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 ?
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