and R. D. Everett
Comparison of the predicted amino acid sequence of
Vmw175 with those of the related proteins expressed by
varicella-zoster virus (VZV) (McGeoch et al., 1986),
pseudorabies virus (PRV) (Vlcek et al., 1989) and equine
herpesvirus (EHV) type 1 (Grundy et al., 1989) reveals
two regions of high conservation (regions 2 and 4)
interspersed with three other regions (1, 3 and 5) that
have fewer similarities. Extensive mutagenic studies
have revealed that region 2 contains sequences that are
essential for Vmw175 functions in early gene activation
and IE gene repression both during virus infection and in
short term transfection assays using isolated genes and
promoter targets (Preston, 1979; Davison et al., 1984;
DeLuca & Schaffer, 1987, 1988; Paterson & Everett,
1988a). The structure of region 4 is also important for
these functions (Paterson & Everett, 1988a; Paterson et
al., 1990). Mutations in both regions 2 and 4 can also
affect the ability of Vmw175 to bind in vitro to D N A
fragments which include the consensus recognition
sequence ATCGTC (Faber & Wilcox, 1986b; Mfiller,
1987; DeLuca & Schaffer, 1988; Paterson & Everett,
1988b; Kattar-Cooley & Wilcox, 1989; Shepard et al.,
1989). Binding to such a consensus sequence at the cap
site of the IE-3 promoter has been directly implicated in
the ability of Vmw175 to repress IE transcription both in
transfection assays and during virus infection (Roberts et
al., 1988; DeLuca & Schaffer, 1988) and binding to a
similar site upstream of the gD promoter contributes to
promoter activation in vitro (Beard et al., 1986; Tedder et
al., 1989).
Apart from the highly conserved regions 2 and 4, there
are other short sections of the Vmw175 polypeptide that
are conserved in the three related gene products so far
sequenced. The most striking of these is a run of serine
residues followed by a short highly acidic block in region
1. Conservation of this region in HSV, VZV and PRV,
and the fact that a stretch of serine residues might be a
major target for phosphorylation, suggests that it may be
important for Vmw 175 function. This paper assesses this
question by the construction of a mutant virus which
lacks the serine-rich region. Surprisingly, the virus was
viable in normal tissue culture cells. The mutant
Vmw 175 polypeptide was capable of activating early and
late gene expression (although rather more slowly than
the wild-type virus) and it could also repress IE gene
expression. The mutant polypeptide was able to bind to
the consensus ATCGTC binding sequence at the IE-3
cap site. We conclude that the serine-rich tract is not
essential for virus growth in tissue culture.
which were grown in Glasgow-modified Eagle's medium (GMEM)
supplemented with 10% newborn calf serum and 10% tryptose
phosphate broth. M64A cells, derived from BHK TK- cells by
biochemical transformation to TK ÷ by transfection of a plasmid
containing the intact HSV-1 TK and IE-3 genes, were grown in
GMEM supplemented with 10 % foetal calf serum. M64A cells express
Vmw175 from integrated IE-3 gene copies and are essentially identical
to the M65 cells described by Davidson & Stow (1985). They were used
for the propagation and titration of HSV-1 mutant inl411, which has
translational termination signals inserted into the IE-3 gene near the 5'
end of the Vmw175 coding sequence (Russell et al., 1987), and the IE-3
deletion mutant D30EBA (see text). Human foetal lung (HFL) cells
were obtained from Flow Laboratories and were grown in GMEM
supplemented with 10% foetal calf serum. 2.5BHK cells were derived
from BHK cells by passage and maintenance in Dulbecco's modified
Eagle's medium containing 2.5% foetal and 2.5% newborn calf sera.
Bacteria and plasmids. All plasmids were maintained in Escherichia
coil strain HB101. Plasmid p175 contains the complete IE-3 coding
region linked to the simian virus 40 promoter and enhancer (Perry et
al., 1986). The deletion mutant plasmids pdelI11 and pD1 were derived
from p175 as described in the text.
Preparation and analysis of viral DNA. Infectious viral DNA was
prepared from cell-released viral stocks by the method of Wilkie (1973).
Viral DNA for Southern blot analysis of the genotype was prepared as
total cellular DNA from infected cell monolayers (Stow et al., 1983).
Viral DNAs were analysed by restriction enzyme digestion, agarose gel
electrophoresis and Southern blotting (Southern, 1975) using nicktranslated probes (Rigby et al., 1977). Viral DNA was also labelled with
[32p]orthophosphate during growth in tissue culture. The labelled
DNA was isolated, cut with restriction enzymes, separated by agarose
gel electrophoresis and detected by autoradiography as described by
Lonsdale (1979).
Transfection of tissue culture cells and isolation of recombinant viruses.
Infectious DNA from viruses inl411 or D30EBA was mixed with calf
thymus carrier DNA and mutant plasmid DNA linearized at the PstI
site and transfected into M64A cells by the method of Stow & Wilkie
(1976). Recombinant viruses were isolated by titrating the progeny
virus from the transfection on M64A cells under agar. Individual
plaques were picked and the viruses grown up in single wells of M64A
cells in 24-well Linbro blocks. When extensive c.p.e, had occurred,
total cellular DNA was prepared from each well and analysed for the
presence of the desired mutation by Southern blotting. Viruses which
carried the required mutation in both copies of IE-3 were plaquepurified three more times on M64A cells. All viral preparations were
analysed by Southern blotting to confirm the presence of the mutation.
After the initial isolation in M64A cells, it was found that virus
I15HBC could be propagated on BHK cells. Because the genotype of
I15HBC was rather unstable in M64A cells (perhaps because of
recombination with the IE-3 gene copies in the cellular genome) it was
plaque-purified once more in BHK cells before large scale stocks were
prepared.
Methods
Labelling of viral polypeptides and their analysis by SDS polyacrylamidegelelectrophoresis. BHK cells (4 x 105 cells in Linbro wells) were
infected with wild-type and mutant viruses at multiplicities of 5 p.f.u.
per cell. At the times indicated in the text, the cells were washed with
phosphate-buffered saline (PBS) and 10 ktCi of [35S]methionine
(Amersham, > 800 Ci/mmol) in PBS was added. After labelling for 2 h,
the cells were harvested in sample buffer and analysed by SDS
polyacrylamide gel electrophoresis as described by Marsden et al.
(1978).
Viruses and cells. HSV-I Glasgow strain 17 syn+ was the wild-type
virus used in this study. Both the wild-type and the mutant virus
I15HBC were routinely propagated and titrated in BHK clone 13 ceils
Infection of cellsfor the preparation of labelled IE protein extracts. BHK
cells (4 × 105 cells in 24-well Linbro dishes) were preincubated for 30
min in the presence of cycloheximide (100 ~tg/ml) and then infected
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
Vmw175 serine-rich region deletion mutant
with the wild-type or mutant viruses at a multiplicity of 5 p.f.u, per cell.
After a 1 h adsorption period, the cells were re-fed with growth medium
containing cycloheximide and incubated at 37 °C for a further 4 h. The
cells were then washed three times with PBS, then once with PBS
containing 2.5 ~tg/ml actinomycin D, and then incubated with 10 ~tCi
[3sS]methionine (Amersham, > 800 Ci/mmol) in 0.2 ml PBS plus
actinomycin D at 37 °C for 90 rain. The cells were then washed with
PBS and harvested directly into boiling mix (100 ~tl). This infection and
labelling procedure maximizes the production of the IE proteins of
HSV-1 (Preston et al., 1978).
Preparation of nuclear extracts from infected cells. Nuclear extracts of
infected cells were prepared by a modification of the procedure of
Dignam et al. (1983). HeLa cells were infected with 5 p.f.u, per cell of
wild-type and mutant viruses at either permissive or non-permissive
temperature. Five h after a I h adsorption period the cells were scraped
into PBS, pelleted and resuspended in 2 volumes of buffer A (10 mMHEPES pH 7'9, 1.5 mM-MgC12, 10 mM-KC1, 0.5 mM-DTT) followed by
the addition of NP40 to 0.5%. After 10 min on ice the nuclei were
pelleted by centrifugation in the Sorvall SS34 rotor at 2000 r.p.m, for 10
min, using 1.5 ml reaction vials set in rubber adaptors for this rotor. The
supernatant was discarded and then the nuclei were further compacted
by spinning at 12000 r.p.m, for 20 min in the same rotor. Proteins were
eluted from the nuclei by incubation on ice in 2 volumes of buffer C (20
mM-HEPES pH 7-9, 25% glycerol, 0.42 M-NaCI, 1.5 mM-MgC12, 0'2
mM-EDTA, 0.5 mM-PMSF, 0-5 mM-DTT) for 30 min with frequent
mixing. The nuclear debris was pelleted by centrifugation in the Sorvall
SS34 rotor at 15 000 r.p.m, for 30 min. The supernatants containing the
nuclear DNA-binding proteins were frozen in dry ice/ethanol and
stored at - 1 4 0 °C. The Vmw175 contained in such extracts retained
the ability to bind to DNA, without any loss of activity, over several
months and freezing and thawing cycles.
Analysis of the DNA-binding capability of Vmw175 by gel retardation.
The ability of Vmw175 to bind to the IE-3 cap site region was assayed
using the conditions and probe described by Miiller (1987). The 45 bp
BamHI-AvaI fragment spanning the IE-3 cap site was end-labelled by
filling in with T4 DNA polymerase, separated on an 8% polyacrylamide gel, eluted and purified through a Sephadex G-50 column.
Binding reactions were carried out for 20 rain at the temperatures
indicated in the text in 20 gl volumes containing 1 I~g poly(dI).
poly(dC), 0.1 ng of probe, and 1 to 4 gl of nuclear extract (5 to 20 l~g of
protein) in a buffer containing 10 mM-Tris-HCl pH 7.6, 1 mM-EDTA
and 0.1% NP40. The variations in salt concentration (20 to 80 mMNaCI) due to the occasional variation of the amounts of nuclear extract
used does not affect the formation of the Vmw175-specific complex
which is stable up to at least 300 mM-NaCI (Paterson et al., 1990). The
complexes were resolved on 4% polyacrylamide gels run in 0-5 x Trisborate-EDTA and were detected by autoradiography of the dried gels.
Confirmation that complexes contained Vmw175 was obtained by
incubating the binding reaction for a further 10 min prior to
electrophoresis with monoclonal antibody 58S (Showalter et al., 1981),
which recognizes an epitope near the carboxy terminus of Vmw175
(Paterson & Everett, 1988a). Migration of the specific complexes on
the polyacrylamide gels was further retarded by binding of the
antibody.
Results
Construction and isolation o f an HSV-1 strain 17 mutant
with a large deletion in IE-3
We have previously described the construction and
characterization of a large number of insertion and
1777
deletion mutations in a plasmid-borne copy of IE-3,
which encodes Vmw175. In order to assess the biological
significance of the effects of these mutations, it is
important to transfer selected mutations from the
plasmid into the viral genome. As a first step in this
procedure, it was desirable to construct a virus derived
from strain 17 that lacks a large portion of the Vmw175
coding region to serve as the parent virus in the
recombination experiments. The deletion mutant would
not outgrow any recombinant progeny and rescue of the
deletion would ensure inheritance of the desired mutation if it lay in the deleted region. Although deletion
mutants of IE-3 are available in strain KOS (DeLuca et
al., 1985), they have not been described in strain 17. We
decided to construct a large deletion mutant in the strain
17 IE-3 coding sequences using plasmid pdelI11, which
has lost sequences from codons 83 to 1236. To minimize
the problem of the selective advantage of wild-type virus,
we used infectious DNA from virus inl411 (Russell et al.,
1987) as the parental virus DNA for the deletion
mutagenesis. Virus inl411 has a translational termination signal at codon 83 of the Vmw175 coding sequence
and does not express Vmw175. The position of IE-3 in
the HSV-1 genome, and the locations of the mutations
used in this study are shown in Fig. 1.
Viral inl411 DNA was transfected into M64A cells
with linearized pdelI 11 plasmid DNA and progeny virus
harvested 4 days later. The resulting virus stock was
titrated on M64A cells under agar. A number of plaque
isolates were picked and inoculated into Linbro wells of
M64A cells and 4 days later the cell-released virus from
each was harvested and total cellular DNA prepared
from the cell monolayer. This DNA was digested with
BamHI and subjected to agarose gel electrophoresis and
Southern blotting. Using nick-translated plasmid p175
(Perry et al., 1986) as a probe, the isolates were screened
for those having the required deletion in the joint
fragment BamHI k. One isolate with a deleted B a m H I k
fragment was further plaque-purified three times (checking the restriction pattern on each occasion) and then a
stock of the mutant virus D30EBA was prepared. The
DNA of this virus was further analysed and compared to
that of the parental virus inl411 by the technique of
Lonsdale (1979). The results after digestion of the viral
DNA with BamHI are shown in Fig. 2. The B a m H I k
joint fragment of inl411 has been lost from D30EBA
(lane 1), as has the terminal fragment B a m H I y (which
comigrates with B a m H I z). There is a faint novel B a m H I
band of the expected size (4.3 kb) between fragments n
and o in D30EBA. It was difficult to detect all the
predicted restriction fragments of D30EBA in this
manner probably because of variations in the numbers of
'a' sequences and other reiterations in the deleted short
repeat terminal and joint regions, and also because the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
1778
I---3
T. Paterson and R. D. Everett
.//
I
IE-1
IE-2
I
1
IE-1 IE-3
IE-3
IE-4
1
o
H
3
4
5
6
a
it
S
2
IE-5
S
I
t
2
ttt
S
S
bc
de fgh
ij
"qk
lmn
t!
BB
BE
0
pq
r
RNA ,
1298
Region 4
ATG
Ut3
W
X
Region 2
yz
1270
D30EBA
84
I
J
229
ll5HBC
162
[]
206
Serine-rich/acidic region
st
1
TAA
176
m
Fig. 1. A map of the HSV-1 genome showing the positions of the IE
genes and a partial restriction map of the IE-3 region. S, SstI; H,
HinclI; B, BamHI; E, EcoRl. The transcribed region of IE-3 is
indicated by the arrow with the 1298 amino acid coding region shown
below. The regions of Vmw175 most highly conserved in the
alphaherpesviruses, regions 2 and 4, are also shown. The plasmids used
for this work were based on p175, which contains the IE-3 region
(linked to the SV40 promoter and enhancer) from the BamHI site at
+ 27 in the IE-3 transcription unit to the SstI site on the far side of the
'a' sequence cloned into a pBR322 vector. The deleted region in the
Vmw 175 mutant D30EBA and the extent of the deletion in I 15HBC (in
plasmid pD1) are shown as open boxes. The serine-rich and acidic
regions are located within the stippled box. The numbers refer to
codons from the N-terminal end of Vmw175.
m e t h o d necessarily results in lower yields of the t e r m i n a l
fragments. However, further analysis of D 3 0 E B A by
digestion with HindlII, E c o R I a n d KpnI gave results
entirely consistent with the predicted g e n o m e structure
(data not s h o w n ; Paterson, 1989).
D u e to r e c o m b i n a t i o n b e t w e e n D 3 0 E B A a n d the IE-3
sequences i n t e g r a t e d into the M 6 4 A cell line, stocks of
D 3 0 E B A c o n t a i n e d a p p r o x i m a t e l y 0-001 ~ to 0.01 ~ of
a p p a r e n t l y wild-type virus. However, the presence of
such r e c o m b i n a n t g e n o m e s was n o t a serious p r o b l e m for
the uses of D 3 0 E B A described i n this paper. W h e n
D 3 0 E B A was used to infect B H K cells, the p a t t e r n of
viral polypeptide synthesis was very s i m i l a r to that
observed with the p a r e n t i n l 4 1 1 a n d other m u t a n t s with
large deletions in IE-3 ( D e L u c a et al., 1985). T h e r e was
a n a c c u m u l a t i o n of the I E polypeptides (with the
exception of Vmw175), a n d also the large s u b u n i t of
r i b o n u c l e o t i d e reductase, b u t there was n o e v i d e n c e of
synthesis of other early or late proteins (data not shown).
/i
Fig. 2
Fig. 3
Fig. 2. Analysis of the genomic viral DNA of D30EBA by the
Lonsdale (1979) method. Viral DNA was labelled in vivo with
[32p]orthophosphate, extracted, digested with BamHI and analysed by
agarose gel electrophoresis and autoradiography. Lane 1, D30EBA;
lane 2, parent inl411. The BamHI k band of inl411 (5.9 kb) and one of
the bands in the BamH! x and y doublet (1-95 kb and 1-84kb) are not
present in D30EBA. A faint additional band in D30EBA between
BamHl n and o probably represents the deleted BamHI k band (4.3 kb).
The restriction fragments of D30EBA that lie outside the short repeat
region are identical to those of the parent inl411.
Fig. 3. Analysis of the genomic viral DNA of I15HBC and rescued
viruses. Viral genomic DNA was labelled, extracted, digested with
EcoRI and analysed as described in Methods. Lanes 1 to 6 contain
DNA from strain 17, I15HBC, rl-10, r2-1 and rescued viruses of
D30EBA and inl411 respectively. The loss of the EcoRI k band (5.5 kb
in strain 17) in I15HBC and its replacement by smaller bands (4-4 kb
and 0-8 kb) are discussed in the text. The band marked ? is of unknown
origin but is frequently observed in HSV-1 EcoRI digests (A. Davison,
personal communication). For comparison, EcoRI bands rn and o are
4.07 kb and 1.49 kb respectively.
Construction o f a virus lacking the serine-rieh region o f
Vmw175
P l a s m i d p D 1 encodes a deleted IE-3 gene l a c k i n g codons
162 to 229 a n d includes a n E c o R I l i n k e r at the site of the
deletion (Fig. 1; P a t e r s o n & Everett, 1988a). T h i s
deletion s p a n s the serine-rich a n d acidic region from
codons 176 to 206, w h i c h is conserved i n the corresponding P R V , E H V a n d V Z V polypeptides. I n order to
construct a virus with a n IE-3 gene l a c k i n g this d o m a i n ,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
1779
Vmw175 serine-rich region deletion mutant
linearized plasmid pD1 DNA was cotransfected with
D30EBA viral DNA into M64A cells. As described
above for the construction of D30EBA, the progeny virus
were plated onto M64A cells and single plaques were
screened by Southern blotting for transfer of the plasmid
pD1 deletion into the viral genome. A number of
candidate isolates were picked for further purification.
After three rounds of plaque purification (and a further
purification on BHK cells; see below) stocks of isolate
I15HBC, which exhibited the expected restriction
profile on Southern blots, were prepared. The genomic
DNA of I15HBC was further characterized by the
technique of Lonsdale (1979) (Fig. 3). The results clearly
show that I15HBC (lane 2), in comparison with strain 17
(lane 1) and rescued and parental viruses (lanes 3 to 6),
has the expected EcoRI restriction profile. The terminal
fragment EcoRI k has been lost and novel fragments,
migrating above EcoRI m and below EcoRI o, result from
the EeoRI linker which replaces the deleted sequences in
I15HBC.
Virus I15HBC grows on a non-complementing cell line
When the growth characteristics of I15HBC were
investigated, it was surprising to find that the virus
formed plaques on BHK cells although the plaques were
somewhat smaller than those of strain 17. The plaquing
ability of I15HBC was slightly temperature-sensitive (a
threefold to sevenfold reduction in titre at 38.5 °C
compared to 31 °C) and at the higher temperature it had
a tendency to produce syncytial plaques. This latter
effect was dependent on the stock of BHK cells used for
titration and was not always observed. To test whether
the syncytial plaque phenotype of I 15HBC was due to the
deletion in Vmw175, or to some other adventitious
change in the viral genome, the wild-type IE-3 gene was
recombined into I15HBC by cotransfection of viral
I15HBC DNA with either pGX58 (containing XhoI
fragment c; Everett, 1984) or the EcoRI-SalI fragment
spanning the region -129 to +2041 of the IE-3
transcription unit. Rescued viruses were picked on the
basis of a non-syncytial plaque morphology and then
tested for their genome structure. Rescued viruses (rl-10
and r2-1 respectively) from both experiments contained
the wild-type IE-3 gene, thus strongly suggesting that the
syncytial phenotype of I15HBC was due to the deletion
in Vmw175 (Fig. 3).
After it became clear that I 15HBC was able to plaque
on BHK cells, the virus was routinely propagated on this
cell type. Such stocks of the virus had particle to p.f.u.
ratios in the range of 200 : 1 which is slightly higher than
those observed with similar stocks of strain 17. The
plating efficiency of I15HBC was similar on BHK,
2.5BHK and HFL cells, which illustrates that unlike
viruses unable to express functional Vmwll0 (Stow &
I
I
I
I
|
10 9
108
.........o
.~ 10 7
/"
6 ~
106
105
3
6
9
12
Time post-adsorption (h)
I
24
Fig. 4. Single-step growth curve comparison of wild-type (E]) and
I15HBC (©) viruses. B H K cells were infected in parallel in 35 m m
plates with strain 17 and I15HBC at a multiplicity of about 2 p.f.u, per
cell. After a 1 h adsorption period the cells were washed and re-fed with
2 ml of medium. At the indicated times, the cells were scraped into the
m e d i u m and sonicated and total virus was determined by plaque assay.
Stow, 1985; Everett, 1989) there was no cell typedependent effect. However, the syncytial plaque morphology of I15HBC was particularly marked in HFL
cells.
The growth efficiency of I15HBC relative to strain 17
in BHK cells was tested in a single-step growth curve
experiment. The results (Fig. 4) illustrate that the yield of
I 15HBC is about 10-fold less over the time course of this
experiment. Therefore, although the serine-rich region of
Vmw175 is not essential, it does contribute to fully
efficient viral growth.
Viral polypeptide expression of I15HBC
Viral polypeptide expression induced by I15HBC was
compared to that of the wild-type virus in BHK cells in a
time course experiment. Cells were infected as described
in Methods and subsequently labelled with [3SS]methio n_
ine from 0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10 and 14 to 16 h
post-adsorption. The labelled proteins were analysed by
SDS-gel electrophoresis (Fig. 5). Compared to the wildtype virus, viral polypeptide synthesis by I15HBC is
slightly delayed (which is consistent with its smaller
plaques and reduced yields) but apparently normal
amounts of all of the obvious viral polypeptides are
eventually produced. These results are consistent with
previous transfection experiments which showed that
Vmw175 expressed by plasmid pD1 was located in the
nucleus and could trans-activate the gD promoter
(although about three times less efficiently than the wild-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
1780
T. Paterson and R. D. Everett
(a)
1
2
3
4
•
.5
6
7
(b)
1
2
3
4
5
6
7
Fig. 5. Viral polypeptide synthesis in BHK cells infected with 17+ and 115HBC. BHK cells were infected with strain 17 (a) and I 15HBC
(b) at a multiplicity of 5 p.f.u, per cell and labelled with [35S]methionine at various times thereafter. Lanes 1 to 7, mock-infected cells,
and cells labelled at 0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10 and 14 to 16 h after adsorption, respectively. The four most prominent high M r viral
polypeptides are the major capsid protein, the large subunit of ribonucleotide reductase (RR1), glycoprotein gB and the major DNAbinding protein. Further down the gel Vmw65 is seen accumulating in large amounts late in infection.
1
2
3
4
late gene activation or the repression o f I E gene
expression in tissue culture.
Experiments using lower multiplicities o f infection
showed that I 1 5 H B C (again unlike viruses with defects
in V m w 1 1 0 ; Everett, 1989) did not have a multiplicitydependent defect in viral gene expression (data not
shown). Labelling o f the viral I E polypeptides after
reversal of a cycloheximide block revealed that I 1 5 H B C
expressed a V m w 175 polypeptide which was a p p r o x i m a tely the expected size, some 6 K smaller than the wildtype protein (Fig. 6).
Polypeptide Vmw75 expressed by H 5 H B C binds to the
IE-3 cap site recognition sequence in vitro
Fig. 6. Virus I15HBC produces a Vmw175 polypeptide of reduced M r.
BHK ceils were infected with strain 17, mock-infected and infected
with r2-1 and I15HBC (lanes 1 to 4 respectively) in the presence of
cycloheximide. After reversal of the cycloheximide block viral IE
proteins were labelled and separated by SDS-gel electrophoresis. The
IE proteins are identified on the left and the smaller Vmw 175 produced
by II5HBC is indicated on the right.
type protein) and repress the IE-3 p r o m o t e r (Paterson &
Everett, 1988 a). W e conclude that, in the b a c k g r o u n d o f
the virus, the serine-rich h o m o l o g y region is not required
for the efficient function o f Vmw175 in either early and
Vmw175 is a sequence-specific D N A - b i n d i n g protein
w h i c h recognizes a n u m b e r o f different sequences o f
w h i c h m a n y contain the consensus A T C G T C (Faber &
Wilcox, 1986b; Beard et al., 1986; Michael et al., 1988).
The sequence at the cap site o f the IE-3 p r o m o t e r is
strongly b o u n d by Vmw175 (Mfiller, 1987). W e have
previously confirmed these results, finding that while
extracts f r o m mock-infected cells give two complexed
bands in gel retardation experiments, infected cell
extracts form a third b a n d which can be further retarded
by addition o f the anti-Vmw175 monoclonal a n t i b o d y
58S (Paterson & Everett, 1988b). This Vmw175-specific
complex is not formed by extracts from cells infected
with inl411 (Paterson & Everett, 1988 b) or D 3 0 E B A (see
below). W e have investigated whether a similar
Vmw 175-specific complex can be formed with extracts
f r o m cells infected with I 1 5 H B C . T h e results (Fig. 7)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
Vmw175 serine-rich region deletion mutant
(a)
1
2
3
(b)
4
1
2
3
4
1781
176
206
HSV-I SASSTSSDSGSSSSSSASSSSSSSDEDEDDD
353
A
373
SSSSSSSW G SSSE D E D D E
VZV
B
377
C
PRV
Fig. 7. Deletion of the serine-rich region of Vmw175 does not affect its
ability to bind to the IE-3 cap site. Nuclear extracts were prepared from
cells infected with D30EBA, I15HBC, rl-10 and r2-1 and used in gel
retardation assays with the IE-3 cap site probe as described in the text
and Methods. Binding reactions were performed at (a) 0 °C, (b) 39.5 °C.
Lanes 1 to 4; D30EBA, I15HBC, rl-10 and r2-1 respectively. The host
bands B and C and the Vmw175-specific band A are marked. The
uncomplexed probe band has been cut from the bottom of the
autoradiogram.
show that whereas extracts from D30EBA-infected cells
gave only the two host bands B and C (lane 1), extracts
from I15HBC-infected cells (lane 2) and rescued viruses
rl-10 and r2-1 (lanes 3 and 4) gave the Vmw175-specific
band A. The band A produced with I15HBC-infected
extracts could be further retarded with monoclonal
antibody 58S (data not shown). The amount of this band
formed with I15HBC extracts was reproducibly less than
that formed with either wild-type or rescued virus
extracts (data not shown and Fig. 7) and this correlated
with lower amounts of Vmw175 in the I15HBC extracts
as detected by ELISA (data not shown). However, as
extracts were routinely made 5 h post-infection with all
viruses, the lower amounts of Vmw175 in I15HBCinfected extracts may simply represent the rather slower
onset of infection with this virus (Fig. 3). Alternatively,
the lack of the serine-rich homology region may reduce
the stability of Vmw175. Despite the reduced amounts of
the I15HBC complex it was as thermostable as the
complexes formed with the rescued virus extracts
(compare panels a and b, Fig. 7). These results using
Vmw175 prepared from cells infected with I15HBC are
consistent with the results using extracts from cells
transfected with plasmid pDl (Paterson & Everett,
1988b).
These results strongly suggest that the serine-rich
homology region does not contribute to the DNAbinding domain of Vmw175. In further support of this
hypothesis, we have shown that a small proteolytic
subfragment of Vmw175 can form a complex with the
IE-3 cap site probe and that the size of this complex is
unaffected by the deletion of the serine-rich tract
(unpublished results).
401
S A S S S S S S S S S S S S S S S S S S S E G EEDE
Fig. 8. Comparison of the amino acid sequences of the serine-rich and
acidic regions of HSV-1, VZV and PRV. The numbers refer to the
number of amino acids from the N-terminal ends of the three proteins.
The similarity between the sequences, with the run of serines followed
by five to seven acidic residues, is obvious. The single-letter amino acid
code has been used.
Discussion
This report clearly demonstrates that a prominent serinerich tract and the surrounding sequences which include a
highly acidic region (Fig. 8) are not required for the
function of Vmw175 during HSV growth in tissue
culture. This conserved region extends from codon 176 to
codon 206, which is entirely within the deletion in
I15HBC (codons 162 to 229). There is strong evidence
that this region is a target for the normal phosphorylation
of Vmw175. A mutant polypeptide including only the
first 171 codons is not phosphorylated while another
expressing the first 251 codons is (DeLuca & Schaffer,
1988). However, an in-frame deletion mutant from
which residues 185 to 309 have been removed can be
phosphorylated (DeLuca & Schaffer, 1988). This suggests that either the six remaining serine residues
between 176 and 184 can be phosphorylated in the
absence of the rest of the conserved region, or that other
sites in the protein (including threonine residues; Faber
& Wilcox, 1986a) are also targets for phosphorylation.
Whatever the role of this conserved sequence in the
phosphorylation of Vmw175, it is very surprising that it
is not essential for the function of the protein. The region
is conserved in the corresponding genes of all the
alphaherpesviruses so far sequenced and it might be
expected that such a strongly conserved feature would
have an important function. Indeed, other conserved
segments of Vmw175 such as regions 2 and 4 have been
shown to be functionally important. This is further
underlined by the observation that the amino acid
residues that have been mutated in temperaturesensitive alleles of Vmw175 are precisely conserved in
the alphaherpesvirus family (Paterson et al., 1990).
Therefore, although amino acid conservation within the
coding regions of related proteins may be a good
indicator of important or functional domains, virus
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
1782
T. Paterson and R. D. Everett
I15HBC illustrates that this may not always be the case
(at least for virus growth in tissue culture).
Shepard et al. (1989) have also investigated the effect
of mutations in region 1 on the function of Vmw175.
Using a truncated Vmw175 protein including only the
first 774 amino acid residues, they found that further
deletion of residues 31 to 210, 31 to 274, and 143 to 210
(all of which remove the serine-rich region) did not affect
autoregulation or DNA-binding to ATCGTC consensus
sites. However, these deletions reduced transactivation
of the TK promoter by the truncated polypeptide, and
the largest deletion inhibited DNA-binding to the nonconsensus sites in the TK promoter (Shepard etal., 1989).
These deletions generally remove more coding sequence
than the deletion in I15HBC and since they are in a
molecule already substantially deleted, their phenotype
may not reflect that pertaining to the complete protein.
Indeed, these authors cite unpublished work entirely in
agreement with the data presented here (Shepard et al.,
1989).
The cell growth-dependent syncytial plaque phenotype of I15HBC is potentially interesting. Rescue of the
deletion by transfection with a DNA fragment internal
to IE-3 and screening for normal plaque morphology
strongly indicates that the syncytial phenotype is due to
the altered Vmw175 in I15HBC. This could result from
slight alterations in the amount of expression of the viral
envelope proteins, which implies that the extent of
phosphorylation of Vmw175 could affect its ability to
activate different viral genes. Interestingly, another
viable virus with a small deletion in Vmw175 between
codons 209 and 236 had a similar phenotype of small
syncytial plaques (Schr6der et al., 1985). However, in this
case the phenotype could not be attributed to the deletion
in Vmw175.
Despite the results presented in this paper, which
illustrate that the serine-rich region is not essential for
virus growth in tissue culture, it seems inconceivable that
such a highly conserved region within the alphaherpesvirinae has no role in the virus life cycle. It remains to be
seen whether the I15HBC mutation affects other
properties of the virus such as latency or pathogenesis.
The authors thank Professor J. H. Subak-Sharpe for his continuing
interest in this work and Dr Nigel Stow for helpful comments on the
manuscript. T.P. was the recipient of a Medical Research Council
studentship.
References
BEARD, P., FAILER,S., WILCOX,K. W. & PIZER, L. I. (1986). Herpes
simplex virus immediate early infected cell polypeptide 4 binds to
DNA and promotes transcription. Proceedings of the National
Academy of Sciences, U.S.A. 83, 4016-4020.
CABRAL, G. A., COURTNEY, R. J., SCHAFFER, P. A. & MARCIANO-
CABRAL, F. (1980). Ultrastructural characterization of an early
nonstructural polypeptide of herpes simplex virus type 1. Journal of
Virology 33, 1192-1198.
CAMPBELL, M. E. M., PALrREYMAN,J. W. & PRESTON, C. M. (1984).
Identification of herpes simplex virus DNA sequences which encode
a trans-acting polypeptide responsible for stimulation of immediate
early transcription. Journal of Molecular Biology 180, 1-19.
COSTANZO,F., CAMPADELLI-FIuME,G., FOA-TOMASI,L. & CASSM, E.
(1977). Evidence that herpes simplex virus DNA is transcribed by
cellular RNA polymerase B. Journal of Virology 21, 996-1001.
DAVIDSON,I. & STOW, N. D. (I 985). Expression of an immediate-early
polypeptide and activation of a viral origin of DNA replication in
cells containing a fragment of herpes simplex virus DNA. Virology
141, 77-88.
DAVISON, M. J., PRESTON, V. G. & MCGEOCH, D. J. (1984).
Determination of the sequence alteration in the DNA of the herpes
simplex virus type 1 temperature-sensitive mutant ts K. Journal of
General Virology 65, 859-863.
DELUCA, N. A. &SCHAFFER,P. A. (1987). Activities of herpes simplex
virus type 1 (HSV-1) ICP4 genes specifying nonsense polypeptides.
Nucleic Acids Research 15, 4491-4511.
DELUCA, N. A. & SCHAEFER, P. (1988). Physical and functional
domains of the herpes simplex virus transcriptional regulatory
protein ICP4. Journal of Virology 62, 732-743.
DELUCA,N. A., McCARTHY,A. M. &SCHAFFER,P. A. (1985). Isolation
and characterization of deletion mutants of herpes simplex virus type
1 in the gene encoding immediate eady regulatory protein ICP4.
Journal of Virology .56, 558-570.
DIGNAM,J. D., LEBOVITZ,R. M. & ROEDER,R. G. (1983). Accurate
transcription initiation by RNA polymerase II in a soluble extract
from isolated mammalian nuclei. Nucleic Acids Research ll, 14751489.
EVERETT, R. D. (1987). The regulation of transcription of viral and
cellular genes by herpesvirus immediate early gene products.
Anticancer Research 7, 589-604.
EVERETT, R. D. (1987). The regulation of transcription of viral and
cellular genes by herpesvirus immediate early gene products.
Anticancer Research 7, 589-604.
EVERE'I'r, R. D. (1989). Construction and characterization of herpes
simplex type 1 mutants with defined lesions in immediate early gene
1. Journal of General Virology 70, 1185-1202.
FABER, S. W. & WILCOX,K. W. (1986a). Characterization of a herpes
simplex virus regulatory protein: aggregation and phosphorylation
of a temperature-sensitive variant of ICP4. Archives of Virology 91,
297-312.
FABER, S. W. & WILCOX, K. W. (1986b). Association of the herpes
simplex virus regulatory protein ICP4 with specific nucleotide
sequences in DNA. Nucleic Acids Research 14, 6067-6083.
GRUNDY, F. J., BAUMANN,R. P. & O'CALLAGHAN,D. J. (1989). DNA
sequence and comparative analysis of the equine herpesvirus type 1
immediate early gene. Virology 172, 223-236.
HAY, R. T. & HAY, J. (1980). Properties of herpesvirus-induced
"immediate early" polypeptides. Virology 104, 230-234.
KATTAR-COOLEY,P. & WILCOX,K. W. (1989). Characterization of the
DNA-binding properties of herpes simplex virus regulatory protein
ICP4. Journal of Virology 63, 696-704.
LONSDALE,n. M. (1979). A rapid technique for distinguishing herpes
simplex virus type 1 from type 2 by restriction enzyme technology.
Lancet i, 849-852.
McGEOCH, D. J., DOLAN,A., DONALD,S. & BRAUER,D.' H. K. (1986).
Complexe DNA sequence of the short repeat region in the genome of
herpes simplex virus type 1. Nucleic Acids Research 14, 1727-1764.
McGEOCH, D. J., DALRYMPLE,M. A., DAVISON,A. J., DOLAN, A.,
FRAME, M. C., MCNAB, D., PERRY, L. J., SCOTT,J. E. & TAYLOR,P.
(1988). The complete DNA sequence of the long unique region in the
genome of herpes simplex virus type 1. Journal of General Virology 69,
1531-1574.
MCKNIGHT,J. L., KRISTIE,T. M. & ROIZMAN,B. (1987). Binding of the
virion protein mediating ct gene induction in herpes simplex virus
type 1-infected cells to its cis site requires cellular proteins.
Proceedings of the National Academy of Sciences, U.S.A. 84, 70617065.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59
Vmw175 serine-rich region deletion mutant
MARSDEN, H. S., STOW, N. S., PRESTON, V. G., T1MBURY,M. C. &
WILKIE, N. i . (1978). Physical mapping of herpes simplex virusinduced polypeptides. Journal of Virology 28, 624-642.
METZLER,D. W. & WILCOX,K. W. (1985). Isolation of herpes simplex
virus regulatory protein ICP4 as a homodimeric complex. Journal of
Virology 55, 329-337.
MICHAEL,N., SPECTOR,D., MAVROMARA-NAZOS,P., KRISTIE,T. M. &
ROIZMAN, B. (1988). The DNA-binding properties of the major
regulatory protein ct4 of herpes simplex virus. Science 239, 15311534.
Mf3LLER, M. T. (1987). Binding of the herpes simplex virus immediate
early gene product ICP4 to its own transcription start site. Journalof
Virology 61, 858-865.
O'HARE, P. & GODING, C. R. (1988). Herpes simplex virus regulatory
elements and the immunoglobulin octamer domain birfd a common
factor and are both targets for virion transactivation. Cell 52, 435445.
PATERSON,T. (1989). A mutational analysis of the structure andfunction
of the herpes simplex virus immediate early protein Vmw175. Ph.D.
thesis, University of Glasgow,
PATERSON,T. & EVERETT,R. n. (1988a). Mutational dissection of the
HSV-1 immediate early protein Vmw175 involved in transcriptional
transactivation and repression. Virology 166, 186-196.
PATERSON, T. & EVERETT, R. n. (1988b). The regions of the herpes
simplex virus type 1 immediate early protein Vmw175 required for
site-specific DNA-binding closely correspond to those involved in
transcriptional regulation. Nucleic Acids Research 16, 11005-11025.
PATERSON,T., PRESTON,V. G. & EVERETT,R. n. (1990). A mutant of
herpes simplex virus type 1 immediate early polypeptide Vmw175
binds to the cap site of its own promoter in vitro but fails to
autoregulate in FiFo.Journal of General Virology 71, 851-861.
PEREIRA, L., WOLFF, M. H., FENWICK, M. & ROIZMAN, B. (1977).
Regulation of herpesvirus macromolecular synthesis. V. Properties
of polypeptides made in HSV-1- and HSV-2-infected cells. Virology
77, 733-749.
PERRY, L. J., RIXON, F. J., EVERETT, R. D., FRAME, M. C. &
McGEOCH, D. J. (1986). Characterization of the IEll0 gene of
herpes simplex virus type 1. Journal of General Virology 67, 23652380.
POWELL, K. L. & PURIFOY,D. J. M. (1976). DNA-binding proteins of
ceils infected by herpes simplex-virus types 1 and 2. lntervirology 7,
225-239.
PRESTON,C. M. (1979). Control of herpes simplex virus type l mRNA
synthesis in cells infected with wild-type virus or the temperaturesensitive mutant tsK. Journal of Virology 29, 275-284.
PRESTON,C. M. & NOTARIANNI,E. L. (1983). Poly(ADP-ribosyl)ation
of a herpes simplex virus immediate early polypeptide. Virology 131,
492-501.
PRESTON, C. M., FRAME, M. C. & CAMPBELL, M. E. M. (1988). A
complex formed between cell components and an HSV structural
polypeptide binds to a viral immediate early gene regulatory
sequence. Cell 52, 425-434.
PRESTON, V. G., DAVISON, A., MARSDEN, H. S., TIMBURY, M. C.,
SUBAK-SHARPE, J. H. & WILKIE, N. M. (1978). Recombinants
between herpes simplex virus types 1 and 2: analysis of genome
structures and expression of immediate early polypeptides. Journalof
Virology 28, 499-517.
RIGBY, P. W. J., DIECKMAN, M., RHODES, C. & BERG, P. (1977).
1783
Labelling deoxyribonucleic acid to high specific activity in vitro by
nick translation with DNA polymerase I. Journal of Molecular
Biology 113, 237-251.
ROBERTS,M. S., BOUNDY,A., O'H~d~E, P., PIZZORNO,M. C., CIUFO,D.
M. & HAYW~tD, G. S. (1988). Direct correlation between a negative
autoregulatory response element at the cap site of the herpes simplex
virus type 1 IE175 (c~4)promoter and a specific binding site for the
IE175 (ICP4) protein. Journal of Virology 62, 4307-4320.
RUSSELL, J., STOW, E. C., STOW, N. D. & PRESTON, C. M. (1987).
Abnormal forms of the herpes simplex virus immediate early
polypeptide Vmw175 induce the cellular stress response. Journal of
General Virology68, 2397-2406.
SCHRODER, C. I"I., DEZAZZO, J., KNOPF, K. W., KAERNER, H. C.,
LEVlNE, M. & GLORIOSO,J. (1985). A herpes simplex virus type 1
mutant with a deletion in the polypeptide-coding sequences of the
ICP4 gene. Journal of General Virology66, 1589-1593.
SHEPARD, A. A., IMBALZANO, A. N. & DELUCA, N. A. (1989).
Separation of primary structural components conferring autoregulation, transactivation, and DNA-binding properties to the herpes
simplex virus transcriptional regulatory protein ICP4. Journal of
Virology 63, 3714-3728.
SHOWALTER, L. D., ZWEIG, M. & HAMF~d~, B. (1981). Monoclonal
antibodies to herpes simplex type 1 proteins including the immediate
early protein ICP4. Infection and Immunity 34, 684-692.
SOUTHERN,E. M. (1975). Detection of specific sequences among DNA
fragments separated by gel electrophoresis. Journal of Molecular
Biology 98, 503-517.
STOW, N. D. & STOW, E. C. (1986). Isolation and characterization of a
herpes simplex virus type 1 mutant containing a deletion within the
gene encoding the immediate early polypeptide Vmw110. Journal of
General Virology 67, 2571-2585.
STOW, N. D. & WILKIE, N. M. (1976). An improved technique for
obtaining enhanced infectivity with herpes simplex virus type 1
DNA. Journal of General Virology 33, 447-458.
STOW, N. D., McMONAGLE,E. C. & DAVlSON,A. J. (1983). Fragments
from both termini of the herpes simplex virus type 1 genome contain
signals required for the encapsidation of viral DNA. Nucleic Acids
Research 11, 8205-8220.
TEDDER, D. G., EVERETT,R. D., WILCOX, K. W., BEARD, P. & PIZER,
L. I. (1989). ICP4-binding sites in the promoter and coding regions of
the herpes simplex virus gD gene contribute to activation of in vitro
transcription by ICP4. Journal of Virology 63, 2510-2520.
VLCEK,C., PACES,V. & SCHWYZER,M. (1989). Nucleotide sequence of
the pseudorabies virus immediate early gene, encoding a strong
trans-activator protein. Virus Genes 2, 335 346.
WAGNER, E. K. (1985). Individual HSV transcripts: characterization
of specific genes. In The Herpesviruses, vol. 3, pp. 45-104. Edited by
B. Roizman. New York & London: Plenum Press.
WILCOX, K. W., KOHN, A., SKLYANSKAY,E. & ROIZMAN,B. (1980).
Herpes simplex virus phosphoproteins. I. Phosphate cycles on and
off some viral polypeptides and can alter their affinity for DNA.
Journal of Virology 33, 167 182.
WILKIE, N. M. (1973). The synthesis and substructure of herpesvirus
DNA : the distribution of alkali-labile single strand interruptions in
HSV-1 DNA. Journal of General Virology 21, 453-467.
(Received 23 November 1989; Accepted 27 March 1990)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:15:59