Journal
of General Virology (1998), 79, 2765–2775. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Translational control of viral and host protein synthesis during
the course of herpes simplex virus type 1 infection : evidence
that initiation of translation is the limiting step
Anne-Marie Laurent, Jean-Jacques Madjar and Anna Greco
Immuno-Virologie Mole! culaire et Cellulaire CNRS UMR 5537, Faculte! de Me! decine Lyon-R. T. H. Laennec, Rue Guillaume Paradin,
69372 Lyon CEDEX 08, France
Herpes simplex virus type 1 (HSV-1) infection
induces the selective shut-off of host protein synthesis, other than ribosomal proteins, and the
successive synthesis of viral proteins. Because viral
mRNAs persist in the cytoplasm after viral protein
synthesis has been inhibited, we hypothesized that
viral gene expression may be under translational
control. Expression of genes encoding immediate
early ICP27, early DBP and late US11 proteins,
together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was monitored over the
course of infection at the level of mRNA and protein
synthesis. After an efficient synthesis beginning with
the appearance of successive viral mRNAs in the
cytoplasm, synthesis of viral proteins was shut off
similarly to the synthesis of GAPDH. This shut-off
Introduction
During the course of lytic infection by herpes simplex virus
type 1 (HSV-1), host protein synthesis is inhibited, while three
groups of viral genes, immediate early (IE), early and late, are
successively expressed. HSV-1-induced inhibition of host
protein synthesis is a multistep process which involves several
mechanisms. The primary shut-off of host protein synthesis
does not require de novo protein synthesis and is, in part, the
consequence of a non-specific degradation of all cellular and
viral mRNAs, induced by the virion-associated host shut-off
(VHS) protein, leading to a disruption of polyribosomes. VHS
protein, encoded by the viral UL41 true late gene, is a viral
structural component released into infected cells by the virion
(Sydiskis & Roizman, 1967 ; Nishioka & Silverstein, 1977 ;
Fenwick & Clark, 1982 ; Read & Frenkel, 1983 ; Schek &
Bachenheimer, 1985 ; Strom & Frenkel, 1987 ; Fenwick &
Author for correspondence : Jean-Jacques Madjar.
Fax ­33 4 78 77 87 18. e-mail madjar!laennec.univ-lyon1.fr
0001-5701 # 1998 SGM
was not achieved by mRNA degradation but by
progressive shifts of viral mRNAs from large polyribosomes to smaller ones, then to 40S ribosomal
subunits. Transient expression of the UL41 gene
alone, directing synthesis of virion-associated host
shut-off (VHS) protein, induced efficient mRNA
degradation, but did not impair recruitment of the
remaining GAPDH and β-actin mRNAs into polyribosomes. These results indicate that HSV-1 induces a selective repression of initiation of mRNA
translation which is probably the main cause of
the shut-off of viral protein synthesis, and which
contributes to the repression of host protein synthesis. VHS protein is not directly involved in this
repression, at least in the absence of other viral
proteins.
Owen, 1988 ; Oroskar & Read, 1989 ; Jones et al., 1995).
However, the shut-off of host protein synthesis that follows
infection appears to be selective and under translational
control. Synthesis of β-actin becomes much less efficient, while
that of ribosomal proteins continues almost unaffected, despite
a similar degradation of their mRNAs (Simonin et al., 1997).
This is made possible by more efficient translation of ribosomal
protein mRNAs immediately after infection (Greco et al.,
1997).
A secondary shut-off of protein synthesis occurs later in the
course of infection, and requires viral gene expression. ICP27
IE protein and UL13 early protein contribute to this process
(Nishioka & Silverstein, 1977 ; Fenwick & Clark, 1982 ; Read &
Frenkel, 1983 ; Sacks et al., 1985 ; McMahan & Schaffer, 1990 ;
Hardwicke & Sandri-Goldin, 1994 ; Overton et al., 1994).
ICP27 inhibits gene expression at the transcriptional and posttranscriptional levels. This includes the selection of alternative
termination sites of transcription, inhibition of RNA splicing
and destabilization of mRNAs. This delayed inhibition of
protein synthesis appears to result from selective virus-induced
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A.-M. Laurent, J.-J. Madjar and A. Greco
translational control. During the course of infection, initiation
of translation of the remaining β-actin mRNAs is selectively
and progressively inhibited, while that of the remaining
ribosomal protein mRNAs becomes more efficient (Greco et al.,
1997 ; Simonin et al., 1997).
The successive expression of viral genes results primarily
from transcriptional control (Batterson & Roizman, 1983 ;
Campbell et al., 1984 ; Godowski & Knipe, 1986 ; Weinheimer
& McKnight, 1987 ; Homa et al., 1991 ; Guzowski & Wagner,
1993 ; Roizman & Sears, 1993). Transcription of all IE genes is
specifically induced by VP16, another structural protein
delivered into cells by invading virions. With the exception of
ICP47, all other IE proteins have been shown to be involved in
the regulation of transcription of the three groups of viral
genes. Transition from the IE to early phase of viral gene
expression is accompanied by the shut-off of IE protein
synthesis and induction of early protein synthesis. Depression
of IE protein synthesis is, at least in part, controlled at the posttranscriptional level, and again involves both VHS and ICP27
viral proteins (Sacks et al., 1985 ; Oroskar & Read, 1989 ; Rice
& Knipe, 1990 ; McLauchlan et al., 1992 ; Sandri-Goldin &
Mendoza, 1992 ; Hardwicke & Sandri-Goldin, 1994).
Because IE mRNAs persist in the cytoplasm late in infection
when IE protein synthesis is almost completely inhibited, the
hypothesis of translational control of viral gene expression has
been suggested by various groups (Honess & Roizman, 1974 ;
Silverstein & Engelhardt, 1979 ; Schek & Bachenheimer, 1985 ;
Weinheimer & McKnight, 1987 ; Elshiekh et al., 1991).
Although this hypothesis remains unproved, it is supported by
other data. For example, inhibition of IE protein synthesis still
occurs in enucleated cells (Honess & Roizman, 1973 ; Fenwick,
1977). Also, the rate of synthesis of some early and late
proteins is not strictly correlated to the amount of their
mRNAs during the course of infection (Sandri-Goldin et al.,
1983 ; Johnson & Spear, 1984 ; Arsenakis et al., 1988 ; Yager et
al., 1990 ; Elshiekh et al., 1991).
Consistent with the hypothesis of translational control of
viral and host protein synthesis is the observation that infection
by HSV-1 leads to specific alterations in the translational
apparatus (Fenwick & Walker, 1979 ; Kennedy et al., 1981 ;
Garcin et al., 1990 ; Masse! et al., 1990 a, b ; Simonin et al., 1995 a ;
Greco et al., 1997). The ribosomal protein S6 undergoes an
irreversible phosphorylation during the adsorption step of
virus infection (Kennedy et al., 1981 ; Masse! et al., 1990 a, b). An
increase in S6 phosphorylation is always correlated with the
preferential recruitment and translation of ribosomal protein
mRNAs (Jefferies et al., 1994). Other ribosomal proteins, Sa, S2,
S3a and L30, are unusually phosphorylated after infection, and
two other phosphorylated proteins are associated with the
ribosomal fraction (Johnson et al., 1986 ; Masse! et al., 1990 a, b ;
Roller & Roizman, 1992). One of these proteins has been
identified as the product of the viral US11 true late gene (Diaz
et al., 1993). US11 RNA-binding protein appears to be involved
in post-transcriptional regulation of gene expression (Diaz et
CHGG
al., 1996). In addition, some cellular translation factors are
modified in HSV-1-infected cells (Chou et al., 1995 ; Kawaguchi
et al., 1997). These data and our recent results demonstrating
that HSV-1 infection induces a selective translational control of
host protein synthesis (Greco et al., 1997 ; Simonin et al., 1997)
reinforce the hypothesis that viral protein synthesis is regulated
at the translational level.
In order to verify this hypothesis we measured the rate of
synthesis and the amount of cytoplasmic mRNA of ICP27 IE
protein, double-stranded DNA-binding early protein (DBP)
and US11 true late protein at different times after infection. No
direct correlation was found between the rates of synthesis of
these proteins and the amounts of their mRNAs. Analysis of
the distribution of viral and cellular mRNAs among polyribosomes showed that all mRNA molecules, initially
associated with large polyribosomes, progressively shifted to
smaller polyribosomes, and then to free inactive particles
during the course of infection. These results indicate that
translation of the mRNAs studied was progressively inhibited
at the initiation step, before binding of the 60S ribosomal
subunit.
Methods
+ Cell lines and virus strain. HeLa and Vero cells were grown as
monolayers in Eagle’s minimum essential medium (MEM) supplemented
with 10 % heat-inactivated newborn calf serum (NBCS). The HSV-1
macroplaque strain (MP), obtained from B. Jacquemont (Lyon, France),
was a gift of B. Roizman (Chicago, IL, USA) and was used throughout this
study. Viruses were grown in Vero cells.
+ Infection of cells and 35S-labelling. HeLa cells were infected just
before confluence with a m.o.i. of 30 p.f.u. per cell in medium 199 (ICN
Flow Laboratories) supplemented with 1 % heat-inactivated NBCS. After
adsorption for 1 h at 33 °C, the inoculum was replaced with medium 199
containing 1 % inactivated NBCS, and cells were incubated at 37 °C for
different times as indicated in the figure legends. Times post-infection
(p.i.) were calculated from the time of addition of the inoculum. For $&Slabelling in vivo, the medium was removed 1 h before harvesting and
replaced with methionine- and cysteine-free MEM (ICN Flow Laboratories), supplemented with 1 % inactivated and dialysed NBCS, and
incubated for 30 min at 37 °C. The medium was removed and replaced
with the same medium supplemented with a mixture of -[$&S]methionine
and -[$&S]cysteine at final concentrations of 2±75 MBq}ml (75 µCi}ml)
and 0±6 MBq}ml (16 µCi}ml), respectively (NEN). In all experiments
control mock-infected and infected cells were submitted to exactly the
same experimental conditions.
+ Estimation of viral and host protein synthesis throughout
infection. Cells were washed three times with ice-cold PBS after
labelling (130 mM NaCl, 4 mM Na HPO .2H O, 1±5 mM KH PO ),
#
% #
# %
scraped into PBS, and collected by centrifugation at 500 g. Cells were
then lysed in ice-cold buffer (0±1 % SDS, 1 % Triton X-100, 1 % sodium
deoxycholate, 150 mM NaCl, 10 mM Tris–HCl pH 7±4, 1 mM EDTA,
0±2 mM PMSF). Insoluble material was removed by centrifugation at
12 000 g. Immunoprecipitations of proteins were performed as previously
described (Simonin et al., 1995 b). Aliquots of lysates prepared from 10&
cells were incubated for 18 h at 4 °C with one of the following : rabbit
polyclonal anti-ICP27 antibody, rabbit polyclonal anti-US11 antibody
(Diaz et al., 1993), mouse monoclonal anti-DBP antibody or mouse
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Control of host and HSV-1 mRNA translation
monoclonal antibody directed against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Interchim), each diluted 50-fold in lysis buffer.
Anti-ICP27 and anti-DBP antibodies (antibody 42 and antibody Z1F11
directed against the DBP of 65 kDa, respectively) were kindly provided
by H. Marsden (MRC, Glasgow, UK) (Schenck et al., 1988 ; Sinclair et al.,
1994). Antigen–antibody complexes were collected with protein A
coupled to Sepharose CL-4B beads (Pharmacia) for 30 min at 4 °C.
Immunoprecipitated proteins were then analysed by SDS–PAGE
(Laemmli, 1970). Dried gels were submitted to autoradiography. The
corresponding immunoprecipitated proteins were quantified by scanning
densitometry of gels with a Storm 840 PhosphorImager (Molecular
Dynamics).
+ Measurement of the amounts of viral and host mRNAs in
the cytoplasm. Cytoplasmic RNAs from 5¬10& control mock-infected
and infected HeLa cells or from transfected HeLa cells were purified after
lysis of the cells with NP40 at a final concentration of 0±1 % followed by
proteinase K digestion and phenol–chloroform extraction. RNA was
analysed on Northern blots as described (Greco et al., 1997).
Hybridization was performed at 65 °C with different $#P-labelled DNA
probes by random priming. For detection of ICP27, ICP22 and DBP
mRNAs, $#P-labelled probes were prepared from the 746 bp SalI–EcoNI
fragment of pSG28, the 469 bp XhoI–PvuI fragment of pSG25 and the
1308 bp MluI fragment of pSG124 (Goldin et al., 1981), respectively.
UL34 and US11 mRNAs were detected with $#P-labelled probes prepared
from the 472 bp EcoRI–BamHI fragment of pG51 and the 230 bp XhoI
fragment of pHSV-Us11, respectively (Diaz et al., 1996). The probes used
to detect the cytoplasmic GAPDH and β-actin mRNAs were prepared by
labelling the 318 bp SacI–BamHI fragment of pTRI-hGAPDH (Ambion)
and the 1100 bp PstI fragment of plasmid pAL41-β (Hanahan, 1983),
respectively. Hybridized mRNAs were revealed by autoradiography and
quantified by scanning densitometry of the blots with a Storm 840
PhosphorImager.
Results
Synthesis of viral proteins during the course of
productive infection
In order to investigate whether expression of viral genes
was under translational control, it was first necessary to
determine the precise pattern of synthesis of viral proteins
during the course of HSV-1 infection. The synthesis of proteins
representing the three groups of viral gene products was
investigated : ICP27, the product of an IE gene ; DBP, which is
the product of the UL42 early gene ; and US11, the product of
a true late gene. As a control for HSV-1-induced shut-off of
host protein synthesis, the synthesis of GAPDH was followed
in HeLa cells that were either mock-infected or infected for the
times indicated in the legend of Fig. 1. Proteins were $&Slabelled during the last 30 min before harvesting of the cells.
The level of protein synthesis was estimated by measurement
+ Distribution of mRNAs among polyribosomal fractions.
Polyribosomes were prepared from about 8¬10' mock-infected and
infected HeLa cells or from about 3¬10' transfected HeLa cells.
Fractionation of polyribosomes on sucrose gradients was carried out
from post-mitochondrial supernatants as previously described (Greco et
al., 1997). In experiments with mock-infected and infected cells, twenty
fractions of identical volume were collected from the top of the gradients,
whereas in experiments with transfected cells, ten fractions of identical
volume were collected. Each fraction was treated with 100 µg}ml
proteinase K and 1 % SDS for 15 min, before phenol–chloroform
extraction and ethanol precipitation of the RNA. RNA from each fraction
was resuspended in 10 mM Tris–HCl pH 7±4, 1 mM EDTA, 1 U RNase
inhibitor (Pharmacia). The quantitative distribution of the various mRNAs
among the different fractions was then analysed on Northern blots as
described above.
+ Construction and transfection of a plasmid expressing the
UL41 gene. pUL41 was prepared from pCMVβ (Clontech) by replacing
the 3474 bp NotI fragment carrying the β-galactosidase gene with the
2188 bp EcoNI fragment of pSG-124 (Goldin et al., 1981), containing the
entire ORF and polyadenylation signal of the UL41 gene. HeLa cells were
transfected by the calcium phosphate precipitation procedure with 5 µg
pUL41 to which 10 µg pUC18 was added (Sambrook et al., 1989).
Control HeLa cells were transfected with 15 µg pUC18 only. After 20 h,
cells were harvested and lysed to estimate the amount of GAPDH and βactin mRNAs present in the cytoplasm and the distribution of mRNA
among polyribosomal fractions as described above. Under our experimental conditions, the yield of transfection reached almost 100 %
(Greco et al., 1994).
Fig. 1. Synthesis of ICP27, DBP, US11 protein and GAPDH during the
course of HSV-1 infection. HeLa cells were either mock-infected (M) or
infected for 1 to 15 h. Proteins were labelled with a mixture of
[35S]methionine and [35S]cysteine during the last 30 min before
harvesting of the cells. After immunoprecipitation with the relevant
antibodies, proteins were separated by SDS–PAGE and analysed by
autoradiography. Positions of proteins of interest are indicated by arrows.
Times (h) after infection are indicated above each lane. In the panel DBP,
lane C is a control taken at 5 h p.i. in the absence of anti-DBP antibody.
This control indicates that the protein which appeared 2 h p.i. is not DBP,
but the result of non-specific adsorption to the protein A–Sepharose
beads. US11 protein appears as a doublet of 24 and 26 kDa (Diaz et al.,
1993). No labelled protein was detected after a second round of
immunoprecipitation.
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A.-M. Laurent, J.-J. Madjar and A. Greco
Fig. 2. Viral and GAPDH mRNAs during the course of HSV-1 infection. Total cytoplasmic RNAs of HeLa cells either mockinfected (M) or infected for 3, 6, 9 and 15 h were extracted from post-mitochondrial supernatants, then separated by
electrophoresis through a 1±2 % agarose gel containing 0±22 M formaldehyde, and blotted onto positively charged nylon
membranes for Northern blot analysis. Positions of mRNAs are indicated by arrows. US11 and ICP27 mRNAs were probed on
the same membrane, while DBP and GAPDH mRNAs were probed on a second membrane.
of the radioactivity incorporated into each protein separated
by SDS–PAGE after immunoprecipitation.
Synthesis of ICP27 protein, which migrates with an
apparent molecular mass of 63 kDa, started rapidly after
infection, reached a maximum between 3 and 4 h p.i., and then
declined, becoming almost undetectable after 7 h (Fig. 1). As
expected for a protein involved in DNA synthesis, DBP was
detected for the first time at 3 h p.i. (Goodrich et al., 1989). Its
synthesis reached a maximum between 5 and 7 h p.i. and then
decreased progressively, but persisted up to 15 h p.i. Synthesis
of the US11 late protein was detected only at 4 h p.i. US11
protein synthesis was greatest at 7 h p.i. and then decreased
slowly to the end of the investigation. These three viral
proteins exhibited the expected patterns of synthesis, which
are typical of expression of their groups of genes (Honess &
Roizman, 1974 ; Elshiekh et al., 1991).
Synthesis of GAPDH was maximal in mock-infected cells
(Fig. 1). Synthesis decreased progressively during the first 9 h
p.i., and was undetectable after this time. The kinetics of
GAPDH synthesis during the course of the replicative cycle
was similar to that found previously for β-actin (Greco et al.,
1997 ; Simonin et al., 1997).
Viral and GAPDH mRNAs during the course of HSV-1
infection
To determine whether the levels of protein synthesis
measured above reflect the levels of mRNA in the cytoplasm,
we estimated the amount of these mRNAs, together with
amounts of ICP22 and UL34 mRNA, at different times after
infection. Total cytoplasmic RNA was purified from either
mock-infected cells or cells that had been infected for 3, 6, 9, 12
or 15 h, and RNA was analysed by Northern blotting using
specific $#P-labelled DNA probes (Fig. 2).
ICP27 and ICP22 IE mRNAs exhibited similar kinetics of
appearance in the cytoplasm. Present in the cytoplasm as early
CHGI
as 3 h p.i., they reached their maximal level at 6 h for ICP27
and between 6 and 9 h for ICP22. Levels then declined for the
remainder of the experiment, but remained detectable after
15 h p.i. The early protein mRNAs DBP and UL34 also
exhibited similar kinetics of appearance in the cytoplasm,
starting at 6 h p.i. They reached their maximal level at 9 h, then
declined slowly, and remained present in significant amounts
after 15 h p.i. The use of a common probe to detect both ICP47
and US11 protein mRNAs (2 and 1±3 kb, respectively)
allowed us to detect simultaneously a typical IE mRNA and a
true late mRNA (Fig. 2, top right panel). The kinetics of
appearance and accumulation of these mRNAs, as well as the
kinetics of disappearance of ICP47 mRNA, were in complete
agreement with those of other mRNAs analysed in this
experiment, and are consistent with the reported kinetics of
accumulation of other HSV-1 mRNAs (Weinheimer &
McKnight, 1987 ; Goodrich et al., 1989 ; Oroskar & Read,
1989 ; Elshiekh et al., 1991 ; Johnson et al., 1991). As expected,
the amount of GAPDH mRNA decreased progressively
throughout infection, similarly to amounts of β-actin and
ribosomal protein mRNAs (Greco et al., 1997 ; Simonin et al.,
1997). However, GAPDH mRNA was still detectable after
15 h p.i. (Fig. 2, bottom right panel). Finally, the sizes of viral
and GAPDH mRNAs apparently remained unchanged
throughout the course of infection.
This qualitative analysis alone does not allow us to conclude
definitively whether a correlation exists throughout infection
between the rates of synthesis of these proteins and the
amounts of the mRNA by which they are encoded.
Comparison of the levels of the newly synthesized
proteins and of their mRNAs during the course of
infection
Quantification of the $&S-labelled proteins and $#P-labelled
probes depicted in Figs 1 and 2 was performed by scanning
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Control of host and HSV-1 mRNA translation
after 6 (ICP27) and 9 h (DBP). In contrast, GAPDH synthesis
was subject to a rapid and continuous repression which was
accompanied by a simultaneous degradation of GAPDH
mRNA. By comparing carefully the level of GAPDH synthesis
with the amount of GAPDH mRNA remaining at each timepoint, it is clear that after 9 h of infection GAPDH synthesis
was almost completely turned off, although some GAPDH
mRNA remained in the cytoplasm. These results emphasize
the existence of translational control of both viral and host
protein synthesis.
Distribution of viral and cellular mRNAs among
polyribosomes during the course of HSV-1 infection
Fig. 3. Comparison of the levels of newly synthesized proteins with those
of their mRNAs during the course of infection. Results are expressed as
pixels¬mm2. The amount of 35S incorporated into the specified proteins
during the last 30 min before cell harvest is indicated to the left
(histograms). The amount of bound 32P-labelled probe, proportional to the
amount of the corresponding mRNA at the time of harvest, is indicated to
the right (*). For each lane, the background was calculated and
subtracted from the amount of radioactivity measured for each protein or
mRNA. M, mock-infected.
densitometry of dried gels and of membranes, using a
molecular imager system (Fig. 3). The superposition of the
amounts of proteins synthesized during the last 30 min before
harvesting of cells, and of the amounts of mRNA present in the
cytoplasm at each time of harvesting, revealed an unexpected
degree of translational control of gene expression. Distinct
differences could be detected between viral and host protein
synthesis. Viral protein synthesis started as soon as viral
mRNAs appeared in the cytoplasm, and was later repressed,
even though substantial amounts of mRNA remained in the
cytoplasm. This was true for IE, early and late proteins, with
the beginning and end of synthesis appearing sequentially
delayed. In addition, ICP27 and to a lesser extent DBP
mRNAs, but not US11 mRNA, underwent some degradation
To investigate at which step of translation viral and host
protein synthesis were controlled, we traced the distribution of
viral and GAPDH mRNAs among ribosomes and polyribosomes throughout infection. To the aim, postmitochondrial supernatants of HeLa cells, either mock-infected
or infected for 3, 6, 9 and 15 h, were separated into twenty
fractions after sucrose-gradient centrifugation. As expected,
ribosomes were shifted from polyribosomes to 80S ribosomes
as early as 3 h p.i. (Fig. 4 A). This altered distribution of
ribosomes did not change significantly up to 15 h p.i. (not
shown). The distribution of mRNAs of interest among the
different fractions was assessed by Northern blot with specific
$#P-labelled DNA probes, and quantification of mRNA was as
described above. Fig. 4 (B) shows the results of a typical
experiment ; identical results were obtained from at least two
independent experiments for each mRNA. The distributions of
viral and GAPDH mRNAs among the different fractions of the
sucrose gradients were not identical. This distribution varied
not only with the time of infection, but also with the size of the
mRNA. Table 1 summarizes the sizes of these mRNAs.
After 3 h of infection, most of the ICP27 and ICP22 IE
mRNAs (65 and 83 %, respectively) were associated with 80S
ribosomes and polyribosomes (fractions 8 to 20) (Fig. 4 B).
More than 30 % of the ICP27 and ICP22 mRNAs were within
large polyribosomes containing five or more ribosomes per
mRNA molecule (fractions 15 to 20). From the early (6 h) to the
late stage (9 and 15 h) of infection, these IE mRNAs were
progressively shifted to smaller polyribosomes containing
fewer than 5 ribosomes per mRNA molecule (fractions 8 to 14),
and to particles sedimenting in the 40–60S region of the
gradient (fractions 4 to 7). After 9 h of infection, less than 10 %
of the remaining mRNAs were within large polyribosomes.
The DBP and UL34 early mRNAs behaved similarly to the
IE mRNAs, although changes in their distribution occurred 3 h
later (Fig. 4 B). After 6 h of infection, 53 % of UL42 and 70 % of
UL34 mRNAs were associated with 80S ribosomes and
polyribosomes. From 9 to 15 h p.i., these two mRNAs were
progressively shifted from large polyribosomes to smaller
polyribosomes, and again to particles sedimenting in the
40–60S region of the gradient.
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A.-M. Laurent, J.-J. Madjar and A. Greco
Fig. 4. For legend see facing page.
CHHA
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Control of host and HSV-1 mRNA translation
Table 1. Sizes of HSV-1 viral and host mRNAs
Lengths of mRNAs, ORFs and 5« UTRs are given in nucleotides. The
exact size of DBP and UL34 mRNAs and 5« UTRs are not known
(McGeoch et al., 1985, 1988 ; Ercolani et al., 1988).
Gene
mRNA
ORF
5« UTR
ICP27
ICP22
DBP
UL34
ICP47
US11
GAPDH
1683
1815
C 1715
C 1900
1967
1320
1284
1536
1260
1464
825
264
483
1005
138
518
C 195
C 600
530
214
75
The ICP47}US11 probe revealed both the 2 kb ICP47 IE
mRNA and the 1±3 kb true late US11 mRNA (Fig. 4 B). ICP47
mRNA behaved similarly to ICP22 and ICP27 mRNAs.
Association of US11 mRNA with 80S ribosomes and polyribosomes decreased from 70 % after 6 h of infection to 55 %
after 9 h. Although US11 mRNA cannot accommodate a large
number of ribosomes because of its short ORF (483 nucleotides), most US11 mRNA was associated with six to eight
ribosomes at 6 and 9 h p.i. (fractions 16 to 20). At the end of
the experiment, US11 mRNA was shifted to smaller polyribosomes ; only 15 % of the remaining US11 mRNA was still
associated with five or more ribosomes, while 42 % was bound
to one to four ribosomes, the other US11 mRNA being no
longer translated (fractions 4 to 7).
The behaviour of GAPDH mRNA was followed as a
marker of host mRNA translation throughout infection (Fig.
4 B). In mock-infected cells, about 70 % of GAPDH mRNA was
associated with 80S ribosomes and polyribosomes, 30 % of it
being associated with six or more ribosomes (fractions 17 to
20). Distribution of GAPDH mRNA through the gradient
changed progressively during the course of infection as shown
by a decrease in the amount of the remaining GAPDH mRNA
found in fractions containing large polyribosomes. After 9 h of
infection, just 9 % of GAPDH mRNA remained associated with
these large polyribosomes, while about 25 % was within
smaller polyribosomes. More importantly, more than half of
the remaining mRNA was found among particles sedimenting
in the 40S region of the gradient (fractions 4 and 5).
The partitioning of viral and GAPDH mRNAs through the
gradients was compared for the two infection time-points that
exhibited the largest differences (Fig. 4 C). In each case
analysed, viral mRNAs were initially associated with a large
number of ribosomes. They were then shifted from large
polyribosomes to smaller polyribosomes and also to particles
sedimenting in the 40–60S region of the gradient. This
observed shift in the distribution of the different mRNAs
occurred simultaneously with a progressive decrease in
synthesis of the proteins they encode. These results, observed
for IE as well as for early and late viral proteins, demonstrate
that protein synthesis was under translational control. In
addition, they indicate that initiation of translation of the viral
mRNAs becomes the limiting step for viral protein synthesis
(Mathews et al., 1996). This was also true for GAPDH
synthesis, the translation of its mRNA being similarly inhibited
at the initiation step, as previously reported for β-actin (Greco
et al., 1997).
Behaviour of GAPDH and β-actin mRNA following
UL41 transient expression
VHS protein, the product of the true late UL41 gene, is
thought to be responsible for the primary shut-off of host
protein synthesis by non-selective degradation of mRNA. To
determine whether this degradation also led to a shift of
mRNA from polyribosomes to 40S ribosomal subunits, we
investigated the behaviour of GAPDH and β-actin mRNA after
transfection of the UL41 gene, allowing the transient synthesis
of the VHS protein. Twenty hours after transfection, postmitochondrial supernatants of control and of UL41-transfected
HeLa cells were separated into ten fractions after sucrosegradient centrifugation. Distribution of GAPDH and β-actin
mRNAs among the different fractions was then assessed by
Northern blot. Quantification of these mRNAs was as
described above.
In the presence of UL41 gene expression, the amounts of
cytoplasmic GAPDH and β-actin mRNAs were reduced to
about 25 % of the control (Fig. 5 A). As expected, ribosomes
were shifted from polyribosomes to 80S ribosomes, giving a
sedimentation profile very similar to that observed after
Fig. 4. Distribution of viral and host mRNAs among polyribosomes. The post-mitochondrial supernatants used for mRNA
analysis in Fig. 2 were fractionated by centrifugation on sucrose gradients. Twenty fractions of identical volume were collected.
Distribution of the indicated mRNAs was analysed by Northern blot. Numbering of the twenty fractions is from top to bottom of
the gradient. (A) UV absorbance profiles at 254 nm of polyribosomes from HeLa cells either mock-infected (thin line) or
infected for 6 h (6 h p.i.) (thick line). The top and bottom of the gradient, as well as the positions of 40S, 60S, 80S ribosomal
particles and of polyribosomes are shown. (B) Autoradiograms of membranes after hybridization with 32P-labelled DNA probes.
Time p.i. (h) is indicated to the left. Northern blots were carried out in duplicate for each experiment. UL34, ICP27 and GAPDH
mRNAs were probed sequentially on one set of membranes, while ICP22, DBP and ICP47/US11 mRNAs were probed
sequentially on a second set. Exposures of the autoradiograms were : ICP27, 2 days for 3 and 6 h p.i., 5 days for 9 and 15 h
p.i ; ICP22, 3 days for 3, 6 and 9 h p.i., 5 days for 15 h p.i. ; DBP, 3 days for 3 and 6 h p.i., 1 day for 9 and 15 h p.i. ; UL34,
3 days ; ICP47/US11, 7 days for 3 and 6 h p.i., 2 days for 9 and 15 h p.i. ; and GAPDH, 3 days. (C) Quantification of viral and
GAPDH mRNAs. After autoradiography of the blots in (B), radioactivity was quantified by scanning densitometry. Results are
expressed as the percentage of the sum of the radioactivity present in the twenty fractions.
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A.-M. Laurent, J.-J. Madjar and A. Greco
was responsible for the degradation of mRNAs, but not for
the HSV-1-induced accumulation of mRNAs out of the polyribosomes. Therefore, VHS protein does not affect the
efficiency of host mRNA translation.
Discussion
Fig. 5. Analysis of GAPDH and β-actin mRNAs in cells transiently
expressing the UL41 gene. HeLa cells were transfected for 20 h with
either pUL41, directing synthesis of VHS protein, or with pUC18 as a
control. (A) Amount of GAPDH and β-actin cytoplasmic mRNAs in cells
transfected either with pUL41 (labelled VHS) or pUC18 (labelled C).
Results are expressed as the percentages of the control. (B) Distribution
of GAPDH and β-actin mRNAs among polyribosomes. Analyses were
performed as indicated in Fig. 4. Top graph ; UV absorbance profiles of
polyribosomes from HeLa cells transfected either with pUL41 (thin line) or
with pUC18 (thick line). Lower two graphs ; quantification of GAPDH and
β-actin mRNAs. Results are expressed as the percentage of the sum of the
radioactivity present in the ten fractions.
HSV-1 infection (Fig. 5 B). These results indicate that VHS
protein was indeed synthesized and possessed mRNAdegrading activity. However, the distribution of GAPDH and
β-actin mRNAs among the different fractions of the sucrose
gradients was very similar for UL41-transfected cells and for
control cells (Fig. 5 B). These results indicate that VHS protein
CHHC
These results demonstrate that HSV-1 viral protein synthesis begins as soon as viral mRNAs appear in the cytoplasm.
They also show that the synthesis of viral proteins is
progressively turned off during the course of infection, as is
that of most host proteins. Host proteins such as GAPDH and
β-actin (Greco et al., 1997 ; Simonin et al., 1997) exhibit a virusinduced shut-off of their synthesis beginning immediately after
infection. This virus-induced shut-off of protein synthesis
appears to be the consequence of translational controls
affecting both host and viral proteins. The efficiency of mRNA
translation is first reduced by a decrease in the number of
ribosomes translating the same mRNA molecule, which occurs
at the early stage of infection for host mRNAs and a few hours
after their appearance in the cytoplasm for viral mRNAs. At
the late stage of infection, the remaining host and viral mRNAs
are generally no longer translated. They appear to be
sequestered in complexes probably containing 40S ribosomal
subunits, sedimenting in the 40–60S region of the sucrose
gradients because of the size of the mRNAs and of their 5«
UTRs. The 5« UTRs of the viral mRNAs examined here range
from 138 (ICP27) to about 600 nucleotides (UL34), while that
of GAPDH is only 75 nucleotides long (Ercolani et al., 1988)
(Table 1). Therefore, whereas the 5« UTRs of viral mRNAs can
probably accommodate more than one 40S ribosomal subunit,
it is unlikely that the 5« UTR of GAPDH mRNA can bind more
than a single 40S ribosomal subunit because of its small size.
This probably explains why viral mRNAs were found mainly
in fractions 5 to 7 at the late stage of infection, while most
GAPDH mRNAs were found in fractions 4 and 5 (see Fig. 4).
Under these conditions, the shut-off of viral and host protein
synthesis appears to result from a common mechanism leading
to an inhibition of mRNA translation at the initiation step. This
repression of mRNA translation is not caused by VHS protein,
however. Indeed, in the absence of other viral proteins VHS
protein does not impair efficient mRNA recruitment by
ribosomes, as shown by transient expression of the UL41 gene
encoding VHS protein. VHS protein contributes to the shut-off
of host protein synthesis only by decreasing the amount of
mRNAs available for translation. Similarly, degradation of viral
mRNAs does not appear to be the major cause of inhibition of
viral protein synthesis. Indeed, during the course of infection
only the amount of IE mRNAs declined significantly but, in all
cases, viral protein synthesis was turned off well before the
decrease in the amount of viral mRNAs was apparent. This was
particularly clear for US11 mRNA, the amount of which was
still increasing between 9 and 15 h p.i., while US11 protein
synthesis was drastically reduced during this period.
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Control of host and HSV-1 mRNA translation
The shut-off of host protein synthesis is selective, as
previously demonstrated (Greco et al., 1997). mRNAs
harbouring a polypyrimidine tract at the 5« terminus (5«terminal oligopyrimidine ; 5« TOP), such as those encoding
ribosomal proteins, are translated better after HSV-1 infection
than before (Meyuhas et al., 1996 ; Amaldi & PierandreiAmaldi, 1997 ; Greco et al., 1997). Even though these 5« TOP
mRNAs undergo a similar pattern of degradation to that of
other host mRNAs, synthesis of ribosomal proteins is not
inhibited, and new ribosomes are still efficiently assembled
during the course of HSV-1 infection (Greco et al., 1997 ;
Simonin et al., 1997). These data suggest that two groups of
mRNAs are affected differently by HSV-1 infection : initiation
and re-initiation of the 5« TOP mRNAs remains efficient and is
even stimulated after HSV-1 infection, whereas re-initiation of
other viral and host mRNAs becomes inefficient. However, the
first initiation of translation of all viral mRNAs appears to be
carried out efficiently. On the basis of the observation that new
ribosomes are still synthesized and assembled after HSV-1
infection, it is tempting to speculate that initiation of translation
of viral mRNAs, followed by their efficient translation, takes
place on these new ribosomes that may be free of virusinduced modifications, such as phosphorylation of Sa, S2, S3a
and L30 ribosomal proteins (Masse! et al., 1990 b). In addition,
these new ribosomes contain under-phosphorylated S6 ribosomal protein when they are first made, probably making them
ineffective for 5« TOP mRNA translation (Greco et al., 1997).
If viral mRNAs are translated by newly assembled
ribosomes, there may be a specific way for these mRNAs and
ribosomes to interact properly in an infected cell. Moreover,
after a few rounds of translation, re-initiation of translation
would appear to become progressively less likely. The
mechanism involved in this inhibition of translation initiation is
probably identical for viral mRNAs and for host mRNAs
devoid of 5« TOP. Therefore, selective initiation of mRNA
translation would be expected to be restricted to 5« TOP
mRNAs through a step that distinguishes them from other
mRNAs. Because initiation of translation appears to be blocked
after the binding of 40S ribosomal subunits but before
association of the 60S ribosomal subunit, it is likely that the
small ribosomal subunit remains associated with the 5« UTR
except when the 5« UTR contains a 5« TOP region. To our
knowledge, general initiation factors have not been clearly
implicated in the shut-off of protein synthesis resulting from
HSV-1 infection (Mathews, 1996). The best documented
modification of the translational apparatus is the unusual
phosphorylation of a few proteins of both ribosomal subunits
after infection, making these proteins potential candidates to
inhibit the initiation of translation of viral and host mRNAs,
except for those harbouring a 5« TOP tract.
We are grateful to H. Marsden (MRC, Glasgow, UK) for the generous
gift of anti-ICP27 and anti-DBP antibodies. This work was supported by
the Centre National de la Recherche Scientifique and Universite! Claude
Bernard Lyon 1. A.-M. Laurent was supported by a fellowship from the
Ministe' re de la Recherche et de l’Enseignement Supe! rieur and by the
Association de Recherche sur le Cancer.
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Received 20 May 1998 ; Accepted 16 July 1998
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