Previous Article | Next Article 
Journal of Virology, August 2000, p. 7362-7374, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Conserved Carboxyl-Terminal Half of Herpes Simplex Virus
Type 1 Regulatory Protein ICP27 Is Dispensable for Viral Growth in
the Presence of Compensatory Mutations
Scott M.
Bunnell1 and
Stephen A.
Rice1,2,*
Department of Biochemistry, University of
Alberta, Edmonton, Alberta, Canada T6G 2H7,1 and
Department of Microbiology, University of Minnesota Medical
School, Minneapolis, Minnesota 554552
Received 9 March 2000/Accepted 24 May 2000
 |
ABSTRACT |
ICP27 is an essential herpes simplex virus type 1 (HSV-1)
immediate-early protein that regulates viral gene expression by poorly
characterized mechanisms. Previous data suggest that its carboxyl
(C)-terminal portion is absolutely required for productive viral
infection. In this study, we isolated M16R, a second-site revertant of
a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion,
as demonstrated by the fact that its cloned ICP27 allele can complement
the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing
demonstrated that the intragenic reversion is a frameshift alteration
in a homopolymeric run of C residues at codons 215 to 217. This results
in the predicted expression of a truncated, 289-residue molecule
bearing 72 novel C-terminal residues derived from the +1 reading frame.
Consistent with this, M16R expresses an ICP27-related molecule of the
predicted size in the nuclei of infected cells. Transfection-based
viral complementation assays confirmed that the truncated, frameshifted
protein can partially substitute for ICP27 in the context of viral
infection. Surprisingly, its novel C-terminal residues are required for
this activity. To see if the frameshift mutation is all that is
required for M16R's viability, we re-engineered the M16R ICP27 allele
and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed
which possesses the frameshift in the context of an ICP27 gene with the
C terminus deleted. We found that both M16exC and exCd305
are nonviable in Vero cells, suggesting that one or more extragenic
mutations are also required for the viability of M16R. Consistent with
this interpretation, we isolated two viable derivatives of
exCd305 which grow productively in Vero cells despite being
incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the
truncated, frameshifted ICP27 protein can enhance viral DNA
replication. In summary, our results demonstrate that the C-terminal
portion of ICP27, conserved widely in herpesviruses and previously
believed to be absolutely essential, is dispensable for HSV-1 lytic
replication in the presence of compensatory genomic mutations.
| |
INTRODUCTION |
Herpes simplex virus type 1 (HSV-1),
one of the most intensively studied and best-characterized
herpesviruses, serves as a prototype for understanding how these
important DNA viruses replicate in their host eukaryotic cells. The
HSV-1 genome is composed of ~152,000 bp of double-stranded DNA
and encodes approximately 80 proteins (for a review, see
reference 32). During lytic infection, the
viral genes are transcribed in the cell nucleus by cellular RNA
polymerase II. However, despite its dependence on the host cell,
HSV-1 is able to impose tight regulatory control on its genes
such that they are expressed in a coordinately activated cascade
consisting of three temporal phases known as the immediate-early (IE,
also known as 
), delayed-early (DE, also known as 
), and late (L,
also known as 
) phases (reviewed in reference
40).
Approximately half of the HSV-1 genes can be considered essential in
that they are absolutely required for productive infection of cultured
cells (32). One gene which is clearly in this category is
that encoding infected cell protein ICP27. ICP27 is a 512-residue IE
protein which is localized predominantly to infected cell nuclei. Viral
mutants that make temperature-sensitive forms of ICP27 fail to
replicate at the nonpermissive temperature (33).
Furthermore, mutants with the ICP27 gene deleted are completely
nonviable in tissue culture but can be propagated in engineered cell
lines which possess a stably transfected ICP27 gene (14,
27).
Characterization of a number of viral ICP27 mutants has indicated that
ICP27 performs several regulatory functions during viral lytic
infection. First, it is a critical activator of viral gene expression.
In the absence of functional ICP27, many DE and L viral genes are not
expressed efficiently as mRNAs or proteins (14, 15, 27, 29, 33,
39). Second, ICP27 represses viral IE and DE genes at late times
after infection (14, 27, 33). Third, ICP27 contributes to
the shutoff of host gene expression, as both cellular protein synthesis
and mRNA levels are elevated in ICP27 mutant infections compared to
wild-type (WT) HSV-1 infection (8, 33, 38). Fourth, ICP27
stimulates viral DNA replication by approximately 10-fold (14,
27). This function is likely attributable to ICP27's ability to
transactivate several DE genes which encode viral DNA replication
factors (15, 39). Fifth, in human cells, ICP27 prevents
virus-induced apoptosis (2). It is not known whether
this function results from ICP27's ability to modulate viral or
cellular genes.
Despite its many potent regulatory effects, the molecular mechanism(s)
by which ICP27 carries out its activities is largely uncharacterized.
However, considerable evidence indicates that at least some of its
effects on gene expression occur at the posttranscriptional level. A
variety of studies have demonstrated that under certain experimental
conditions, ICP27 can (i) modulate the efficiency of pre-mRNA
polyadenylation (15, 16, 36), (ii) alter the intranuclear
distribution of pre-mRNA splicing factors (23, 35),
(iii) inhibit pre-mRNA splicing (10), (iv) increase the stability of specific cellular mRNAs (4), and (v)
inhibit the nuclear export of intron-containing viral transcripts
(25). Furthermore, ICP27 has some of the characteristics of
a posttranscriptional regulatory protein. It is able to bind to RNA
both in vitro (4, 12, 20) and in vivo (34),
although it is unclear whether it recognizes a specific target
sequence. Moreover, similar to some other posttranscriptional
regulators, ICP27 shuttles continuously between the nucleus and the
cytoplasm (19, 24, 34, 38). Recently, it has been
suggested that ICP27 has a role in the nuclear export of
intronless viral mRNAs (34, 38).
The functional domains of ICP27 have not been clearly delineated, but
numerous studies suggest that the C-terminal half of the protein (from
approximately residue 260 to the C terminus at residue 512) is
especially critical. Mutagenesis experiments have indicated that stop
codon insertions, in-frame linker insertions, in-frame deletions, or
alterations of selected codons in the C-terminal half of the ICP27 gene
all disrupt the protein's ability to modulate reporter genes in
transfection assays (9, 17, 31). Moreover, many C-terminal
mutations are lethal when introduced into recombinant viruses (17,
27, 28) or when tested in plasmid-based viral complementation
assays (17, 31). Interestingly, genomic sequence analyses
have suggested that all mammalian and avian herpesviruses encode a
protein with homology to the C-terminal ~200 residues of ICP27
(3, 4, 22). In contrast, the N-terminal portion of ICP27 is
not closely conserved among herpesviruses. Taken together, these
studies suggest that the C-terminal half of ICP27 has a fundamental
role in one or more of its essential regulatory functions.
This report describes a genetic study which was initiated to focus on
the C-terminal region of ICP27. We utilized the HSV-1 ICP27 mutant M16,
which fails to replicate in cultured cells due to an amino acid
alteration at residue 488, near ICP27's C terminus (28).
Our goal was to isolate second-site revertants with the idea that such
mutants might provide clues to ICP27's tertiary structure and possibly
identify viral proteins that interact with the C terminus. We were
successful in isolating a viable second-site revertant, designated
M16R. However, analysis of M16R led to a very surprising finding: this
mutant harbors a frameshift mutation in the N-terminal half of the
ICP27 gene and thus does not apparently express the C-terminal half of
ICP27. Further analysis of M16R and other, similar mutants has led us
to the quite unexpected conclusion that the C-terminal half of ICP27 is
not absolutely required for the lytic growth of HSV-1 in cultured
cells. This finding provides novel insight into ICP27's functions and
functional domains.
| |
MATERIALS AND METHODS |
Cells and viruses.
Vero cells, obtained from the American
Type Culture Collection, and V27 cells, Vero cell derivatives which
contain a stably transfected copy of the ICP27 gene (27),
were maintained in Dulbecco's minimal essential medium (DMEM)
supplemented with 5% fetal bovine serum (FBS) and 5% FBS plus
geneticin (G418; Gibco-BRL) at 100 µg/ml, respectively. WT HSV-1
strain KOS1.1 (11) was propagated in Vero cells. The HSV-1
ICP27 mutants d27-1 (27) and M16 (28)
were propagated in V27 cells. The isolation and propagation of other
HSV-1 mutants are described below.
For isolation of M16R, six M16 plaques were isolated in V27 cells.
These were subjected to three rounds of freeze-thawing, and a portion
of each was passaged in Vero cells growing in 25-cm2
flasks. After 48 h, the cells were subjected to freeze-thawing and
the resulting lysates were passaged on 25-cm2 flasks of
Vero cells. Cytopathic effects (CPE) indicative of HSV-1 infection
appeared in one flask 3 days postinfection. A viral isolate from this
flask was plaque purified one time in Vero cells and designated M16R.
Restriction analysis of a PCR fragment derived from M16R DNA confirmed
that the virus retains the XhoI site which marks the
original M16 mutation. M16R was further plaque purified in Vero cells,
and a low-titer stock was prepared in Vero cells. High-titer stocks
were prepared in V27 cells.
The HSV-1 ICP27 mutants M16exC,
exCd305, and
n217d were engineered by in vivo homologous recombination
using a previously
described protocol (
27,
28), with the
exception that transfections
were carried out using the Lipofectamine
reagent (Gibco-BRL) as
described below. For each engineered virus, two
viral isolates
were obtained from independent transfections. The ICP27
gene structures
of all isolates were confirmed by extensive PCR
analysis.
For isolation of R1exCd305 and R2exCd305, four
exCd305
plaques were isolated in V27 cells. These were amplified once by
passage
in V27 cells growing in 3.8-cm
2 wells of a 12-well
plate. At 6 days postinfection, virus was
released by freeze-thawing
and a portion of each lysate was passaged
in 75-cm
2 flasks
of Vero cells for 2 days. Infected cell lysates were prepared
by
freeze-thawing and passaged sequentially up to three times
in
25-cm
2 flasks of Vero cells for 4 to 5 days per
passage. By passage
three, three of the four lysates had yielded
cultures which exhibited
characteristic HSV-1 CPE. DNA was
isolated from the positive flasks,
and the ICP27 gene was amplified
from the DNA samples by PCR.
One sample yielded an ICP27 gene PCR
fragment with no deletion,
suggesting that the virus present had a WT
ICP27 allele, possibly
obtained by recombination of
exCd305
with the stably transfected
ICP27 gene of V27 cells. However, the
other two DNA samples yielded
ICP27 gene PCR products which still
possessed the
exCd305 deletion,
indicating the
presence of second-site revertants. Viral isolates
from each of
the two positive lysates were plaque purified three
times in V27 cells
and designated R1exCd305 and R2exCd305. Mutants
stocks were prepared in
V27
cells.
Plasmid constructs.
All plasmids used for complementation
experiments possess the ICP27 gene within a
BamHI-SacI HSV-1 fragment. In the case of the WT
virus, this fragment is 2.4 kb. The plasmids were generated by the
following manipulations. Plasmids p27 and pM1627 contain the ICP27
alleles of WT HSV-1 (strain KOS1.1) and M16, respectively, and were
constructed by cloning the 2.4-kb BamHI-SacI
fragments from pM27 (28) and pM16 (28) into
pUC19. Plasmid pM16R was created by cloning the 2.4-kb
BamHI-SacI fragment of M16R, isolated from viral
genomic DNA, into pUC19. Plasmids pWTexC and pM16exC are derivatives of
plasmid pM27exC. pM27exC was constructed by oligonucleotide-directed
mutagenesis (Altered Sites system; Promega) starting from pM27.
Relative to pM27, it possesses a single base pair insertion at codons
215 to 217, converting the run of eight coding strand cytosines to
nine. pWTexC was created by three-fragment ligation involving the
2.7-kb SacI-BamHI fragment of pUC19, the 1.2-kb
BamHI-SalI ICP27 gene fragment from pM27exC, and
the 1.2-kb SalI-SacI ICP27 allele fragment from
p27. pM16exC was engineered using the same strategy, except that the
1.2-kb SalI-SacI fragment was derived from
pM1627. To construct plasmid pexCn406, a 12-bp NheI linker
containing stop codons in all three reading frames (New England
Biolabs) was cloned into the StuI site of pWTexC. pWTn406
was constructed by a three-fragment ligation involving the 2.7-kb
SacI-BamHI fragment from pUC19, the
BamHI-SalI fragment from p27, and the
SalI-SacI fragment from pexCn406. Plasmids
pexCd305 and pn217d possess a C-terminal deletion in the ICP27 coding
region. To create this deletion, plasmid pPsd10-11 (V. Leong and
S. A. Rice, unpublished data, 2000), was utilized. This plasmid
contains an ICP27 allele which has an engineered XhoI site
at codon 304 and lacks codons 306 to 339. The C-terminal deletion was
engineered by digesting pPsd10-11 with XhoI and
EcoNI, which cuts at the 3' end of the ICP27 coding region.
The 3'-recessed ends were filled in using the large fragment of
Escherichia coli DNA polymerase I and then ligated together.
This gave rise to pPsd305, in which the ICP27 gene is missing codons
306 to 512. Plasmid pexCd305 is the product of three-fragment ligation
of the 2.7-kb SacI-BamHI fragment from pUC19, the
1.2-kb BamHI-SalI fragment from pM27exC, and the
0.6-kb SalI-SacI fragment from pPsd305. To create
pn217d, oligonucleotide-directed mutagenesis was first used to alter
pM27 such that an ochre termination codon was created in the ICP27 coding region at codon 218, resulting in plasmid pMn217d. To generate pn217d, three-fragment ligation was carried out using the 1.2-kb BamHI-SalI fragment of pMn217d, the 0.6-kb
SalI-SacI fragment from pPsd305, and the 2.7-kb
SacI-BamHI fragment from pUC19. Plasmids pd305R1
and pd305R2 were created by cloning the ICP27 gene-containing BamHI-SacI fragments from the R1exCd305 and
R2exCd305 viral DNAs, respectively, into pUC19.
The plasmids employed for recombinant HSV-1 production possess the
ICP27 gene on a
PstI HSV-1 DNA fragment, which in the case
of WT virus is 6.0 kb. The plasmids used to engineer the virus
mutants
M16exC,
exCd305, and
n217d were pPsM16exC,
pPsexCd305,
and pPsn217d, respectively. They were constructed by
cloning the
ICP27 gene-containing
BamHI-
SacI
fragments from pM16exC, pexCd305,
and pn217d, respectively, into
pPs27pd1 (
31) in place of the
corresponding WT
fragment.
The
BamHI-
SacI fragments of pM16R, pd305R1, and
pd305R2 were sequenced in their entirety. In addition, the structures
of the
engineered ICP27 gene mutations in pMn217d and pM27exC were
confirmed
by DNA sequencing. Sequencing was performed at the
Biochemistry
Core DNA facility of the University of Alberta using an
Applied
Biosystems 373A DNA
Sequencer.
Complementation assay.
The d27-1 viral
complementation assays were carried out as described previously
(29, 31), except that transfections were carried out with 1 µg of plasmid DNA using the Lipofectamine reagent (Gibco-BRL).
Lipofectamine transfections for complementation assay and virus
constructions were carried out as follows. Purified DNA was mixed with
serum-free DMEM to give a final volume of 200 µl. Separately, 10 µl
of Lipofectamine reagent per µg of DNA was mixed with serum-free DMEM
to give a volume of 200 µl. The two mixtures were combined and
allowed to sit for 15 to 45 min at room temperature. Mixtures were then
made up to a final volume of 2 ml with serum-free DMEM and added to
25-cm2 flasks of Vero (for complementation assays) or V27
(for virus construction) cells that had been rinsed with serum-free
DMEM. After a 5-h incubation at 37°C, the transfection mixtures were removed and replaced with DMEM containing 5% FBS.
Analysis of mutant viruses.
To study the growth of mutant
viruses, viral plaque assays and single-cycle yield analyses were
performed. For HSV-1 plaque assays, virus stocks were titrated on Vero
or V27 monolayers using an overlay of medium 199 (Gibco-BRL) containing
1% heat-inactivated calf serum and 1% pooled normal human serum
(ICN). Representative plaques or foci were photographed using an
Olympus SC 35 Type 12 camera mounted on an Olympus CK40 microscope.
Single-cycle growth assays were carried out as described previously
(28, 29). Briefly, parallel cultures of Vero and V27 cells
were infected with virus at a multiplicity of infection (MOI) of 10 and
treated with a glycine-saline solution (pH 3.0) at 2 h
postinfection (hpi) as already described to inactivate unadsorbed virus
(5). Following incubation for 24 h, the virus yield was
determined by plaque assay of the cell lysates on V27 cells.
An immunofluorescence assay was used to determine the localization of
mutant ICP27 proteins. Vero cells growing on coverslips
were infected
at an MOI of 10. At 4 hpi, these were processed
for indirect
immunofluorescence assay as described previously
(
26).
Monoclonal antibody H1113 (
1) (Goodwin Institute for
Cancer
Research, Plantation, Fla.), which recognizes residues
109 to 137 of
ICP27 (
18), was used at a dilution of 1:600. The
secondary
antibody was tetramethyl rhodamine isothiocyanate-conjugated
goat
anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories
Inc.,
Mississauga, Ontario, Canada) diluted 1:200. The cells were
visualized
with a Zeiss Axioskop 20 fluorescence microscope equipped
with a
Plan-Neofluar 63× objective
lens.
Mutant ICP27 polypeptides were characterized by immunoblotting. Vero
cells growing in 25-cm
2 flasks were infected at an MOI of
10 and harvested at 4 or 6
hpi as described previously (
30).
Total proteins were separated
by sodium dodecyl sulfate (SDS)-15%
polyacrylamide gel electrophoresis
and transferred to nitrocellulose
membranes. The membranes were
blocked with 10% skim milk in TBST (1×
Tris-buffered saline, 0.2%
Tween 20), washed twice with TBST, and then
probed with a 1:1,000
dilution of H1113 in TBST for 45 min. After
washing of the filters
twice with TBST, immunoreactive proteins were
visualized using
a horseradish peroxidase-conjugated secondary antibody
diluted
1:3,000 in TBST and an enhanced chemiluminescence system
(Amersham).
Viral DNA for PCR and Southern analysis was prepared as follows. Cell
monolayers growing in wells of 12-well plates were infected
at an MOI
of 10. For analysis of viral genome structures, V27
cells were used
whereas Vero cells were used for the DNA replication
experiments. At
various times after infection, the infected cells
were lysed by the
addition of 400 µl of 10 mM Tris (pH 8.0)-10
mM EDTA-2% SDS-100
µg of proteinase K per ml. Following incubation
at 37°C for a day,
48 µl of 3 M sodium acetate (pH 5.2) was added
to the lysates, which
were then extracted once with phenol-chloroform-isoamyl
alcohol
(25:24:1) and once with chloroform-isoamyl alcohol (24:1).
DNA was
precipitated with 95% ethanol, resuspended in 10 mM Tris
(pH 7.6)-1
mM EDTA, and subjected to RNase A (50 µg/ml) digestion
at 37°C for
1
h.
Southern analyses were carried out as follows. Equal masses of DNA
extracted from infected cells were cleaved with
PstI and
SalI and separated on a 1% agarose gel. Following acid
cleavage
and alkali denaturation, DNA was transferred to a nylon
membrane
and hybridized with a
32P-labeled probe generated
by random-primer labeling. Hybridization
was carried out in 250 mM
sodium phosphate (pH 7.2)-7% SDS-1%
bovine serum albumin-1 mM EDTA
at 65°C for approximately 12 h.
The ICP27 gene N
terminus-specific probe was a 510-bp PCR fragment
corresponding to
ICP27 gene N-terminal codons 90 to 259. The C
terminus-specific probe
was a 588-bp PCR fragment corresponding
to codons 308 to 503. The probe
used for analysis of viral DNA
replication was a 480-bp PCR fragment
corresponding to a sequence
at the 5' end of the UL55 gene. This
sequence encompasses 55 bp
upstream of the UL55 coding region, as well
as codons 1 to 142.
Membranes were washed twice for 15 min (each time)
at 68°C in
2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate)-0.1%
SDS and twice for 15 min (each time) at 68°C in 0.1×
SSC-0.1%
SDS prior to film exposure. Signal quantitation was done by
phosphorimage
analysis using a Fujix BAS100 bioimaging analyzer with
MacBAS
imaging
software.
| |
RESULTS |
Isolation of M16R.
As reviewed in the introduction, much
previous work suggests that the C-terminal portion of HSV-1 ICP27 is
essential for its function. To investigate this important region of the
protein, we set out to isolate second-site revertants of M16, an HSV-1 mutant with a C-terminal ICP27 mutation (28). The
dinucleotide alteration in M16 changes residue 488 of ICP27 from
cysteine to leucine (Fig. 1A and B). As a
result, M16 is completely unable to replicate in Vero cells but can
replicate efficiently in V27 cells, which possess a stably transfected
ICP27 gene (27). To select revertants, six M16 plaques were
generated in V27 cells and used to infect small cultures (approximately
3 × 106 cells) of Vero cells. After 2 days, the cells
were disrupted by freeze-thawing and a portion of each lysate was
passaged on a new set of Vero cells. These cultures were monitored over
several days for the appearance of CPE characteristic of HSV-1
infection. Such CPE were observed in one culture. A virus from this
culture was plaque purified in Vero cells and designated M16R. To test whether M16R is a second-site revertant, PCR analysis of its genome was
carried out. This analysis showed that the 3' end of the M16R ICP27
gene retains the original dinucleotide mutation of M16, which is marked
by an XhoI site (Fig. 1B; data not shown). Therefore, M16R
is a second-site revertant of M16.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 1.
Structures of ICP27 alleles and the proteins they
encode. Each line denotes an ICP27 allele used in this study. The bars
above the lines represent the encoded ICP27 polypeptide; white bars
indicate ICP27 sequences, whereas gray bars denote sequences translated
from the +1 reading frame.
|
|
The growth properties of M16R were tested in a viral plaque assay, with
WT HSV-1 and M16 serving as controls (Table
1, experiment
1). Consistent with
previous results (
28), WT HSV-1 formed plaques
efficiently
on both Vero and V27 cells whereas M16 was unable
to form plaques on
Vero cells. M16R formed plaques efficiently
in both Vero and V27 cells,
similar to the WT virus. However,
the M16R plaques were considerably
smaller than WT plaques (Fig.
2A),
suggesting that M16R does not replicate or spread as well
as WT HSV-1
in Vero cells.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 2.
Growth of M16R in Vero cells. (A) Monolayers of Vero
cells were infected with serial dilutions of WT HSV-1 or M16R. After 4 days, photographs of individual plaques were taken. (B) Replicate
cultures of approximately 3 × 106 Vero or V27 cells
were infected in duplicate at an MOI of 10 with the viral strains
indicated. At 24 hpi, the infections were terminated by freezing and
infectious virus was released by three cycles of freeze-thawing. The
amount of infectious virus in each lysate was determined by plaque
assay of the lysate on V27 cells and divided by the number of infected
cells to determine the yield per cell.
|
|
To further study M16R's growth properties, single-cycle viral yield
assays were carried out. Vero or V27 cells were infected
in duplicate
with WT HSV-1, M16, or M16R at an MOI of 10, and
the infections were
allowed to proceed for 24 h. Viral yields
were determined by
plaque assay of the cell lysates on V27 cells
(Fig.
2B). As
expected, WT HSV-1 replicated efficiently in either
cell line
whereas M16 could only replicate in V27 cells. The growth
phenotype of
M16R was distinct from that of the two other viruses.
In Vero cells,
its yield was ~100-fold higher than that of M16,
consistent with its
ability to form plaques in these cells. However,
its yield was
~100-fold reduced compared to that of the WT. M16R
replicated
~10-fold more efficiently in V27 cells than in Vero
cells, indicating
that expression of WT ICP27 can enhance its
growth. Taken together, the
data from the plaque formation and
yield experiments demonstrate that
M16R can grow productively
in Vero cells, albeit not as efficiently as
WT HSV-1.
Identification of a frameshift mutation in M16R.
A second-site
reversion mutation in the M16R genome could be either in the ICP27 gene
(intragenic) or outside of it (extragenic). To test whether M16R has an
intragenic reversion, we cloned its ICP27 allele and tested it in a
viral complementation assay for the ability to complement the growth of
d27-1, a viral ICP27 null mutant (27, 29). To
carry out the assay, Vero cells were transfected in duplicate with
pUC19 or a pUC19 derivative bearing the WT, M16, or M16R ICP27 allele.
One day after transfection, the cells were infected with
d27-1. Progeny virus were harvested after an additional day,
and titers were determined by plaque assay on V27 cells (Fig.
3A). Consistent with previous assays
(28), the WT ICP27 plasmid dramatically enhanced the growth
of d27-1 (>105-fold increase in viral titer).
In contrast, the M16 allele was completely unable to support
d27-1 growth. The M16R ICP27 plasmid exhibited significant
complementation activity, increasing the growth of d27-1 by
~1,000-fold. Since the ICP27 gene is the only intact HSV-1 gene on
the transfected plasmid, this experiment suggests that M16R contains an
intragenic reversion. To confirm this, we sequenced the entire 2.4-kb
viral insert of the M16R plasmid. Only two differences from the
sequence of WT strain KOS1.1 (which was determined in parallel) were
found. First, as expected from the PCR analysis, the M16R ICP27 allele
retains the original M16 dinucleotide alteration at codon 488. Second,
whereas the WT gene has a run of eight C residues in the coding strand
at codons 215 to 217, the M16R gene has a run of nine C's (Fig.
4A). The insertion results in a
frameshift mutation, such that the M16R ICP27 allele is expected to
encode a 289-residue polypeptide consisting of the first 217 residues
of ICP27 fused to 72 novel C-terminal residues derived from the +1
reading frame (Fig. 4B).
View larger version (54K):
[in this window]
[in a new window]
|
FIG. 3.
Complementation of d27-1 by cloned ICP27
alleles. Replicate cultures of Vero cells were transfected in duplicate
with pUC19 or pUC19 derivatives bearing various ICP27 alleles. One day
after transfection, the cultures were infected with the HSV-1 deletion
mutant d27-1 and incubated for a further day. Virus was
released from the cultures by three cycles of freeze-thawing, and the
titers of the resulting lysates were determined by plaque assay of the
lysates on V27 cells.
|
|
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 4.
The M16R frameshift mutation leads to the expression of
a truncated, nucleus-localized ICP27 molecule. (A) The frameshift
mutation of M16R. The coding strand sequences of codons 214 to 218 are
shown for both the WT and M16R ICP27 alleles. The WT gene has a run of
eight C residues; the M16R gene has nine. This leads to alteration of
the encoded protein at residue 218 as shown. (B) Amino acid sequence
(in single-letter code) of the frameshifted portion of the M16R ICP27
protein (residues 218 to 289). (C) Immunoblot analysis of the M16R
protein. Vero cells were mock infected or infected with the viruses
indicated. At 4 hpi, total proteins were harvested and analyzed by
Western blot analysis using monoclonal antibody H1113, which recognizes
residues 109 to 137 of ICP27. The molecular masses of marker proteins
are indicated at the right. (D) Immunofluorescence analysis of the M16R
protein. Vero cells were mock infected or infected with the WT, M16, or
M16R virus, as indicated. At 4 hpi, the cells were fixed,
permeabilized, and processed for immunofluorescence using the H1113
antibody.
|
|
This result was quite surprising, as much previous work suggests that
the C-terminal portion of ICP27 is required for viral
growth. To see if
M16R expresses the predicted 289-residue protein,
we carried out an
immunoblot analysis. Vero cells were mock infected
or infected with WT,
M16, or M16R. Total proteins were isolated
at 4 hpi and subjected to
immunoblotting using H1113, a monoclonal
antibody which recognizes
residues 109 to 137 of ICP27 (
18).
As can be seen in Fig.
4C, WT and M16 express intact ICP27 molecules
of ~63 kDa whereas M16R
expresses a truncated molecule of ~38
kDa, a size consistent with
that expected for the 289-residue
protein. Immunofluorescence analysis
was also carried out to determine
the intracellular localization of the
M16R protein. Vero cells
were mock infected or infected with WT HSV-1,
M16, or M16R. At
4 hpi, the cells were fixed and processed for
immunofluorescence
using the H1113 antibody. The polypeptides expressed
by all three
viruses were predominantly nuclear but differed in
intranuclear
localization (Fig.
4D). WT ICP27 was distributed
throughout the
nucleus in a speckled pattern, whereas the M16 protein
was localized
more diffusely and was largely excluded from nucleoli.
The M16R
protein showed a distinct localization pattern in that it was
distributed diffusely in the nucleus but was preferentially localized
to nucleoli. In summary, the results of these analyses indicate
that
M16R expresses a truncated ICP27 molecule which localizes
to the
nucleus in an altered
pattern.
Ability of frameshifted ICP27 alleles to complement
d27-1.
As noted above, it was surprising that the
frameshifted M16R ICP27 gene was able to enhance the growth of
d27-1 in the viral complementation assay. To investigate
this phenomenon further, we carried out a series of experiments using
the viral complementation assay. First, to confirm that the frameshift
is the bona fide intragenic reversion, we used site-directed
mutagenesis to engineer this mutation into a plasmid bearing the M16
mutation. This construct, which we designated pM16exC (Fig. 1D), should
be identical to the original M16R allele. In addition, we introduced
the frameshift mutation into an otherwise WT ICP27 allele, generating
WTexC (Fig. 1E). When tested in the viral complementation assay, both
the M16R and M16exC alleles complemented d27-1 growth by
~1,000-fold (Fig. 3B). This confirms that the frameshift mutation is
the only change required in the M16 allele to endow it with
enhanced complementation activity. The WTexC allele also showed
~1,000-fold complementation, demonstrating that the M16 alteration at
codon 488 is not required.
The above-described results are consistent with the hypothesis that the
complementation activity is mediated by the 289-residue
protein.
However, given the large amount of data which suggest
that the
C-terminal portion of ICP27 is essential for growth,
we wished to rule
out the possibility that this part of the protein
was being expressed
by some mechanism and contributing to the
observed complementation. For
example, the C-terminal region might
be expressed via translational
frameshifting, mRNA splicing, or
the utilization of an internal
ICP27 gene promoter. To help exclude
these possibilities, we engineered
a downstream nonsense mutation
into the frameshifted gene at codon 406. This allele was designated
exCn406 (Fig.
1F). We also constructed a
gene, designated WTn406,
in which the stop codon was engineered into
the otherwise WT gene
(Fig.
1G). When tested in the viral
complementation assay, the
nonsense mutation completely inactivated the
WT ICP27 gene, consistent
with previous results (
17,
31).
However, it had little, if
any, effect on the frameshifted allele,
which again showed ~1,000-fold
complementation (Fig.
3C). The results
suggest that the C-terminal
portion of the ICP27 gene is not involved
in the complementation
activity. To address this issue more
definitively, we engineered
a frameshifted gene in which nearly all of
the C-terminal coding
region (codons 306 to 512) is deleted. This
allele was designated
exCd305 (Fig.
1H). When tested in the
complementation assay, the
exCd305 gene complemented
d27-1 as efficiently as the comparable
non-deletion-containing allele, WTexC (Fig.
3D). Therefore, the
complementation activity of the frameshifted alleles is mediated
by the
289-residue protein, with no contribution from the C-terminal
portion
of
ICP27.
To see if the novel C-terminal residues of the 289-residue protein
(Fig.
4B) play a role in complementation, we engineered
an ICP27 allele
which specifically lacks these residues. This
was done by introducing a
nonsense mutation into the ICP27 gene
at codon 218, which is the site
at which the novel C-terminal
residues begin (Fig.
4A). The
mutation was made in the context
of the codon 306 to 512 deletion, to rule out any possible effect
of the C-terminal portion of
ICP27. The altered gene was designated
n217d (Fig.
1I). When
tested for complementation,
n217d showed
only minimal
activity (~5-fold) whereas the
exCd305 allele again
showed nearly 1,000-fold complementation (Fig.
3E). These results
indicate that the novel 72 C-terminal residues play a critical
role in
the function of the 289-residue
protein.
One or more extragenic mutations are required for the viability of
M16R.
Based on the above findings, we hypothesized that the
intragenic frameshift mutation is sufficient to explain M16R's
viability in Vero cells. To test this, we engineered a recombinant
virus, designated M16exC, in which the WT ICP27 gene was replaced with the M16exC ICP27 allele (Fig. 1D). The M16exC allele was engineered to
be identical to that of M16R. Therefore, if the frameshift mutation is
sufficient for the viability of M16R, then the M16exC virus should be
viable also. Two other recombinant viruses, exCd305 and
n217d, were additionally constructed. These possess the
ICP27 alleles shown in Fig. 1H and 1I, respectively. The recombinant viruses were isolated and amplified in V27 cells to ensure that no
selection would be applied for a functional ICP27 molecule. For each
mutant, two genetically independent isolates were obtained. One isolate
was arbitrarily designated the primary isolate, to be used for
extensive further analysis. The other isolate was designated the
secondary isolate (and given the suffix b), to be used to confirm key
findings. Extensive PCR analysis of all six isolates confirmed that
their ICP27 alleles had the expected genomic structures (not shown).
Experiments were performed to characterize the ICP27-related proteins
expressed by the various mutants. Immunoblotting analysis
was used to
characterize the sizes of the polypeptides. Total
protein samples were
prepared from infected cells at 6 hpi and
analyzed by immunoblotting
using the H1113 antibody (Fig.
5A).
As
expected, both M16exC and
exCd305 expressed a major
ICP27-related
polypeptide which comigrated with that of M16R. In
contrast,
n217d
expressed two major proteins smaller in
size, the larger of which
was roughly consistent with the size expected
for its allele.
In this experiment, both WT and mutant samples showed
multiple
protein bands. We suspect that the faster-migrating species
are
derived from the slower-migrating species by proteolysis, as their
abundance varied between experiments (for example, compare the
M16R
patterns in Fig.
4C and
5). To characterize the localization
of the
mutant polypeptides, immunofluorescence analysis was performed
using
the H1113 antibody. At 4 hpi, all three polypeptides showed
the same
pattern of localization as the M16R protein (shown in
Fig.
4D), i.e.,
predominant nuclear localization with preferential
accumulation in
nucleoli (data not shown). In both the immunoblotting
and
immunofluorescence experiments, the steady-state levels of
the
truncated proteins expressed by M16R, M16exC,
exCd305, and
n217d all appeared approximately equal (Fig.
5A and data not
shown).
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 5.
ICP27-related proteins expressed by mutant viruses. Vero
cells were mock infected or infected with the viruses indicated. At 6 hpi, total proteins were harvested and analyzed by immunoblot analysis
using monoclonal antibody H1113, which recognizes residues 109 to 137 of ICP27. The molecular masses of marker proteins are indicated at the
right. (A) Analysis of M16exC, exCd305, and
n217d. (B) Analysis of R1exCd305 and R2exCd305.
|
|
Next, the growth properties of the engineered viruses was
examined by plaquing analysis (Table
1, experiment 2).
M16exC,
exCd305, and
n217d all formed
plaques efficiently on V27 cells
but were unable to form readily
discernible plaques on Vero cells.
This was in sharp contrast to M16R,
which efficiently formed plaques
on Vero cells. The three new mutants
did form some small foci
of infected cells that could be seen under
microscopic examination
but only when relatively low dilutions of the
viral stocks were
plated. Representative
exCd305 and
n217d foci are shown in Fig.
6C and D, respectively. The foci produced
by
n217d were slightly
smaller than those made by M16exC and
exCd305. Plaque formation
analysis of the secondary isolates
of all three viruses gave essentially
identical results (data not
shown).
View larger version (126K):
[in this window]
[in a new window]
|
FIG. 6.
Morphology of mutant plaques. Vero cell monolayers were
mock infected (A) or infected with serial dilutions of the following
viruses: M16R (B), exCd305 (C), n217d (D),
R1exCd305 (E), and R2exCd305 (F). After 4 days, photographs of
individual plaques or foci were taken.
|
|
Single-cycle viral yield assays were also carried out. Cultures of Vero
cells were infected in duplicate at an MOI of 10 with
WT HSV-1,
d27-1, M16R, M16exC,
exCd305, or
n217d. As controls,
each inoculum was also used to infect
cultures of V27 cells. The
cultures were harvested at 24 h, and
the yields were determined
by plaque assay of the infected cell lysates
on V27 cells (Fig.
7A). In V27 cells, all
infections were comparably productive,
achieving yields of ~100
PFU/cell. In Vero cells, however, the
yields differed dramatically. As
expected, WT HSV-1 replicated
efficiently, producing ~100 PFU/cell;
M16R replicated modestly,
producing ~1 PFU/cell; and
d27-1
did not replicate at all, producing
<0.001 PFU/cell. Consistent with
the results of the plaque formation
analysis, M16exC,
exCd305 and
n217d all replicated
significantly
less efficiently than M16R. The yield of M16exC was
~500-fold
reduced from that of M16R, whereas the yield of
exCd305 was ~100-fold
reduced. The mutant
n217d displayed the most severe growth impairment,
being
essentially indistinguishable from
d27-1 in its inability
to
produce infectious progeny.
View larger version (57K):
[in this window]
[in a new window]
|
FIG. 7.
Replication ability of mutant viruses. Replicate
cultures of Vero or V27 cells were infected in duplicate at an MOI of
10 with the viral strains indicated. At 24 hpi, the infections were
terminated and infectious virus was released by three cycles of
freeze-thawing. The amount of infectious virus in each lysate was
determined by plaque assay on V27 cells. (A) Analysis of M16exC,
exCd305, and n217d. (B) Analysis of R1exCd305 and
R2exCd305.
|
|
Together, the results of the plaque formation and yield analyses lead
to two important insights. The first is based on the
comparison of M16R
and M16exC. Although M16exC was engineered
to have the same ICP27
allele as M16R, it clearly is not viable
in Vero cells. The simplest
interpretation of this finding is
that M16R possesses an extragenic
mutation which is required for
its viability in addition to the
intragenic frameshift alteration.
The second insight is based on a
comparison of the growth phenotypes
of
exCd305 and
n217d. Although neither virus is able to efficiently
form
plaques in Vero cells,
exCd305 produces significantly
higher
yields than
n217d. M16exC, which also expresses
the 289-residue
protein, also replicates significantly better than
n217d. Thus,
it appears that the 289-residue, but not the
217-residue, ICP27
protein is able to enhance viral replication in
infected cells,
although not to a level sufficient for viability. This
finding
is consistent with the results of the viral complementation
assay
(Fig.
3E), which also indicated that the C-terminal frameshift
segment is critical to the function of the truncated
protein.
Isolation of viable revertants of exCd305.
The
above-described results suggest that M16R harbors one or more
extragenic mutations which are required, in addition to its intragenic
frameshift mutation, for viability. The relative ease with which M16R
was isolated suggests that the frequency of such extragenic mutations
is high. To test this hypothesis, we attempted to select viable
revertants of exCd305, which possesses the frameshift
mutation in the context of the C-terminally deleted ICP27 gene.
Four exCd305 plaques, generated on V27 cells, were amplified into small stocks. These were then passaged several times in Vero cells. Using this protocol, two exCd305
plaques eventually yielded cultures that showed HSV-1 CPE and
tested positive in a PCR assay for the presence of the ICP27 gene
with a C-terminally deletion. Viral isolates from these cultures
were purified and designated R1exCd305 and R2exCd305.
Southern blotting was carried out to characterize the ICP27 alleles of
the two new revertants (Fig.
8A). A blot
of
PstI-
SalI-digested
viral DNAs was
hybridized with a probe specific for the N-terminal
portion of the
ICP27 gene (Fig.
8B). As expected, all viruses
except
d27-1
showed a hybridizing 1.8-kb fragment. However, when
a C
terminus-specific probe was used (Fig.
8C), no hybridization
was
observed for either
exCd305 or its revertants whereas
appropriate
hybridizing bands were seen for the other viruses.
Note that
d27-1
shows a hybridizing band of 4.3 kb, similar
to the WT band, since
its deletion removes the
SalI site in
the ICP27 gene and fuses
the shortened
PstI fragments
together. From these data, we conclude
that both R1exCd305 and
R2exCd305 retain the C-terminal deletion
of their parent.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 8.
Southern blot analysis of exCd305 revertants.
(A) Schematic diagram of expected Southern blot results. The arrow at
the top indicates the ICP27 coding region, and the lines denote
relevant restriction fragments in the ICP27 region of the WT and mutant
HSV-1 genomes. (B and C) Viral genomic DNAs were digested with
PstI and SalI and hybridized with a radioactive
probe specific for the N-terminal (B) or C-terminal (C) region of the
ICP27 gene (denoted by gray bars in panel A). The positions of DNA size
standards are shown at the right of each autoradiogram.
|
|
Immunoblotting was used to characterize the ICP27-related polypeptides
synthesized by the revertants (Fig.
5B). Both revertants
expressed
truncated ICP27-related proteins that comigrated with
those
of
exCd305 and M16R and that were expressed at similar
levels.
In addition, immunofluorescence analysis showed that the ICP27
proteins of R1exCd305 and R2exCd305 localized similarly to that
of
their parent; i.e., they exhibited nuclear localization with
preferential accumulation in nucleoli (data not
shown).
Next, the growth phenotypes of R1exCd305 and R2exCd305 were analyzed by
plaque assay. Both mutants differed dramatically from
their parent in
the ability to form plaques on Vero cells (Table
1, experiment 3).
Whereas
exCd305 had a Vero/V27 cell plaque
formation ratio
of <0.000024, R1exCd305 and R2exCd305 had ratios
that approximated 1, similar to that of M16R. This indicates that
the two mutants are viable
in Vero cells. The R1exCd305 plaques
on Vero cells (Fig.
6E) were
approximately the same size as those
made by M16R (Fig.
6B), whereas
the R2exCd305 plaques were somewhat
smaller (Fig.
6F). A minor fraction
of the R2exCd305 plaques displayed
a syncytial morphology; the
significance of this observation is
unknown.
To further characterize the growth abilities of R1exCd305 and
R2exCd305, single-cycle growth assays were carried out (Fig.
7B). The
WT,
d27-1, M16R, and
exCd305 viruses were
included for
comparison. As expected, all grew efficiently in V27
cells. In
Vero cells, R1exCd305 and R2exCd305 produced approximately
100-
and 10-fold more progeny than their parent, respectively. The
growth of R1exCd305 was comparable to that of M16R, consistent
with the
fact the two viruses make plaques similar in size on
Vero cells.
R2exCd305, on the other hand, grew somewhat less efficiently
than
either R1exCd305 or M16R, consistent with its smaller plaque
size.
Together, the data from the plaque formation and yield experiments
demonstrate that both
exCd305 revertants have acquired
mutations
which allow them to grow more efficiently and form plaques in
Vero
cells.
The Southern blot analysis indicates that R1exCd305 and R2exCd305
retain the ICP27 gene C-terminal deletion, but it is possible
that they
have mutations elsewhere in their ICP27 coding sequences.
To exclude
this possibility, the ICP27 coding regions of both
mutants were cloned
and sequenced in their entirety. No mutations,
relative to the
exCd305 allele, were present in either revertant.
Therefore, we conclude that the reversion mutations of both mutants
are
extragenic. We also sequenced 413 bp upstream and 471 bp downstream
of
each coding region. No changes were found for R1exCd305, but
R2exCd305
possessed a single nucleotide mutation 214 bp upstream
of the
ICP27 gene transcriptional start site (a C-to-A alteration).
The
significance of this difference is currently under
investigation.
Viral DNA replication in mutant-infected cells.
Viral DNA
replication is a central event in viral lytic infection, amplifying
viral genomes for later packaging into progeny particles. In addition,
the process of DNA replication confers a cis-acting change
in the viral template which is associated with a switch from DE to L
gene expression (13). ICP27 has an important role in
activating viral DNA replication, as it stimulates the levels of
replicated DNA by 5- to 10-fold (14, 27, 39). As an initial
step in understanding how the intra- and extragenic mutations of M16R
and the other relevant mutants affect the outcome of infection, we
examined the ability of various mutants to replicate their DNA. In the
first experiment, Vero cells were mock or HSV-1 infected and total DNA
was purified from the cultures at either 1 or 12 hpi. Equal amounts of
the DNA were double digested with PstI and SalI
and subjected to Southern blot analysis using an HSV-1-specific probe
(Fig. 9A). To quantitate the degree of
DNA replication by each mutant, the hybridization signal at 12 hpi was
divided by the signal at 1 hpi to determine the fold DNA amplification (Fig. 9B). The results indicated that WT HSV-1 replicated its DNA
to the greatest degree, followed in order by R2exCd305,
M16R, exCd305, and M16exC, which all showed lower but
significant DNA replication. In contrast, R1exCd305 showed very low
(and possibly insignificant) DNA amplification whereas
d27-1 and n217d exhibited essentially no DNA
replication.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 9.
Viral DNA synthesis in mutant-infected Vero cells.
Replicate cultures of Vero cells were mock infected or infected with WT
HSV-1 or ICP27 mutants at an MOI of 10. At 1 hpi and various times
thereafter, total DNA was isolated from the cultures. Five micrograms
of each DNA sample were double digested with PstI plus
SalI, and the digests were subjected to Southern blot
analysis using a probe specific for the UL55 open reading frame. This
probe detects a 4.15-kb fragment in the WT HSV-1, M16, M16R, and M16exC
genomes. In d27-1, due to the deletion in the ICP27 (UL54)
gene which removes a SalI site, the probe detects a 4.3-kb
fragment. In exCd305, n217d, R1exCd305, and
R2exCd305, due to the deletion of the C terminus-encoding region, the
probe detects a 3.5-kb fragment. (A) Viral DNA accumulation at 12 hpi.
An autoradiograph of the Southern blot is shown, and the positions of
DNA size standards are indicated to the right. (B) Quantitation of fold
DNA amplification at 12 hpi. The filter used for panel A was analyzed
by phosphorimager analysis. For each infecting virus, the amount of
hybridization signal at 12 hpi was divided by the 1-hpi signal to
derive the fold DNA accumulation. (C) Quantitation of fold DNA
amplification at 16 hpi. (D) Quantitation of fold DNA amplification in
a time course (8, 12, 16, and 24 hpi).
|
|
We hypothesized that some of the mutants have delayed DNA replication
relative to that of the WT virus. Therefore, we carried
out a
second experiment in which DNA was prepared at 16 rather
than 12 hpi.
The results are shown in Fig.
9C. As before, WT HSV-1
replicated its
DNA the most efficiently whereas
d27-1 was unable
to
replicate its DNA to any significant extent. M16R, M16exC,
exCd305, R1exCd305, R2exCd305, and
n217d
all replicated their
DNAs to intermediate levels. Thus, R1exCd305,
which was borderline
for DNA replication in the first experiment,
clearly is able to
replicate more DNA than
d27-1. The virus
that differed the most
between the two experiments was
n217d. In this experiment, it
replicated a significant
amount of viral DNA whereas it was negative
for DNA replication at 12
hpi.
The above-described results suggest that
n217d is competent
for DNA replication but delayed in the kinetics of DNA accumulation
compared to the other mutants. To test this hypothesis, we performed
a
time course analysis of DNA replication comparing
n217d, the
WT,
d27-1, and
exCd305 at 8, 12, 16, and 24 hpi.
The results,
shown in Fig.
9D, confirm that
n217d is able to
replicate DNA
more efficiently than
d27-1, by about
threefold at 16 to 24 hpi.
Of note was the fact that
exCd305
replicated its DNA comparably
to the WT virus in this experiment.
However, we note that since
our procedure only measures cell-associated
DNA, it may underestimate
the amount of DNA at late times for the WT
virus, since a significant
amount of viral progeny may be secreted into
the
medium.
Three conclusions can be drawn from the DNA replication experiments.
The first is based on the observation that all viruses
expressing
the 289-residue frameshifted protein, including nonviable
mutants such
as M16exC, are able to replicate appreciably more
viral DNA than
d27-1. Therefore, we conclude that the 289-residue
protein
is able to significantly enhance viral DNA replication.
Second, there
is no clear correlation between the ability of the
various frameshift
mutants to replicate DNA and their viability
in Vero cells. It
follows, therefore, that the extragenic mutations
of the
revertants, which are required for viability, must affect
an
essential function other than DNA replication. Third,
n217d
replicates slightly more viral DNA than an ICP27 null mutant.
Based on this, we conclude that the N-terminal 217 residues of
ICP27
have an inherent but weak ability to stimulate viral DNA
replication.
| |
DISCUSSION |
HSV-1 mutants that replicate in the absence of the C-terminal half
of ICP27.
In this paper, we describe the isolation of three HSV-1
mutants (M16R, R1exCd305, and R2exCd305) which grow productively in cultured cells despite the fact that they do not express an intact ICP27 molecule. Instead, due to a frameshift mutation, they produce an
aberrant and highly truncated form of ICP27 that completely lacks its
C-terminal half. In the case of M16R, we cannot exclude the possibility
that the C-terminal portion of ICP27 is expressed via some mechanism
such as mRNA splicing or utilization of an alternate promoter. We
do not have an antibody which recognizes the C-terminal part of the
molecule, so it is difficult to exclude this possibility. However, in
R1exCd305 and R2exCd305, the coding sequences for the C-terminal
segment have been completely deleted. Therefore, we can conclude that
the C-terminal half of ICP27 is not absolutely required for productive
growth of HSV-1 in cultured cells, although as discussed below, it
appears that compensatory mutations are required for the viability of
these unusual mutants.
It is accurate to describe M16R, R1exCd305, and R2exCd305
as viable for at least two reasons. First, they efficiently form
plaques on Vero cells, as demonstrated by the fact that mutant
stocks
give nearly equal titers on Vero and V27 cells. Second,
when seeded at
a low MOI on Vero cells, they efficiently spread
across the monolayer
to infect and destroy all of the cells. Of
course, viability is a viral
phenotype that is highly dependent
on the host cells used. Vero cells
are particularly good hosts
for HSV-1, possibly due to their inability
to produce type I interferons
(
6,
7). It will be interesting
to see whether the revertants
grow productively in other types of
cultured cells or in animal
models of HSV-1
infection.
The finding that the C-terminal half of ICP27 is dispensable for viral
growth, even in the presence of compensating mutations,
was quite
unexpected because a large amount of previous data implies
that this
part of the polypeptide is critical for its regulatory
functions. For
example, numerous mutations in the C-terminal half
of ICP27 destroy the
protein's ability to positively or negatively
regulate cotransfected
genes in transient-transfection assays
(
4,
9,
17,
31). Based
on the results of such experiments,
Sandri-Goldin and colleagues
proposed several years ago that the
C-terminal 249 and 62 residues of
ICP27 comprise its "activation"
and "repression" domains,
respectively (
9). Mutations in the
C-terminal repressor
domain destroy ICP27's abilities to induce
the nuclear redistribution
of cellular splicing factors (
35),
inhibit pre-mRNA
splicing (
8,
36), and shuttle between the
nucleus and
cytoplasm (
19). Moreover, there is strong genetic
evidence
for the importance of the C-terminal portion of ICP27,
as many
mutations in the C-terminal half of the ICP27 gene are
lethal when
introduced into recombinant viruses (
17,
27,
28).
Despite the fact that the revertants are viable, they are attenuated in
their growth compared to WT HSV-1. The mutant plaques
are small, and
their yields in single-cycle growth assays are
reduced ~100-fold
compared to that of the WT. This growth defect
can be attributed to a
defect in ICP27, since expression of WT
ICP27 from the stably
transfected gene of V27 cells significantly
complements the growth of
the mutants in plaque formation and
yield assays (Table
1; Fig.
2B and
7). Therefore, although our
studies indicate that the C-terminal part
of ICP27 is dispensable
in some circumstances, they also demonstrate
that it plays a significant
role in viral
growth.
The truncated, frameshifted ICP27 molecule significantly enhances
viral growth.
The frameshift mutation in M16R and the other
mutants in this study results in the expression of a truncated
289-residue polypeptide consisting of the N-terminal 217 residues of
ICP27 fused to 72 novel C-terminal residues. Our results indicate that
this protein exhibits a significant regulatory function in infected
cells. First, it can enhance the growth of the viral ICP27 null mutant d27-1 by ~1,000-fold in a transfection-based
complementation assay. Second, the engineered mutants M16exC and
exCd305, which express the 289-residue protein, exhibit
significantly higher viral yields in Vero cells than does
d27-1. It was somewhat surprising that the M16R ICP27 allele
was able to complement d27-1 growth by ~1,000-fold in the
transfection assay (Fig. 3) but only led to an ~10- to 100-fold
increase in viral yield relative to d27-1 when introduced into a recombinant virus (Fig. 7A). We speculate that artificially high
expression of the mutant protein in transfected cells enhances its
ability to stimulate the production of viral progeny.
What function does the 289-residue protein perform in infected cells?
Our analysis of viral DNA replication provides a likely
answer.
Although there was some variability in the overall levels
of viral DNA
replication in our experiments, we consistently found
that all of the
mutants expressing the 289-residue protein, including
the nonviable
mutants, replicated significantly more viral DNA
than did
d27-1 (Fig.
9). Thus, the 289-residue protein is able
to
mediate the DNA replication enhancement function of ICP27.
Although the
source of the variability in the DNA replication
experiments is
unknown, it may relate to the fact that fold DNA
replication in our
assay is determined by dividing a relatively
large number (the units of
accumulated DNA at a late time point)
by a much smaller number (units
of DNA at 1 hpi) which, in some
cases, is close to the background
signal and thus difficult to
measure
accurately.
The finding that an N-terminal form of ICP27 can stimulate viral DNA
replication is consistent with some of our past results.
We previously
found that an N-terminal ICP27 mutant,
d1-2, is
deficient
for viral DNA synthesis but shows only modestly reduced
expression of
several L genes (
29). Conversely, we showed that
several
C-terminal ICP27 mutants have the opposite phenotype;
i.e., they are
proficient at viral DNA replication but are unable
to efficiently
express L genes (
27,
28). Based on these observations,
we
previously proposed that ICP27 mediates two separable functions:
(i) an
N-terminal dependent activity which stimulates viral DNA
synthesis and
(ii) a C-terminal dependent activity which stimulates
L gene expression
independently of the stimulatory effect of viral
DNA replication itself
(
27-29). The results of our present study
are consistent
with this model and further implicate the N-terminal
segment of ICP27
in the DNA replication
function.
An unexpected and still puzzling finding is that the novel 72 residues
at the C terminus of the 289-residue polypeptide, derived
from the +1
reading frame, play a critical role in the function
of the truncated
protein. One possibility is that these residues
carry out a highly
specific function that endows the N-terminal
fragment of ICP27 with a
novel activity. However, a more likely
explanation is that these
residues rescue or stabilize an inherent
but weak activity encoded in
ICP27's N-terminal 217 residues.
Evidence for the latter hypothesis
comes from analysis of the
viral mutant
n217d, which
expresses only the N-terminal 217 residues.
Although this mutant does
not grow in Vero cells, it does accumulate
slightly more viral DNA than
the null mutant, suggesting that
the N-terminal 217-residue fragment
has an inherent but weak ability
to stimulate viral DNA
replication.
How might the 72 novel C-terminal residues enhance the N-terminal
activity? The sequence of the frameshift segment may provide
a clue.
Although BLAST searches fail to reveal similar sequences
in current
protein databases, it is striking that the frameshift
sequence is quite
arginine and glycine rich (Fig.
4B). This makes
it very similar in
composition to ICP27's known RNA-binding domain,
which consists of a
short 15-residue stretch with similarity to
RGG box-type motifs found
in other proteins (
20). This sequence
maps to residues 138 to 152 and is thus present in both the 217-
and 289-residue proteins.
It is possible that the frameshift segment
confers an additional
RNA-binding activity or augments RNA binding
by the RGG box. There are
several other possible mechanisms by
which the frameshift sequence
might enhance the function of the
N-terminal fragment. For example, it
might increase the stability
of the truncated polypeptide or allow it
to localize more efficiently
to the cellular compartment(s) where it
acts. These explanations
appear unlikely, however, since both the 217- and 289-residue
fragments are expressed at comparable levels in
infected cells
and exhibit similar nuclear localization patterns
characterized
by preferential accumulation in nucleoli. Lastly, it is
possible
that the frameshift segment enhances protein multimerization.
This hypothesis is consistent with recent work by Zhi et al. which
demonstrated that ICP27 multimerizes in vivo and that the C-terminal
half is critical for this ability (
42). In this scenario,
the
frameshift segment could allow for the self-association of ICP27,
thereby bypassing the need for the multimerization function normally
supplied by the C-terminal
half.
Extragenic mutations are required for the viability of viruses
expressing frameshifted ICP27.
To test whether the intragenic
frameshift mutation is the only alteration required for M16R's
viability, we re-engineered the M16R ICP27 allele and introduced it
into a new virus, M16exC, in place of the WT gene. However, M16exC is
not viable in Vero cells, nor is another mutant, exCd305,
which was also engineered to express the frameshifted gene. The
simplest interpretation of these results is that M16R possesses one or
more extragenic mutations which are required for viability in
addition to the frameshift mutation. Consistent with this, we were able
to readily select two viable derivatives of exCd305,
designated R1exCd305 and R2exCd305. Sequencing analysis revealed
that these revertants have no unexpected alterations in their ICP27
genes. Therefore, their viable phenotypes must result from extragenic
mutations. It is noteworthy that the growth phenotypes of the two
revertants are distinct, suggesting that their extragenic reversion
mutations are also distinct.
What is the source of the extragenic mutation(s) in M16R? In this
regard, it is instructive to contrast the isolation of M16R,
which
replicates in Vero cells, with that of M16exC, which does
not. In
the case of M16R, multiple passages and plaque purifications
were
performed in Vero cells, as was preparation of the initial
low-titer
stock. In the case of M16exC, however, all genetic manipulations
were carried out in V27 cells to minimize any potential selective
pressure for additional mutations which could enhance growth.
Based on
these considerations, we hypothesize that the extragenic
mutation(s) of
M16R was acquired during its initial, prolonged
selection in Vero
cells, possibly after the acquisition of the
frameshift mutation. This
model is consistent with the proven
sequential acquisition of the
frameshift and extragenic mutations
in the genomes of R1exCd305 and
R2exCd305.
Taken together, our results indicate that at least two mutations are
required to allow HSV-1 to replicate in Vero cells in
the absence of
its C-terminal domain. The first is the intragenic
+1 frameshift
mutation, which expands by one nucleotide a homopolymeric
run of C
residues in the ICP27 coding strand at codons 215 to
217. Sasadeusz et
al. have shown that expansion and contraction
of such homopolymeric
sequences appear to be common mutational
events in HSV (
37).
The frameshift mutation results in expression
of the 289-residue ICP27
protein, which is able to enhance viral
DNA replication. We propose
that the second mutation is an alteration
outside of the ICP27 gene; it
is feasible that multiple extragenic
mutations are required. The
effect of the extragenic change(s)
is unknown. However, since
mutants possessing only the intragenic
change are able to undergo
significant DNA replication, it seems
likely that the extragenic
alterations affect a step in viral
growth after the onset of DNA
synthesis. Given the known role
of ICP27 in L gene expression, we
speculate that this step is
the activation of L genes. Our preliminary
analysis of protein
synthesis patterns of the various mutants supports
this hypothesis,
in that the viable revertants appear to express a
wider range
of viral proteins than do M16exC and
exCd305 (S. Bunnell and S.
Rice, unpublished data,
2000).
To understand the nature and effect of the extragenic mutations, it is
necessary to identify these alterations at the nucleotide
level. We
have attempted to use a marker rescue approach to map
M16R's
extragenic mutation(s) by asking if restriction fragments
derived from
M16R can recombine into the M16exC genome and confer
viability.
However, we have observed that M16exC has a relatively
high rate of
spontaneous reversion, a fact that has hampered our
mapping attempts.
In addition, mapping of the extragenic mutation(s)
of the revertants
will be difficult if multiple extragenic alterations
contribute to
viability.
Does ICP27 mediate distinct N- and C-terminal functions?
Based
on these and our past results, we propose that ICP27 carries out (at
least) two distinct regulatory functions in infected cells, one of
which is mediated by an N-terminal region and the other of which is
dependent upon C-terminal sequences. We propose that the N-terminal
activity is essential and acts to stimulate viral DNA replication,
likely indirectly by transactivating DE genes that encode DNA
replication proteins (15, 39). Although the N-terminal
function can work in the complete absence of C-terminal sequences (at
least in the context of the frameshift mutation), we do not know
whether the C-terminal function depends upon N-terminal regions such as
the RGG box (20) or nuclear export signal (34). This model can help explain why the ICP27 homologues of
varicella-zoster virus and human cytomegalovirus, which bear homology
to the C-terminal, but not the N-terminal, part of ICP27, are unable to
complement the growth of HSV-1 ICP27 mutants (21, 41). That
is, these molecules may be unable to perform the essential N-terminal
function. One finding that our model does not readily explain, however, is why many ICP27 C-terminal mutations (such as M16) are so highly lethal. One possibility is that ICP27 sequences C terminal to residue
217 can inhibit the function of the N-terminal segment, possibly via
direct physical interaction. This inhibition would be relieved by
truncation of the protein by the frameshift mutation. More work is
required to test this hypothesis.
In summary, we report the isolation of novel HSV-1 mutants which
replicate in cultured cells despite the fact that they do
not express
the evolutionarily conserved C-terminal half of ICP27.
We also show
that compensatory mutations are required for the
viability of these
mutants. This study provides further evidence
that ICP27 carries out
multiple independent functions during viral
lytic infection and
indicates that ICP27's ability to enhance
DNA replication is
associated with the N-terminal half of the
molecule. Further
characterization of these unusual mutants is
likely to illuminate the
structure, function, and evolution of
HSV-1
ICP27.
| |
ACKNOWLEDGMENTS |
We are extremely grateful to Jim Smiley (University of Alberta)
for generously providing laboratory space to S.M.B. during the latter
part of these studies, as well as for continuing advice and insight.
Thanks are due to Jim Smiley, Jim Stone, Kim Ellison, and Keith Perkins
for critical reviews of the manuscript. Finally, we acknowledge Leslie
Schiff for many stimulating discussions.
This research was supported by grants from the National Cancer
Institute of Canada and the National Institutes of Health
(RO1-AI42737), as well as a Senior Scholar Research Award from the
Alberta Heritage Foundation for Medical Research (AHFMR). S.M.B. was
supported by a full-time studentship award from the AHFMR. S.A.R. was
an AHFMR Senior Scholar.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Minnesota Medical School, 420 Delaware St. S.E., Box 196 FUMC, Minneapolis, MN 55455. Phone: (612) 626-4183. Fax:
(612) 626-0623. E-mail: stever{at}lenti.med.umn.edu.
| |
REFERENCES |
| 1.
|
Ackermann, M.,
D. K. Braun,
L. Pereira, and B. Roizman.
1984.
Characterization of herpes simplex virus 1 proteins 0, 4, and 27 with monoclonal antibodies.
J. Virol.
52:108-118[Abstract/Free Full Text].
|
| 2.
|
Aubert, M., and J. A. Blaho.
1999.
The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells.
J. Virol.
73:2803-2813[Abstract/Free Full Text].
|
| 3.
|
Bello, L. J.,
A. J. Davison,
M. A. Glenn,
A. Whitehouse,
N. Rethmeier,
T. F. Schulz, and J. Barklie Clements.
1999.
The human herpesvirus-8 ORF 57 gene and its properties.
J. Gen. Virol.
80:3207-3215[Abstract/Free Full Text].
|
| 4.
|
Brown, C. R.,
M. S. Nakamura,
J. D. Mosca,
G. S. Hayward,
S. E. Straus, and L. P. Perera.
1995.
Herpes simplex virus trans-regulatory protein ICP27 stabilizes and binds to 3' ends of labile mRNA.
J. Virol.
69:7187-7195[Abstract].
|
| 5.
|
Cai, W. Z.,
S. Person,
C. DebRoy, and B. H. Gu.
1988.
Functional regions and structural features of the gB glycoprotein of herpes simplex virus type 1. An analysis of linker insertion mutants.
J. Mol. Biol.
201:575-588[CrossRef][Medline].
|
| 6.
|
Diaz, M. O.,
S. Ziemin,
M. M. Le Beau,
P. Pitha,
S. D. Smith,
R. R. Chilcote, and J. D. Rowley.
1988.
Homozygous deletion of the alpha- and beta 1-interferon genes in human leukemia and derived cell lines.
Proc. Natl. Acad. Sci. USA
85:5259-5263[Abstract/Free Full Text].
|
| 7.
|
Emeny, J. M., and M. J. Morgan.
1979.
Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production.
J. Gen. Virol.
43:247-252[Abstract/Free Full Text].
|
| 8.
|
Hardwicke, M. A., and R. M. Sandri-Goldin.
1994.
The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection.
J. Virol.
68:4797-4810[Abstract/Free Full Text].
|
| 9.
|
Hardwicke, M. A.,
P. J. Vaughan,
R. E. Sekulovich,
R. O'Conner, and R. M. Sandri-Goldin.
1989.
The regions important for the activator and repressor functions of herpes simplex virus type 1 protein ICP27 map to the C-terminal half of the molecule.
J. Virol.
63:4590-4602[Abstract/Free Full Text].
|
| 10.
|
Hardy, W. R., and R. M. Sandri-Goldin.
1994.
Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect.
J. Virol.
68:7790-7799[Abstract/Free Full Text].
|
| 11.
|
Hughes, R. G., Jr., and W. H. Munyon.
1975.
Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation.
J. Virol.
16:275-283[Abstract/Free Full Text].
|
| 12.
|
Ingram, A.,
A. Phelan,
J. Dunlop, and J. B. Clements.
1996.
Immediate early protein IE63 of herpes simplex virus type 1 binds RNA directly.
J. Gen. Virol.
77:1847-1851[Abstract/Free Full Text].
|
| 13.
|
Mavromara-Nazos, P., and B. Roizman.
1987.
Activation of herpes simplex virus 1 gamma 2 genes by viral DNA replication.
Virology
161:593-598[CrossRef][Medline].
|
| 14.
|
McCarthy, A. M.,
L. McMahan, and P. A. Schaffer.
1989.
Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient.
J. Virol.
63:18-27[Abstract/Free Full Text].
|
| 15.
|
McGregor, F.,
A. Phelan,
J. Dunlop, and J. B. Clements.
1996.
Regulation of herpes simplex virus poly(A) site usage and the action of immediate-early protein IE63 in the early-late switch.
J. Virol.
70:1931-1940[Abstract].
|
| 16.
|
McLauchlan, J.,
A. Phelan,
C. Loney,
R. M. Sandri-Goldin, and J. B. Clements.
1992.
Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing.
J. Virol.
66:6939-6945[Abstract/Free Full Text].
|
| 17.
|
McMahan, L., and P. A. Schaffer.
1990.
The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection.
J. Virol.
64:3471-3485[Abstract/Free Full Text].
|
| 18.
|
Mears, W. E.,
V. Lam, and S. A. Rice.
1995.
Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27.
J. Virol.
69:935-947[Abstract].
|
| 19.
|
Mears, W. E., and S. A. Rice.
1998.
The herpes simplex virus immediate-early protein ICP27 shuttles between nucleus and cytoplasm.
Virology
242:128-137[CrossRef][Medline].
|
| 20.
|
Mears, W. E., and S. A. Rice.
1996.
The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA-binding activity and determines in vivo methylation.
J. Virol.
70:7445-7453[Abstract].
|
| 21.
|
Moriuchi, H.,
M. Moriuchi,
H. A. Smith, and J. I. Cohen.
1994.
Varicella-zoster virus open reading frame 4 protein is functionally distinct from and does not complement its herpes simplex virus type 1 homolog, ICP27.
J. Virol.
68:1987-1992[Abstract/Free Full Text].
|
| 22.
|
Nicholas, J.
1996.
Determination and analysis of the complete nucleotide sequence of human herpesvirus.
J. Virol.
70:5975-5989[Abstract].
|
| 23.
|
Phelan, A.,
M. Carmo-Fonseca,
J. McLaughlan,
A. I. Lamond, and J. B. Clements.
1993.
A herpes simplex virus type 1 immediate-early gene product, IE63, regulates small nuclear ribonucleoprotein distribution.
Proc. Natl. Acad. Sci. USA
90:9056-9060[Abstract/Free Full Text].
|
| 24.
|
Phelan, A., and J. B. Clements.
1997.
Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm.
J. Gen. Virol.
78:3327-3331[Abstract].
|
| 25.
|
Phelan, A.,
J. Dunlop, and J. B. Clements.
1996.
Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts.
J. Virol.
70:5255-5265[Abstract/Free Full Text].
|
| 26.
|
Quinlan, M. P.,
L. B. Chen, and D. M. Knipe.
1984.
The intranuclear location of a herpes simplex virus DNA-binding protein is determined by the status of viral DNA replication.
Cell
36:857-868[CrossRef][Medline].
|
| 27.
|
Rice, S. A., and D. M. Knipe.
1990.
Genetic evidence for two distinct transactivation functions of the herpes simplex virus protein ICP27.
J. Virol.
64:1704-1715[Abstract/Free Full Text].
|
| 28.
|
Rice, S. A., and V. Lam.
1994.
Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function.
J. Virol.
68:823-833[Abstract/Free Full Text].
|
| 29.
|
Rice, S. A.,
V. Lam, and D. M. Knipe.
1993.
The acidic amino-terminal region of herpes simplex virus type 1 alpha protein ICP27 is required for an essential lytic function.
J. Virol.
67:1778-1787[Abstract/Free Full Text].
|
| 30.
|
Rice, S. A.,
M. C. Long,
V. Lam, and C. A. Spencer.
1994.
RNA polymerase II is aberrantly phosphorylated and localized to viral replication compartments following herpes simplex virus infection.
J. Virol.
68:988-1001[Abstract/Free Full Text].
|
| 31.
|
Rice, S. A.,
L. Su, and D. M. Knipe.
1989.
Herpes simplex virus alpha protein ICP27 possesses separable positive and negative regulatory activities.
J. Virol.
63:3399-3407[Abstract/Free Full Text].
|
| 32.
|
Roizman, B., and A. E. Sears.
1996.
Herpes simplex virus and their replication, p. 1795-1841.
In
B. N. Fields, P. M. Howley, and D. M. Knipe (ed.), Virology, 2nd ed. Raven Press, New York, N.Y.
|
| 33.
|
Sacks, W. R.,
C. C. Greene,
D. P. Aschman, and P. A. Schaffer.
1985.
Herpes simplex virus type 1 ICP27 is an essential regulatory protein.
J. Virol.
55:796-805[Abstract/Free Full Text].
|
| 34.
|
Sandri-Goldin, R. M.
1998.
ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif.
Genes Dev.
12:868-879[Abstract/Free Full Text].
|
| 35.
|
Sandri-Goldin, R. M.,
M. K. Hibbard, and M. A. Hardwicke.
1995.
The C-terminal repressor region of herpes simplex virus type 1 ICP27 is required for the redistribution of small nuclear ribonucleoprotein particles and splicing factor SC35; however, these alterations are not sufficient to inhibit host cell splicing.
J. Virol.
69:6063-6076[Abstract].
|
| 36.
|
Sandri-Goldin, R. M., and G. E. Mendoza.
1992.
A herpesvirus regulatory protein appears to act post-transcriptionally by affecting mRNA processing.
Genes Dev.
6:848-863[Abstract/Free Full Text].
|
| 37.
|
Sasadeusz, J. J.,
F. Tufaro,
S. Safrin,
K. Schubert,
M. M. Hubinette,
P. K. Cheung, and S. L. Sacks.
1997.
Homopolymer mutational hot spots mediate herpes simplex virus resistance to acyclovir.
J. Virol.
71:3872-3878[Abstract].
|
| 38.
|
Soliman, T. M.,
R. M. Sandri-Goldin, and S. J. Silverstein.
1997.
Shuttling of the herpes simplex virus type 1 regulatory protein ICP27 between the nucleus and cytoplasm mediates the expression of late proteins.
J. Virol.
71:9188-9197[Abstract].
|
| 39.
|
Uprichard, S. L., and D. M. Knipe.
1996.
Herpes simplex ICP27 mutant viruses exhibit reduced expression of specific DNA replication genes.
J. Virol.
70:1969-1980[Abstract].
|
| 40.
|
Wagner, E. K.,
J. F. Guzowski, and J. Singh.
1995.
Transcription of the herpes simplex virus genome during productive and latent infection.
Prog. Nucleic Acid Res. Mol. Biol.
51:123-165[Medline].
|
| 41.
|
Winkler, M.,
S. A. Rice, and T. Stamminger.
1994.
UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression.
J. Virol.
68:3943-3954[Abstract/Free Full Text].
|
| 42.
|
Zhi, Y.,
K. S. Sciabica, and R. M. Sandri-Goldin.
1999.
Self-interaction of the herpes simplex virus type 1 regulatory protein ICP27.
Virology
257:341-351[CrossRef][Medline].
|
Journal of Virology, August 2000, p. 7362-7374, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Boyer, J. L., Swaminathan, S., Silverstein, S. J.
(2002). The Epstein-Barr Virus SM Protein Is Functionally Similar to ICP27 from Herpes Simplex Virus in Viral Infections. J. Virol.
76: 9420-9433
[Abstract]
[Full Text]
Top
Abstract
Introduction
