An unusual internal ribosome entry site in the herpes
simplex virus thymidine kinase gene
Anthony Griffiths and Donald M. Coen*
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115
Communicated by Ed Harlow, Harvard Medical School, Boston, MA, May 18, 2005 (received for review January 5, 2005)
low levels of thymidine kinase (TK), a phenotype associated with
the gene. Using a dual-reporter system, a 39-nt sequence including
the mutation was shown to direct expression of the downstream
reporter was not impaired when the mRNA lacked a 5? cap or had
a stable stem loop 5? of the upstream reporter and was relatively
resistant to edeine, an antibiotic that prevents AUG codon recog-
nition by the 40S-eIF2-GTP?Met-tRNAi complex. Twelve nucleo-
tides were as active as the original sequence for translation of the
downstream reporter. Surprisingly, this sequence lacks an AUG
sequence was important. However, many single-base changes had
only limited effects, and introduction of AUG codons did not
increase translation. A mutant virus containing both the single-
in vitro had significantly less TK activity than a virus with the
single-base deletion alone. Thus, a remarkably short internal ribo-
some entry site (IRES) that lacks an AUG codon resides in the viral
tk gene. The IRES appears to be responsible for TK expression from
a drug-resistant mutant that would otherwise express no TK,
which may contribute to pathogenicity. Because we found numer-
ous short sequences with IRES activity, there might be many
hitherto unrecognized polypeptides expressed at low levels from
drug resistance ? pathogenesis ? proteomics ? translation ? acyclovir
structure at the 5? end of an mRNA and then, through a
mechanism known as scanning, position the 40S ribosome
subunit at the 5?-most AUG codon that is in an appropriate
nucleotide context. At this point, the 60S ribosome subunit is
joined to the 40S subunit, and translation ensues (1). However,
on a minority of eukaryotic mRNAs, translation initiates by
means of a cap-independent mechanism, as first observed with
picornaviruses (2, 3). In picornaviruses, a sequence element
known as an internal ribosome entry site (IRES), which is a long
structured region of RNA, recruits the translational machinery
to the AUG codon, dispensing with the need for the 5? cap
(reviewed in ref. 4). Since their initial discovery, there have been
numerous reports of IRES elements in a variety of viral and
cellular mRNAs, which in most cases appear to employ mech-
anisms similar to those of picornavirus IRESs.
The herpes simplex virus (HSV) gene that encodes thymidine
kinase (TK) has long served as a model for studies of eukaryotic
gene expression (5, 6). The viral TK is also important for the
treatment of HSV infections because this enzyme activates the
antiviral drug acyclovir (ACV). Although ACV is an effective
antiviral agent, drug-resistant virus that causes severe herpetic
disease is sometimes observed in the immunocompromised (7).
The most common mutations observed in these ACV-resistant
(ACVr) clinical isolates are frameshift lesions in the tk gene that
would be expected to abolish TK activity (8). This observation
has raised the question of how these ACVrmutants can cause
n most eukaryotic mRNAs, translational initiation is a
complex process whereby protein factors recognize the cap
severe disease, because TK-negative (TK?) mutants are highly
compromised for pathogenicity in animal models of HSV infec-
tion (9, 10).
Our laboratory has previously investigated one common mu-
tation observed in ACVrclinical isolates, a single guanine
insertion into the tk gene in a run of seven Gs (G7?1G). We
found that viruses carrying this frameshift mutation synthesize
level of TK expression can restore at least some pathogenicity to
the virus (11, 12). This low level of TK, however, still results in
substantial ACV resistance (13). The mechanism that permits
TK expression in this case is an atypical net ?1 ribosomal
frameshift (11, 14).
In this study, we have examined another frameshift mutation
in the tk gene frequently associated with drug-resistant clinical
disease: a deletion in a run of six cytosines known as the C-chord
(C6-1C) (8). We found that a virus containing this mutation
synthesizes low levels of active TK. Analysis of the mechanism
responsible revealed the existence of an unusual IRES in the tk
Materials and Methods
Cells and Viruses. African green monkey kidney (Vero) and TK?
human osteosarcoma (143B) cells (American Type Culture
Collection) were maintained in DMEM supplemented with 10%
FBS at 37°C and 5% CO2. The viruses used in this study were
HSV-1 strain KOS and a series of mutant viruses that express
various levels of active TK: LS-111?-101??-56?-46 (2% of WT
activity), 615.9 (1%), LS-29?-18 (0.5%), and tkLTRZ1 (0%)
(6, 9, 11, 15–19).
Plasmids. Plasmid pAG5 (20) contains the BamHI P fragment of
strain KOS cloned into pBluescript SK (?) (Promega). Plasmid
pAG6.TKC6-1C was made by introducing the C6-1C mutation
into pAG5 by using the QuikChange mutagenesis kit (Strat-
agene), following the manufacturer’s instructions, using two
complementary oligonucleotides (Integrated DNA Technolo-
gies, Coralville, IA) (the sequences for these oligonucleotides
and all others are provided in Table 1, which is published as
supporting information on the PNAS web site). Plasmid pTKC6-
1C.IFS, which contains a stop codon in the TK ORF in addition
to the C6-1C mutation, was made similarly, except that
pAG6.TKC6-1C was used as the template. Plasmid pTKC6-
1C.CUC contains a CUG-to-CUC (both encoding leucine)
Plasmid pAG3 was constructed by using the Renilla luciferase
(Rluc) and firefly luciferase (Fluc) genes from pRL-null and
pGL-basic, respectively (Promega). pRL-null-link was made by
removing the polylinker from pRL-null by digesting with PstI
and BglII. After removing sticky ends with T4 DNA polymerase,
a new polylinker was inserted downstream of the Rluc gene to
Abbreviations: HSV, herpes simplex virus; TK, thymidine kinase; IRES, internal ribosome
entry site; ACV, acyclovir; ACVr, ACV-resistant; Fluc, firefly luciferase; Rluc, Renilla
*To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0504132102 PNAS ?
July 5, 2005 ?
vol. 102 ?
no. 27 ?
make pRL-link by the insertion of a duplex of synthetic oligo-
nucleotides containing BglII, MluI, XhoI, and SmaI recognition
sites into the XbaI site of pRL-null-link, destroying the ensuing
5? XbaI site while retaining the 3? site. The Fluc sequence was
removed from pGL3-basic by digesting with NcoI and XbaI [the
NcoI overhang was removed with mung bean nuclease (NEB,
Beverly, MA), which removes the initiating methionine codon]
and then cloned into SmaI- and XbaI-digested pRL-link to give
pAG1. To remove an in-frame stop codon from the 5? end of the
Rluc gene in pAG1, a fragment containing the gene was gener-
ated by PCR and cloned into NheI- and XhoI-digested pAG1 to
give pAG3. Subsequent pAG3 series plasmids were constructed
by cloning complementary nucleotides into the BglII and XhoI
sites of pAG3. These constructs were designed such that the WT
tk triplets are in the same register as Fluc and the mutant tk
triplets are in the same register as Rluc. To reduce background,
stop codons were placed upstream of the test sequence in the
Fluc frame and downstream of the test sequence in the other two
pAG3.103.SL, which is based on pAG3.103 and has a stable
stem loop (?G ? ?39.2 kcal?mol) inserted upstream of Rluc,
was constructed by inserting an annealed duplex of oligonucle-
otides into the NheI site of pAG3.103.
Correct introduction of all engineered mutations, in the
absence of additional mutations, was confirmed by sequencing.
Construction of Recombinant Viruses. Viruses TKC6-1C, TKC6-
1C.IFS, and TKC6-1C.CUC (Fig. 1) were made after cotrans-
fection of the respective plasmid and infectious DNA from
mutant virus tkLTRZ1 by using Effectene (Qiagen, Valencia,
CA) according to the manufacturer’s instructions. tkLTRZ1
lacks TK activity because of an insertion of LacZ driven by a
strong promoter in tk (18). Screening for recombinant viruses is
described in refs. 20 and 21 and exploits a blue (nonrecombi-
nant)?white (recombinant) screen. The three mutants formed
plaques with WT size and morphology. The correct introduction
of mutations and the absence of unwanted tk mutations were
confirmed by sequencing.
In Situ Measurement of TK Activity. Plaque autoradiography of
infected 143B cells lacking TK was performed as described in
refs. 19 and 20. Viruses that had previously been shown to have
a range of TK activities between 0.5% and 2% were used to
calibrate the assay.
In Vitro Transcription and Translation Assays. pAG3 series plasmids
were linearized with BamHI. Capped transcripts were synthe-
sized by using T7 mMessage mMachine (Ambion, Austin, TX),
and uncapped transcripts were synthesized by using Megascript
T7 (Ambion), both according to manufacturer’s instructions,
except for a 5-min 70% ethanol wash after isopropanol precip-
itation. These RNAs were translated by using rabbit reticulocyte
lysate (Amersham Pharmacia Biotech) according to the manu-
facturer’s instructions (final potassium ion concentration was
130 mM). Fluc and Rluc activities were measured by using
dual-luciferase assay reagents (Promega) and measuring lumi-
nescence on a Victor 2 reader (Wallac, Gaithersburg, MD),
which was generously made available by the Institute of Chem-
istry and Cell Biology, Harvard Medical School. Translations
that were performed in the presence of edeine (a gift from the
National Cancer Institute, Bethesda) were performed as de-
scribed in ref. 22, preincubating the lysate with the drug for 5 min
at 30°C before the addition of mRNA.
Polypeptides were also analyzed by using SDS?PAGE from
[35S]methionine-labeled translations followed by autoradiography.
Active TK Is Synthesized in Cells Infected with Virus TKC6-1C. A
single-base deletion in the C-chord of the tk gene has previously
been identified as a common mutation in ACVrHSV from
patients who suffer herpetic disease despite ACV therapy (7, 8,
23). Gaudreau et al. (8) described two HSV-1 isolates with the
C-chord deletion; by plaque autoradiography, one had a TK-low
phenotype, and the other had a TK?phenotype. To define the
phenotypes resulting from this mutation while avoiding prob-
lems that may be encountered due to using clinical isolates with
poorly defined genotypes, we elected to engineer this mutation
into HSV-1 KOS, a well characterized laboratory strain. To this
end, we recombined a plasmid-borne tk gene carrying this
mutation with virus tkLTRZ1, which is derived from KOS and
has lacZ inserted into tk. This method permits recombinant
viruses to be isolated by using a blue?white screen, which
obviates the need to introduce a selection pressure (ACV is
frequently used to select TK mutants) to help enrich recombi-
nant viruses, because the probability of the recombinant virus
acquiring unwanted mutations is increased. Additionally, this
approach eliminates a potential source of contaminating TK?
virus (12, 20). We named the virus containing the frameshift
mutation TKC6-1C. Plaques from TKC6-1C had ?1.5% of the
TK activity of strain KOS, as measured by quantitative plaque
autoradiography (Fig. 2).
of the gene, the polypeptide that is generated should contain the
nucleoside and ATP binding sites (Fig. 1). We therefore con-
sidered the possibility that this largely out-of-frame polypeptide
was ?1.5% as active as WT TK. To address this question, we
identical non-TK frame polypeptide as TKC6-1C but with a stop
codon introduced into the WT TK frame a short distance
downstream of the frameshift mutation. No detectable TK
activity was observed with TKC6-1C.IFS (Fig. 2). We therefore
concluded that expression of active TK by TKC6-1C requires
synthesis of WT TK amino acids downstream of the frameshift
mutation, raising the question of how this synthesis occurs.
Sequences. We hypothesized that a translational event permitted
the synthesis of active TK. To investigate this possibility, we used
a dual-luciferase reporter system, pAG3 (Fig. 3a), similar to one
previously reported (24). In this system, the test sequence is
placed between a downstream reporter gene (Fluc) and an
upstream reporter (Rluc) that permits an internal control for
translation efficiency. The plasmid is transcribed in vitro and
(WT) TK, the ATP-binding site, and nucleoside-binding sites are marked with
black boxes, and the C-chord is indicated by an arrow. (b) tkLTRZ1 (tk with
LTR-lacZ, indicated by a black box, inserted into the PstI site; the C-chord is
indicated by an arrow). (c) TKC6-1C (tk with a single C, indicated by an arrow,
deleted from the C-chord of KOS tk). (d) TKC6-1C.IFS (tk with a stop codon in
the tk ORF, indicated by an arrow, in addition to the C6-1C mutation). (e)
TKC6-1C.CUC (tk with a CUG-to-CUC change, indicated by an arrow, in addi-
tion to the C6-1C mutation).
www.pnas.org?cgi?doi?10.1073?pnas.0504132102 Griffiths and Coen
then translated in rabbit reticulocyte lysate, a standard system
for studies of translational mechanisms, and Rluc and Fluc
activities measured separately. In our studies, the test sequence
was inserted so that the two reporter genes were out of frame
with each other. Importantly, the initiating AUG codon se-
quence was removed from the Fluc gene. The test sequence was
surrounded by stop codons, upstream in the Fluc frame and
downstream in the other two frames, to reduce the potential
effects of recoding events outside of the test sequence resulting
in expression of Fluc. An in-frame Fluc?Rluc fusion serves as a
normalization control in which the ratio of Fluc to Rluc expres-
sion is set to 100%. The background level for plasmids that did
not promote Fluc expression was ?0.2% relative to the in-frame
Thirty-nine nucleotides including the C-chord of TKC6-1C
were inserted as a test sequence into this system so that the TK
reading frame downstream of the C-chord was out of frame with
Rluc but in-frame with Fluc (pAG3.74). This plasmid directed
expression of the downstream reporter at ?1.5% efficiency
relative to the in-frame fusion control (Fig. 3a), a value remark-
ably similar to the efficiency of TK expression observed from the
TKC6-1C virus. This efficiency of Fluc expression will be
referred to from here on as ‘‘standard levels.’’ We have also
tested reporter constructs containing the tk sequence in trans-
fected Vero cells. In this system, we observe ?0.4% Fluc activity
(a negative control construct expressed levels ?0.08%; unpub-
The tk Sequence Is an IRES. We considered two possible mecha-
nisms to account for Fluc expression downstream of the frame-
shift mutation: ribosomal frameshifting on the test sequence, as
we had previously observed on a run of eight Gs of tk sequence
(11, 14), or translational initiation on the test sequence. To
distinguish between these two mechanisms, we constructed a
plasmid (pAG3.155) in which a stop codon was placed in the
Rluc frame upstream of the test sequence, which would be
expected to prevent Fluc expression as a result of ribosomal
frameshifting but not initiation. Fig. 3b shows that standard
levels of Fluc expression were observed with such a dicistronic
construct, consistent with initiation but not frameshifting. In-
terestingly, increased potassium in the translation reactions
resulted in increased Fluc expression (unpublished data), which
has been observed in IRESs (e.g., 25).
One possible mechanism for initiation on the test sequence is
that translational initiation complexes assemble by means of
interactions with the m7G cap on the 5? end of mRNA, and a
small fraction of these complexes ‘‘skip’’ upstream AUG codons
and continue scanning until they initiate on the test sequence, or
complexes that did initiate translation on upstream AUG codons
then reinitiate after translation termination, or both. A second
potential cap-dependent mechanism is ribosomal shunting, in
which signals in the mRNA direct the scanning complex to
bypass regions of mRNA (1). Alternatively, initiation could
occur by means of an IRES-mediated event, independently of a
5? cap and ribosomal scanning or shunting (reviewed in ref. 26).
To ask whether a 5? cap was important for the expression of Fluc,
uncapped mRNA synthesized from pAG3.103 was translated,
and Rluc and Fluc activities were compared with those from
capped pAG3.103 message (Fig. 3c). Despite an ?300-fold
reduction in Rluc activity from uncapped mRNA, Fluc was
translated at least as efficiently from uncapped mRNA as from
capped mRNA. As an alternative technique, we generated an
mRNA with a stable stem loop (?G ? ?39.2 kcal?mol) inserted
upstream of the Rluc AUG to occlude scanning ribosomes.
Despite an ?60-fold reduction in Rluc activity, Fluc was syn-
thesized at least as efficiently as from constructs lacking the stem
loop (Fig. 3c), suggesting that Fluc was synthesized indepen-
and stem-loop constructs, the Fluc activity was reproducibly
greater than that of the control construct.
by the test sequence (Lower). To the left of the test sequence is the name of
Fluc activity observed after in vitro translation in rabbit reticulocyte lysate,
with the standard deviation in parentheses. The result in b is presented
similarly. (b) pAG3.155 contains a stop codon (UAG, underlined) in the Fluc
reading frame upstream of the test sequence. (c) The Rluc and Fluc genes are
represented by the boxes, and all of these constructs had identical test
enzyme activity expressed as a percentage of the levels in the capped control
construct (observed Fluc activities were in the thousands of raw light units).
mRNA that has standard levels of Fluc activity and was used as a control. The
from the same template but without the cap. The third line represents an
mRNA that has a cap but has a stable stem-loop structure placed upstream of
the Rluc gene. The fourth line represents the same mRNA as the first line, but
0.25 ?M edeine is added to the translation mix.
An IRES in the tk gene. (a) The Rluc and Fluc genes (Upper) separated
shows the levels of active TK polypeptide expressed by each virus relative to
WT strain KOS. The third line shows the average amounts of radioactivity
measured per plaque as a percentage relative to that measured for KOS. The
next line shows the radiographic images from the plates. Below, the three
study, with the names of the viruses and their activities relative to that of KOS
indicated above the plates. The TK activity levels for TKC6-1C (1.5%) and
TKC6-1C.CUC (0.6%) were estimated from a graph (not shown) of the relative
percentage of active TK polypeptide plotted against the relative percentage
of TK activity in situ for the viruses that have a range of TK activities (LS-111?-
101??-56?-46, 615.9, LS-29?-18, and tkLTRZ1).
Quantitative plaque autoradiography of viruses. The top line shows
Griffiths and Coen PNAS ?
July 5, 2005 ?
vol. 102 ?
no. 27 ?
A third method by which to eliminate cap-dependent trans-
lation is to translate mRNAs in the presence of the antibiotic
edeine. At low concentrations, edeine interferes with the ability
of the 40S-eIF2-GTP?Met-tRNAi complex to recognize the
AUG codon (27, 28). Under these conditions, despite a drastic
reduction in Rluc translation, Fluc expression was similar to that
of the control construct (Fig. 3c).
A fourth possibility is that the message becomes altered in
such a way as the 5? end of a portion of the mRNAs are brought
closer to the 5? end of Fluc (e.g., through a cryptic promoter or
a break in the mRNA). To test for a sequence-specific event that
results in translation initiating somewhere in Fluc, we made a
construct in which the WT tk triplets were not in the same
register as Fluc (pAG3.88). Only background levels of Fluc
activity were observed with this construct (Fig. 4f). We also
purified mRNAs from a denaturing urea gel that were then
Fluc activity was observed with RNA from pAG3.155 but not
from a control construct, and no difference in RNA size
6 and Supporting Materials and Methods, which are published as
supporting information on the PNAS web site).
Taken together, these observations argue strongly that the tk
test sequence does not support ribosomal frameshifting or
internal initiation after ribosomal scanning, shunting, transcrip-
tional initiation, or RNA breakage; rather, they suggest that the
sequence is an IRES.
A 12-nt Segment Suffices for IRES Activity. To define a minimal
sequence sufficient for Fluc expression, a series of deletions in
the test sequence, three bases at a time from the 5? end, was
generated and analyzed (Fig. 4a). Constructs were scored as
expressing standard levels, intermediate levels, or low?
background levels of Fluc activity. Interestingly, removal of part
of the C-chord reduced expression to intermediate levels, but
deletion of the entire C-chord resulted in standard levels. Test
sequences of 12 and 9 bases exhibited intermediate levels of
expression, and Fluc activity was only abolished when the test
sequence was 6 bases long (pAG3.109). A similar series of 3?
test sequence were important for Fluc activity (pAG3.77). These
data suggested that sequences important for activity include CC
GUG CUG G (triplets are in the Fluc and tk frames). However,
this sequence alone supported only intermediate levels of Fluc
expression (pAG3.139). Therefore, we took a short sequence
(UG CUG G) that did not support Fluc expression above
background (pAG3.128) and added back bases to the 5? or 3? end
and measured Fluc activity (Fig. 4c). These data indicated that
the minimal sequence sufficient for standard levels of expression
was CC GUG CUG GCG U (pAG3.154).
Ribosomes Enter the Fluc Frame at or 3? of CUG. To ask at which
codons in that frame with stop codons (Fig. 4d). Expression of
Fluc was not reduced after the addition of stop codons in a
sequential 5?-to-3? fashion until the CUG was changed to UAG.
The introduction of stop codons downstream of the CUG also
prevented expression of Fluc. These data are consistent with the
ribosome entering the Fluc frame at the CUG codon. Alterna-
tively, replacement of the CUG with UAG could have altered a
crucial regulatory sequence.
was cloned into the dual-luciferase vector, and mRNAs from these con-
structs were translated in rabbit reticulocyte lysate. The relative Fluc
activities were then measured. To the left of the test sequence is the
plasmid name. To the right of test sequence is a bar graph in which the
length of the bar indicates the level of Fluc activity, and the error bars
indicate standard deviations. For sequences that support ‘‘standard’’ levels
of Fluc expression (?1%), the bars are black. For sequences that support
‘‘intermediate’’ levels of Fluc expression (?0.5% but ?1%), the bars are
dark gray. For sequences that have low or background levels of recoding
(?0.5%), the bars are light gray. The sequence (pAG3.103) is a tk sequence
expressing standard levels of Fluc expression. (a) A series of 5? three-base
deletions. (b) 3? three-base deletions. (c) Rebuilding the constructs to
regain Fluc activity. (d) Introduction of stop codons (underlined, and in
capital letters when different from the tk sequence) into the Fluc reading
frame. ‘‘. . . ’’ and ‘‘. .’’ denote that the construct has an additional 12 nt of
tk sequence upstream or 3 nt of the tk sequence downstream, respectively,
tk sequence with respect to the Fluc ORF. A base was deleted immediately
www.pnas.org?cgi?doi?10.1073?pnas.0504132102Griffiths and Coen
Point Mutagenesis. The deletion and stop codon analyses sug-
gested that the sequence GUGCUGG was most important. We
therefore mutagenized each of these bases (Fig. 4e). Changing
most of the nucleotides in the sequence had little or no effect.
However, changes to the U or G of the CUG codon (in the tk and
Fluc frames) had more substantial effects on Fluc expression.
Expression of Fluc was reduced to background levels when the
U was changed to an A. The other changes to the U or the G had
Polypeptides Expressed from the Reporter Construct Are Consistent
with Internal Initiation. If Fluc expression is due to internal
translational initiation, then one would expect that constructs
that expressed Fluc activity would direct the synthesis of a
product roughly the size of Fluc. We therefore radiolabeled the
products of cell-free translations from all of our constructs and
analyzed them by SDS?PAGE. Examples from constructs that
had high, intermediate, or low Fluc activities are shown in Fig.
5. An ?60-kDa band that comigrated with authentic Fluc was
expressed from all constructs that expressed Fluc activity (e.g.,
AG3.106 and AG3.121; Fig. 5) and not from those that exhibited
background levels of activity (e.g., AG3.112; Fig. 5). A band of
?100 kDa, which is slightly smaller than one would predict for
the size of an Rluc–Fluc fusion protein, was also observed, but
its presence did not correlate with Fluc enzyme activity. These
data bolster the case for internal initiation of Fluc.
A Mutation That Affects IRES Function in Vitro also Affects TK Activity
of the C6-1C Mutant.IftheIRESthatweidentifiedbyusinginvitro
translation is important for expression of TK from the ACVr
C6-1C virus, then a mutation that reduces IRES activity in vitro
without changing the tk ORF should also reduce TK expression
from the mutant. We therefore constructed a recombinant virus
that carried such a mutation (CUG-to-CUC mutation) that
resulted in an ?3-fold decrease in IRES activity (pAG3.111; Fig.
4; both CUG and CUC encode leucine). Virus C6-1C.CUC was
engineered to have both the C6-1C and CUC mutations (Fig. 1).
This virus had ?0.6% tk activity (Fig. 2). A Northern blot
showed that the tk mRNA from this virus migrated similarly to
those from KOS and TKC6-1C (Fig. 7, which is published as
supporting information on the PNAS web site). These data are
consistent with the TK activity from the TKC6-1C virus being
dependent on the IRES.
In this study, we have found that a virus containing a frameshift
translation lead us to conclude that the mutant sequence is
this sequence is an IRES, what makes it unusual, how it may
function, how it may contribute to TK activity and pathogenesis
by the virus, and its implications for genomic and proteomic
The tk Sequence Is an IRES. To conclude that a sequence functions
as an IRES, it is important to rule out other mechanisms that
have been suggested to explain some reports of IRESs and, in
particular, to rule out ribosome scanning mechanisms (29).
Several results exclude the possibility that a scanning mechanism
was responsible for expression of Fluc in our assays. First, the
downstream gene in the reporter construct lacked an initiating
AUG codon. Second, in experiments where scanning-dependent
translation was perturbed by making uncapped transcripts, by
introducing a stable stem loop, or by translating the mRNA in
the presence of edeine (Fig. 3d), Fluc was expressed as efficiently
as from controls. Indeed, Fluc expression was reproducibly
greater than the controls in these experiments. This finding
would be difficult to explain on the basis of a scanning-
dependent or ribosomal shunting mechanism but could be
entering the test sequence impede IRES-dependent translation
of Fluc. Third, (Fig. 4e) two constructs introduced AUG codons
into the test sequence (pAG3.144 and pAG3.122). If ribosomes
were scanning the test sequence, an increase in expression of
Fluc would be expected (particularly with pAG3.122, because it
has a good Kozak consensus sequence). Such an increase was not
observed. Fourth, although the crucial CUG codon that we
identified is in a reasonably good Kozak consensus, changing to
an unfavorable context did not affect Fluc expression
(pAG3.152; Fig. 4d). Fifth, we observed no evidence for short
transcripts that contained a 5? end near the Fluc ORF that could
account for Fluc expression (Fig. 6); but even if we had, the
results summarized above would not be expected to occur by
means of expression of Fluc from such short transcripts.
The tk IRES Is Unusual. IRESs are typically long, highly structured
RNA sequences that serve to recruit elements of the transla-
tional machinery to the message (reviewed in ref. 30). The tk
IRES is only 12 bases long. RNA folding programs such as
MFOLD (31) predict that this sequence exhibits little, if any,
structure. There have been reports of short sequences of be-
tween 9 and 22 nt that can serve as IRESs (32–35). Unlike the
tk IRES, these sequences were analyzed in reporter constructs
that placed the test sequence in close proximity to the AUG of
the downstream reporter gene.
Although the IRES contains two codons, a GUG and the
crucial CUG codon, that can substitute in cap-dependent initi-
ation for AUG using Met-tRNAi (36), several of the lines of
evidence outlined above suggest that the tk IRES does not use
Met-tRNAi-dependent initiation. There have been reports of
methionine-independent initiation in IRESs (37, 38), but these
IRESs are much longer and more structured than the tk IRES.
A third unusual feature of the tk IRES is that it occurs in a
to contain IRESs are gammaherpesviruses (39–42). In those
cases, unlike the tk sequence, the IRES lies in an intergenic
region rather than in the middle of a coding sequence.
How Might the tk IRES Function? The tk IRES bears certain
similarities to another unusual IRES, that of cricket paralysis
virus (CrPV). Like the tk IRES, it is active under conditions that
disrupt the activity of the eIF2-GTP?Met-tRNAicomplex (in
the presence of low concentrations of the antibiotic edeine) (22).
The tk IRES does not contain an AUG; the CrPV IRES does not
of the translation products of the constructs indicated at the top of the gel
were radiolabeled and analyzed by SDS?PAGE and autoradiography. The
positions of Rluc polypeptide (Rluc), authentic recombinant Fluc polypeptide
(rFluc), and molecular weight markers are indicated.
Griffiths and Coen PNAS ?
July 5, 2005 ?
vol. 102 ?
no. 27 ?
initiate with an AUG codon, but with a GCU codon. Our data
from experiments introducing stop codons suggest that transla-
tion initiates from the tk IRES at or just downstream of a CUG
of any other factors (22). However, the CrPV IRES is much
larger and more complex than the tk IRES, serving to position
the ribosome on the message in such a way that the P site is not
decoded and translation initiates in the A site. It will be
important to learn how a sequence as short as the TK IRES can
recruit the translational machinery to the message.
HSV Employs Unusual Translational Mechanisms to Achieve Patho-
genic Drug Resistance. The C6-1C mutation is the third example
of a frameshift mutation found in an ACVrclinical isolate that
appears to be compensated by an unusual translational mecha-
nism to permit expression of active TK (refs. 11 and 20 and
unpublished work). In the two previous examples, expression of
active TK is explained by ribosomal frameshifting that results in
synthesis of active, full-length enzyme. However, in this case,
synthesis of a C-terminal fragment of TK appears to be involved.
To explain how this fragment leads to active TK, we suggest a
mechanism akin to ?-complementation in Escherichia coli ?-ga-
lactosidase protein (43), in which an N-terminal deletion is
compensated in trans by a corresponding N-terminal fragment
(explained at the atomic level in ref. 44). By analogy, the
out-of-frame C-terminal portion of the mutant TK may be
compensated by the corresponding in-frame C-terminal frag-
ment synthesized by means of the IRES. This compensation may
be assisted by the out-of-frame segment serving as a scaffold and
by the dimerization of TK (45).
Regardless of the precise mechanism by which active TK is
produced, the levels produced (?1.5%) are more than those that
suffice to permit reactivation from latency from mouse ganglia
of a strain that requires TK for reactivation (12). We speculate
that the IRES contributes to the ability of clinical ACVrisolates
carrying the C6-1C mutation to cause disease in humans.
The tk IRES Suggests the Existence of an Expanded Proteome. The
data presented in Fig. 4e show that, at least in vitro, many short
sequences can support IRES-mediated translation. These results
raise the possibility that there may be many hitherto unrecog-
nized polypeptides synthesized from eukaryotic messages, which
could have important implications when considering the coding
potential of genomic DNA.
We thank Fred Wang for suggesting the experiment with C6-1C.1FS, the
Harvard Institute of Chemistry and Cell Biology for use of the lumi-
nometer, and the National Cancer Institute for providing the edeine. We
are grateful to Kevin Bryant for useful discussions and Lee Gehrke for
helpful comments on the manuscript. This work was supported by
National Institutes of Health Grants P01 NS35138, R01 AI26126, and
1. Hershey, J. W. B. & Merrick, W. C. (2000) in Translational Control of Gene
Expression, eds. Sonenberg, N., Hershey, J. W. B. & Mathews, M. B. (Cold
Spring Harbor Lab. Press, Woodbury, NY), pp. 33–88.
2. Pelletier, J. & Sonenberg, N. (1988) Nature 334, 320–325.
3. Jang, S. K., Krausslich, H. G., Nicklin, M. J., Duke, G. M., Palmenberg, A. C.
& Wimmer, E. (1988) J. Virol. 62, 2636–2643.
4. Schneider, R. J. & Mohr, I. (2003) Trends Biochem. Sci. 28, 130–136.
5. McKnight, S. L. (1980) Nucleic Acids Res. 8, 5949–5964.
6. Coen, D. M., Weinheimer, S. P. & McKnight, S. L. (1986) Science 234, 53–59.
7. Gilbert, C., Bestman-Smith, J. & Boivin, G. (2002) Drug Resist. Updat. 5,
8. Gaudreau, A., Hill, E., Balfour, H. H., Jr., Erice, A. & Boivin, G. (1998)
J. Infect. Dis. 178, 297–303.
9. Coen, D. M., Kosz Vnenchak, M., Jacobson, J. G., Leib, D. A., Bogard, C. L.,
Schaffer, P. A., Tyler, K. L. & Knipe, D. M. (1989) Proc. Natl. Acad. Sci. USA
10. Efstathiou, S., Kemp, S., Darby, G. & Minson, A. C. (1989) J. Gen. Virol. 70,
11. Hwang, C. B., Horsburgh, B. C., Pelosi, E., Roberts, S., Digard, P. & Coen,
D. M. (1994) Proc. Natl. Acad. Sci. USA 91, 5461–5465.
12. Griffiths, A., Chen, S. H., Horsburgh, B. C. & Coen, D. M. (2003) J. Virol. 77,
13. Sacks, S. L., Wanklin, R. J., Reece, D. E., Hicks, K. A., Tyler, K. L. & Coen,
D. M. (1989) Ann. Intern. Med. 111, 893–899.
14. Horsburgh, B. C., Kollmus, H., Hauser, H. & Coen, D. M. (1996) Cell 86,
15. Bo ¨ni, J. & Coen, D. M. (1989) J. Virol. 63, 4088–4092.
16. Coen, D. M., Irmiere, A. F., Jacobson, J. G. & Kerns, K. M. (1989) Virology
Virology 168, 210–220.
18. Davar, G., Kramer, M. F., Garber, D., Roca, A. L., Andersen, J. K., Bebrin, W.,
Coen, D. M., Kosz Vnenchak, M., Knipe, D. M., Breakefield, X. O. & Isacson,
O. (1994) J. Comp. Neurol. 339, 3–11.
19. Chen, S. H., Cook, W. J., Grove, K. L. & Coen, D. M. (1998) J. Virol. 72,
20. Griffiths, A. & Coen, D. M. (2003) J. Virol. 77, 2282–2286.
21. Griffiths, A., Renfrey, S. & Minson, T. (1998) J. Gen. Virol. 79, 807–812.
22. Wilson, J. E., Pestova, T. V., Hellen, C. U. & Sarnow, P. (2000) Cell 102,
23. Sasadeusz, J. J., Tufaro, F., Safrin, S., Schubert, K., Hubinette, M. M., Cheung,
P. K. & Sacks, S. L. (1997) J. Virol. 71, 3872–3878.
24. Grentzmann, G., Ingram, J. A., Kelly, P. J., Gesteland, R. F. & Atkins, J. F.
(1998) RNA 4, 479–486.
25. Jackson, R. J. (1991) Biochim. Biophys. Acta 1088, 345–358.
26. Pestova, T. V., Kolupaeva, V. G., Lomakin, I. B., Pilipenko, E. V., Shatsky,
I. N., Agol, V. I. & Hellen, C. U. (2001) Proc. Natl. Acad. Sci. USA 98,
27. Odom, O. W., Kramer, G., Henderson, A. B., Pinphanichakarn, P. & Hardesty,
B. (1978) J. Biol. Chem. 253, 1807–1816.
28. Kozak, M. & Shatkin, A. J. (1978) J. Biol. Chem. 253, 6568–6577.
29. Kozak, M. (2001) Mol. Cell. Biol. 21, 1899–1907.
30. Hellen, C. U. & Sarnow, P. (2001) Genes Dev. 15, 1593–1612.
31. Zucker, M., Mathews, D. H. & Turner, D. H. (1999) in RNA Biochemistry and
Biotechnology, eds. Barciszewski, J. & Clark, B. F. C. (Kluwer, Dordrecht, The
32. Chappell, S. A., Edelman, G. M. & Mauro, V. P. (2000) Proc. Natl. Acad. Sci.
USA 97, 1536–1541.
33. Chappell, S. A. & Mauro, V. P. (2003) J. Biol. Chem. 278, 33793–33800.
34. Owens, G. C., Chappell, S. A., Mauro, V. P. & Edelman, G. M. (2001) Proc.
Natl. Acad. Sci. USA 98, 1471–1476.
35. Zhou, W., Edelman, G. M. & Mauro, V. P. (2003) Proc. Natl. Acad. Sci. USA
36. Kozak, M. (1997) EMBO J. 16, 2482–2492.
37. Sasaki, J. & Nakashima, N. (2000) Proc. Natl. Acad. Sci. USA 97, 1512–
38. Wilson, J. B., Hayday, A., Courtneidge, S. & Fried, M. (1986) Cell 44, 477–487.
39. Coleman, H. M., Brierley, I. & Stevenson, P. G. (2003) J. Virol. 77, 13093–
40. Low, W., Harries, M., Ye, H., Du, M. Q., Boshoff, C. & Collins, M. (2001)
J. Virol. 75, 2938–2945.
41. Isaksson, A., Berggren, M. & Ricksten, A. (2003) Oncogene 22, 572–581.
42. Bieleski, L. & Talbot, S. J. (2001) J. Virol. 75, 1864–1869.
43. Ullmann, A., Perrin, D., Jacob, F. & Monod, J. (1965) J. Mol. Biol. 12, 918–923.
44. Juers, D. H., Jacobson, R. H., Wigley, D., Zhang, X. J., Huber, R. E., Tronrud,
D. E. & Matthews, B. W. (2000) Protein Sci. 9, 1685–1699.
45. Waldman, A. S., Haeusslein, E. & Milman, G. (1983) J. Biol. Chem. 258,
www.pnas.org?cgi?doi?10.1073?pnas.0504132102Griffiths and Coen