to purify endogenous E32PO4^-labeled NSR1-
TAP from both wild-type NSR1-TAP and
ip6kD NSR1-TAP yeast (Fig. 4A). Lack of
IP6K resulted in an almost 60% decline in
phosphorylation of NSR1 in vivo, indicating
that this protein is physiologically phosphoryl-
ated in intact cells by endogenous IP7(Fig. 4,
B and C).
This study establishes that the inositol
pyrophosphate IP7is a physiologic phosphate
donor to a range of proteins in eukaryotic
cells. The proteins we have best characterized
as IP7targets, yeast NSR1 and SRP40 and
mammalian Nopp140 and TCOF1, are nucle-
olar proteins involved in ribosomal biogene-
sis. Additionally, IP7phosphorylation of
proteins involved in endocytosis may me-
diate roles of inositol pyrophosphates in
this process (6), consistent with the phos-
phorylation by IP7of the adaptin b3A sub-
unit (18), a regulator of vesicular trafficking
References and Notes
1. L. Stephens et al., J. Biol. Chem. 268, 4009 (1993).
2. F. S. Menniti, R. N. Miller, J. W. Putney Jr., S. B. Shears,
J. Biol. Chem. 268, 3850 (1993).
4. A. Saiardi, H. Erdjument-Bromage, A. M. Snowman,
P. Tempst, S. H. Snyder, Curr. Biol. 9, 1323 (1999).
5. A. Saiardi, E. Nagata, H. R. Luo, A. M. Snowman,
S. H. Snyder, J. Biol. Chem. 276, 39179 (2001).
6. A. Saiardi, C. Sciambi, J. M. McCaffery, B. Wendland,
S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 99, 14206
7. H. R. Luo et al., Cell 114, 559 (2003).
8. B. H. Morrison, J. A. Bauer, D. V. Kalvakolanu, D. J. Lindner,
J. Biol. Chem. 276, 24965 (2001).
9. T. Laussmann, R. Eujen, C. M. Weisshuhn, U. Thiel,
G. Vogel, Biochem. J. 315, 715 (1996).
10. S. M. Voglmaier et al., Proc. Natl. Acad. Sci. U.S.A.
93, 4305 (1996).
11. Z. Xue, T. Melese, Trends Cell Biol. 4, 414 (1994).
12. T. Stage-Zimmermann, U. Schmidt, P. A. Silver, Mol.
Biol. Cell 11, 3777 (2000).
13. S. M. Wilson, K. V. Datar, M. R. Paddy, J. R. Swedlow,
M. S. Swanson, J. Cell Biol. 127, 1173 (1994).
14. U. T. Meier, J. Biol. Chem. 271, 19376 (1996).
15. S. Ghaemmaghami et al., Nature 425, 737 (2003).
16. S. T. Safrany et al., J. Biol. Chem. 274, 21735 (1999).
17. E. A. Winzeler et al., Science 285, 901 (1999).
18. A. Saiardi, R. Bhandari, S. H. Snyder, unpublished
19. B. Duclos, S. Marcandier, A. J. Cozzone, Methods
Enzymol. 201, 10 (1991).
20. Treacher Collins Syndrome Collaborative Group,
Nature Genet. 12, 130 (1996).
21. W. A. Paznekas, N. Zhang, T. Gridley, E. W. Jabs,
Biochem. Biophys. Res. Commun. 238, 1 (1997).
22. F. Norbis et al., J. Membr. Biol. 156, 19 (1997).
23. M. J. Schell et al., FEBS Lett. 461, 169 (1999).
24. E. C. Dell’Angelica, C. E. Ooi, J. S. Bonifacino, J. Biol.
Chem. 272, 15078 (1997).
25. E. C. Dell’Angelica, V. Shotelersuk, R. C. Aguilar,
W. A. Gahl, J. S. Bonifacino, Mol. Cell 3, 11 (1999).
26. Materials and methods are available as supporting
material on Science online.
27. We thank A. Riccio, B. Wendland, H. R. Luo, and T. R.
Raghunand for suggestions, discussions, and helpful
comments and all the members of the Snyder lab
for creating a stimulating environment. This work
was supported by U.S. Public Health Service Grant
MH18501, Conte Center Grant MH068830-02, and
Research Scientist Award DA00074 (to S.H.S.).
Supporting Online Material
Materials and Methods
Figs. S1 to S7
References and Notes
28 July 2004; accepted 29 October 2004
Regulates SIRT1 Through a
Shino Nemoto, Maria M. Fergusson, Toren Finkel*
Nutrient availability regulates life-span in a wide range of organisms. We dem-
onstrate that in mammalian cells, acute nutrient withdrawal simultaneously
augments expression of the SIRT1 deacetylase and activates the Forkhead
transcription factor Foxo3a. Knockdown of Foxo3a expression inhibited the
starvation-induced increase in SIRT1 expression. Stimulation of SIRT1 tran-
scription by Foxo3a was mediated through two p53 binding sites present in the
SIRT1 promoter, and a nutrient-sensitive physical interaction was observed
between Foxo3a and p53. SIRT1 expression was not induced in starved p53-
deficient mice. Thus, in mammalian cells, p53, Foxo3a, and SIRT1, three
proteins separately implicated in aging, constitute a nutrient-sensing pathway.
In the yeast Saccharomyces cerevisiae and in
the nematode Caenorhabditis elegans, life-
span can be extended by increasing the
expression of the deacetylase Sir2, an enzyme
whose activity depends on the oxidized form
of nicotinamide adenine dinucleotide (NAD)
(1, 2). In these model organisms, the ability
of Sir2 to extend life may be related to its
role in gene silencing. In both the nematode
and yeast, certain simple environmental
stresses can also increase life-span. In yeast,
reducing the amount of available glucose has
this effect. The ability of glucose restriction
to increase the life-span of yeast requires
Sir2 (3). In C. elegans, activation of the
Forkhead transcription factor DAF-16 is also
associated with increased life-span (4) and its
activation depends in part on nutrient avail-
ability (5). Genetic evidence further suggests
that in worms, DAF-16 and Sir2 work
through a common pathway (2), and recent
evidence suggests that their mammalian
counterparts physically interact (6, 7).
Here, we further analyzed the interrela-
tionship of the closest mammalian orthologs
% of wild type
Fig. 4. In vivo 5b[32P]IP7phosphorylation. (A) Presence of an 80-kD phosphorylated band in the
NSR1-TAP strain. Wild-type, nsr1D, and wild-type NSR1-TAP yeast extracts were incubated with
5b[32P]IP7and processed as described in Fig. 2. (B and C) IP6K deletion reduces phosphorylation of
NSR1 by endogenous IP7. (B) Wild-type NSR1-TAP and NSR1-TAP ip6kD yeast were labeled with
[32PO4]iorthophosphate, and TAP-NSR1 was purified as described (26). Yeast homogenates (2 mg)
were subjected to NuPAGE, and the gel was silver stained and autoradiographed to demonstrate
equal levels of basal phosphorylation (left). Silver staining and autoradiogram of purified NSR1-
TAP from wild-type and ip6kD yeast (right). (C) Quantification of the relative phosphorylation of
NSR1-TAP purified from wild-type or ip6kD yeast. Data represent the mean values and SEM from
three independent experiments.
Cardiovascular Branch, National Heart, Lung, and
Blood Institute (NHLBI), Bethesda, MD 20892, USA.
*To whom correspondence should be addressed.
R E P O R T S
www.sciencemag.org SCIENCEVOL 30617 DECEMBER 2004
of DAF-16 and Sir2: the Forkhead transcrip-
tion factor Foxo3a and the mammalian
NAD-dependent deacetylase SIRT1. Using
a mammalian model of nutritional stress (8),
we examined the effects of nutritional with-
drawal on the activity of the SIRT1 promot-
er. When a mammalian cell line (PC12) was
starved overnight of both serum and glucose,
SIRT1 promoter activity increased by a fac-
tor of È4 relative to cells maintained in
complete medium (Fig. 1A). Levels of SIRT1
mRNA (Fig. 1B) (fig. S1) and protein ex-
pression (Fig. 1C) also increased under these
conditions. Under normal nutrient condi-
tions, a fusion protein comprising Foxo3a
and green fluorescent protein (GFP) was pri-
marily cytosolic (Fig. 1D). However, within
1 hour after nutrient withdrawal, the fusion
protein was predominantly found within the
nucleus (Fig. 1D) (fig. S2). Starvation also
increased Foxo3a protein expression (fig. S3).
The activity of a Forkhead-dependent lucifer-
ase reporter increased under starved conditions
in two different mammalian cell lines (PC12
cells, Fig. 1E; HeLa cells, fig. S4). Starvation
also induced the expression of a subset of
other previously identified Forkhead tran-
scriptional targets (fig. S5). When mice were
subjected to an overnight fast, SIRT1 mRNA
increased in numerous tissues including skel-
etal muscle and liver (Fig. 1F) (fig. S6). These
results suggest that in mammalian cells, nutri-
tional stress induces both SIRT1 transcription
and Foxo3a activation.
To determine whether Forkhead activity is
required for the observed induction of SIRT1
expression under starved conditions, we used
small interfering RNA (siRNA) to inhibit
endogenous Foxo3a expression. Transient
expressionofaFoxo3a siRNA reduced SIRT1
promoter activity under starved conditions
(Fig. 1G). In a stable cell line expressing
Foxo3a siRNA constitutively, endogenous
Foxo3a was greatly reduced (Fig. 1H). Sim-
ilarly, whereas the control cell line exhibited
a doubling of SIRT1 protein expression under
starved conditions, cells with decreased
Foxo3a expression had a reduced response.
Transient expression in PC12 cells of a con-
stitutively active mutant of Foxo3a (Foxo3a-
promoter fragment in the presence of nutrients
(Fig. 2A). Successive deletions of the SIRT1
promoter revealed that the stimulatory effect
of Foxo3a-TM was lost between positions –202
and –91. Lack of Forkhead binding sites in this
region suggested that the stimulatory effect
of Foxo3a-TM could be indirect. Interesting-
ly, this region contains two consensus binding
sites for the tumor suppressor protein p53
(Fig. 2B). To determine whether Foxo3a-TM
might stimulate the SIRT1 promoter through
these p53-binding motifs, we compared the
stimulatory effects of Foxo3a-TM on the
wild-type 202–base pair (bp) fragment, or
with promoter fragments in which one or both
of the p53-consensus binding sites were
mutated. Mutation of either p53-binding site
reduced the stimulatory effects of Foxo3a-TM
(Fig. 2C). In the absence of both binding
sites, Foxo3a-TM–dependent stimulation was
reduced more then 90%. Furthermore, a syn-
thetic promoter containing three tandem re-
peats of a 25-bp SIRT1 promoter fragment
containing both p53 binding sites was acti-
vated by Foxo3a-TM to a degree that equaled
or exceeded the observed effects of Forkhead
proteins on the full-length SIRT1 promoter
Fig. 1. SIRT1 induction by nutri-
tional stress requires Foxo3a.
(A) Normalized luciferase ac-
tivity under the control of a
È1.5-kb fragment of the SIRT1
promoter in PC12 cells under
either normal nutrient (fed, F) or
starved (S) conditions. (B) Re-
verse transcription polymerase
chain reaction (RT-PCR) analysis
of SIRT1 mRNA isolated from
PC12 cells under fed or starved
conditions. b-actin transcript
was used as a loading control.
(C) Western blot analysis of
SIRT1 expression in PC12 cells
under fed or starved conditions,
with tubulin as a loading con-
trol. (D) Distribution of a
Foxo3a-GFP fusion protein in
PC12 cells under fed conditions
or 1 hour after nutrient with-
drawal (magnification 400?).
(E) Luciferase reporter construct
under the control of a synthetic
Forkhead promoter in PC12 cells
under fed conditions (F) or after
15 hours of starvation (S). (F)
RT-PCR analysis of SIRT1 mRNA
from mice fed ad libitum or sub-
jected to an overnight fast, with
b-actin transcript as a loading
control (n 0 3 per group). (G)
SIRT1 promoter activity in PC12
cells after transfection with in-
creasing amounts of a siRNA
plasmid directed against Foxo3a
under either fed (F) or starved (S) conditions. (H) Western blot analysis of SIRT1 and Foxo3a ex-
pression, under fed (F) or starved (S) conditions, in two clonal PC12 cell lines, one with antibiotic
resistance alone (–) and one with both antibiotic resistance and stable expression of Foxo3a siRNA
(þ). Tubulin expression was used as a loading control. Error bars are TSD of triplicate determinations.
Fig. 2. The stimulatory effect of Foxo3a on SIRT1 transcription is mediated through two p53
binding sites. (A) Effect of Foxo3a-TM expression (þ or –) in PC12 cells on luciferase activity under
the control of a series of SIRT1 promoter fragments. (B) Schematic diagram of a È200-bp SIRT1
promoter fragment, showing the location of the two predicted p53 binding sites (hatched boxes).
Constructs a to d represent fragments with neither, one, or both of these sites mutated. (C)
Stimulatory effects of Foxo3a-TM expression on luciferase activity under the control of either the
wild-type promoter or fragments shown in (B) containing p53 binding site mutations. (D) Foxo3a-
TM stimulates luciferase activity under the control of a 3? tandem array of a 25-bp fragment
(nucleotides –182 to –158, construct e) of the SIRT1 promoter. Error bars are TSD of triplicate
R E P O R T S
17 DECEMBER 2004 VOL 306SCIENCE www.sciencemag.org
These results raise the possibility that
Foxo3a and p53 might physically and/or
functionally interact. The Forkhead-associated
(FHA) domain may mediate protein-protein
interaction, and some FHA family members
directly interact with p53 (9–13). Using
purified recombinant proteins, we observed
that p53 and Foxo3a appeared to directly in-
teract in vitro (Fig. 3A). To localize the po-
tential region of Foxo3a needed for p53
interaction, we generated a series of truncation
mutants of Foxo3a in which successively
more of the C terminus of the protein was
deleted (Fig. 3B). Full-length and truncated
mutants were expressed as GFP fusion pro-
teins in PC12 cells to assess subcellular dis-
tribution. Under normal nutrient conditions,
Foxo3a-TM was constitutively nuclear,
whereas the distributions of the truncated
Foxo3a mutants, Foxo3a594and Foxo3a465,
were cytosolic (similar to wild-type Foxo3a,
Fig. 1D). In contrast, the location of the
Foxo3a361truncation mutant was constitu-
tively nuclear, presumably because of the
lack of a nuclear export signal.
When overexpressed in HeLa cells, p53
immunoprecipitated with wild-type Foxo3a
and each of the truncation mutants (Fig. 3C).
The amount of associated p53 correlated
better with the amount of nuclear, rather
than total, Foxo3a. For instance, even though
the overall expression level of Foxo3a-TM
was considerably lower than that of wild-type
Foxo3a, the amount of coimmunoprecipitated
p53 was roughly similar. The amount of p53
after Foxo3a immunoprecipitation was great-
est when the constitutively nuclear Foxo3a361
mutant was expressed. This mutant also
showed that the C terminus of Foxo3a is
not required for interaction with p53. The
interaction between Foxo3a and p53 was
also observed when we performed reciprocal
immunoprecipitation of p53 followed by
Western blot analysis for Foxo3a (Fig. 3D).
Previous studies have identified a con-
served histidine as essential for the inter-
action between certain FHA proteins and
phosphopeptides (9, 10). Nonetheless, muta-
genesis of the corresponding histidine resi-
due in Foxo3a did not affect binding to p53,
as assessed by coimmunoprecipitation (fig.
S7). However, the interaction between wild-
type Foxo3a and p53 was strongly dependent
on nutrient availability (Fig. 3E). This may
simply reflect a difference in subcellular dis-
tribution of Foxo3a under normal nutrient or
starved conditions (fig. S2); however, we can-
not rule out the possibility that starvation-
induced posttranslational modifications to
either p53 or Foxo3a are important for their
p53 can function as either a transcrip-
tional repressor or activator (14). Expression
of p53 repressed the transcriptional activity
of a reporter construct under the control of
three tandem copies of a 25-bp element de-
rived from the SIRT1 promoter (Fig. 3F).
Coexpression of Foxo3a-TM relieved this in-
hibition. In contrast, p53 expression modestly
activated transcription of an alternative syn-
thetic p53 response element derived from the
human ribosomal gene cluster. Coexpression
of Foxo3a-TM inhibited p53-dependent tran-
scriptional activity of this synthetic reporter
(Fig. 3G). Thus, in PC12 cells, the physical
interaction between Foxo3a and p53 antago-
nizes p53 function. Functional interaction
between these two proteins was also observed
in HeLa cells (fig. S8).
These in vitro results suggest a complex
nutrient conditions, the predominant effect of
p53 involves repression of SIRT1 (Fig. 3F). In
contrast, under starved conditions, the ability
of activated Foxo3a to stimulate SIRT1
expression requires p53 (Fig. 2C). These data
suggest that in the absence of p53, the basal
expression level of SIRT1 might rise but the
starvation-induced increase would be blunted.
To test this hypothesis, we analyzed SIRT1
Fig. 3. Interaction between
Foxo3a and p53. (A) In
vitro interaction between
wild-type p53 and Foxo3a.
Recombinant proteins were
incubated together, Foxo3a
(IP), and the presence of
p53 was determined by
Western blot analysis with
a p53-specific antibody. Re-
tion of p53 followed by
Foxo3a Western blot is also
shown. (B) Schematic dia-
gram of Foxo3a showing
the nuclear localization se-
quence (NLS), nuclear ex-
port sequence (NES), the
threonine (T) and serine
(S) residues mutated in
Foxo3a-TM, and the sites for truncation (arrows) of
the various Foxo3a-GFP fusion proteins expressed in
PC12 cells. Representative images of the subcellular
distribution of the various GFP fusion proteins are
shown (magnification 100?). (C) Coimmunoprecipi-
tation of wild-type p53 and hemagglutinin (HA)–
Foxo3a constructs overexpressed in HeLa cells. HA-
Foxo3a was immunoprecipitated from lysates of
transfected cells with an HA-specific antibody. The
presence of p53 was determined by Western blot
analysis with a p53-specific antibody. Similarly, in (D)
lysates were immunoprecipitated with a p53-specific
antibody or control isotype immunoglobulin G (IgG)
sera, and the presence of HA-Foxo3a was determined
by Western blot (WB) with an HA-specific antibody.
(E) Interaction between p53 and wild-type Foxo3a
increases after nutrient withdrawal. Lysates from
transfected HeLa cells overexpressing HA-Foxo3a
and wild-type p53 were examined under fed con-
ditions (time 0 0) or at the indicated times after
nutrient withdrawal. (F) Transcriptional activity of a
luciferase reporter under the control of a 3? tandem
array containing the 25-bp p53 binding sites contained in the SIRT1 promoter. In PC12 cells,
expression of wild-type p53 represses luciferase activity, an effect antagonized by Foxo3a-TM
expression. (G) Transcriptional activity of another synthetic p53 response element. Error bars are
TSD of triplicate determinations.
Fig. 4. Role of p53 in basal and starvation-
induced SIRT1 expression. (A) RT-PCR analysis
of basal SIRT1 expression in adipose tissue
from wild-type (WT) or p53–/–mice. b-actin
transcript was used as a loading control. (B)
Lack of starvation-induced SIRT1 induction in
p53–/–mice, as shown by RT-PCR analysis of
SIRT1 mRNA in liver and skeletal muscle of
p53–/–animals under either fed (F; n 0 3) or
starved (S; n 0 3) conditions. b-actin transcript
was used as a loading control.
R E P O R T S
www.sciencemag.org SCIENCEVOL 30617 DECEMBER 2004
expression in mice with a targeted deletion in
p53. Basal SIRT1 expression was higher in
adipose tissue of p53–/–mice than in wild-type
controls (Fig. 4A). This result is particularly
interesting given the recent observation that
SIRT1 plays a prominent role in fat usage
(15). A survey of other tissues revealed a
more modest but consistent increase in basal
SIRT1 expression in the p53–/–mice (fig. S9).
However, SIRT1 mRNA did not appreciably
change in either liver or skeletal muscle of
p53–/–mice after overnight fasting (Figs. 1F
and 4B). This lack of starvation-induced
SIRT1 expression occurred even though,
relative to wild-type animals, p53–/–mice had
an even greater drop in their fasting glu-
cose after food withdrawal (wild-type mice,
102 T 7 mg/dl; p53–/–mice, 76 T 8 mg/dl;
n 0 4 each, P G 0.01). These in vivo results
further support a role for p53 in SIRT1
Our results show that in mammalian
cells, a simple model of acute nutritional
stress results in a Foxo3a-dependent increase
in SIRT1 levels. Interestingly, chronic cal-
oric restriction also increases SIRT1 expres-
sion (16). Foxo3a regulation of SIRT1
expression occurs through an interaction
with p53. In this regard, it is interesting to
note that Forkhead proteins and p53 share a
number of similarities (14, 17). In the worm,
Forkhead proteins respond to nutrient avail-
ability and a homolog of p53 regulates star-
vation response (18). In mammals, although
p53 is often linked to cancer and Forkhead
proteins are commonly associated with aging,
recent evidence has suggested a role for
Forkhead proteins in tumorigenesis (19, 20)
and a role for p53 in life-span (21, 22). Fi-
nally, both Foxo3a and p53 directly and in-
dependently bind to SIRT1 (6, 7, 23, 24).
Taken together, these results suggest a
complicated but undoubtedly important ho-
meostatic regulatory network involving p53,
Foxo3a, and SIRT1. Further analysis of this
network may help us to understand how adap-
tation to certain cellular stresses, including
nutrient availability, may modulate mamma-
References and Notes
1. M. Keaberlein, M. McVey, L. Guarente, Genes Dev. 13,
2. H. A. Tissenbaum, L. Guarente, Nature 410, 227 (2001).
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4. G. I. Patterson, Curr. Biol. 13, R279 (2003).
5. S. T. Henderson, T. E. Johnson, Curr. Biol. 11, 1975
6. M. C. Motta et al., Cell 116, 551 (2004).
7. A. Brunet et al., Science 303, 2011 (2004).
8. See supporting data on Science Online.
9. D. Durocher, J. Henckel, A. R. Fersht, S. P. Jackson,
Mol. Cell 4, 387 (1999).
10. D. Durocher et al., Mol. Cell 6, 1169 (2000).
11. G. S. Stewart et al., Nature 421, 961 (2003).
12. Z. Lou, K. Minter-Dykhouse, X. Wu, J. Chen, Nature
421, 957 (2003).
13. J. Falck et al., Oncogene 20, 5503 (2001).
14. M. Oren, Cell Death Differ. 10, 431 (2003).
15. F. Picard et al., Nature 429, 771 (2004).
16. H. Y. Cohen et al., Science 305, 390 (2004).
17. B. M. Burgering, G. J. Kops, Trends Biochem. Sci. 27,
18. W. B. Derry, A. P. Putzke, J. H. Rothman, Science 294,
19. M. C. T. Hu et al., Cell 117, 225 (2004).
20. J. Seoane et al., Cell 117, 211 (2004).
21. S. D. Tyner et al., Nature 415, 45 (2002).
22. B. Maier et al., Genes Dev. 18, 306 (2004).
23. J. Luo et al., Cell 107, 137 (2001).
24. H. Vaziri et al., Cell 107, 149 (2001).
25. We thank I. Rovira for help in preparing this
manuscript. Supported by NHLBI Intramural Funds.
Supporting Online Material
Materials and Methods
Figs. S1 to S9
21 June 2004; accepted 1 November 2004
Cofolding Organizes Alfalfa
Mosaic Virus RNA and Coat
Protein for Replication
Laura M. Guogas,1,3David J. Filman,2
James M. Hogle,2* Lee Gehrke1,3*
Alfalfa mosaic virus genomic RNAs are infectious only when the viral coat
protein binds to the RNA 3¶ termini. The crystal structure of an alfalfa mosaic
virus RNA-peptide complex reveals that conserved AUGC repeats and Pro-
Thr-x-Arg-Ser-x-x-Tyr coat protein amino acids cofold upon interacting.
Alternating AUGC residues have opposite orientation, and they base pair in
different adjacent duplexes. Localized RNA backbone reversals stabilized by
arginine-guanine interactions place the adenosines and guanines in reverse
order in the duplex. The results suggest that a uniform, organized 3¶
conformation, similar to that found on viral RNAs with transfer RNA–like
ends, may be essential for replication.
A general problem in positive-strand RNA
virology is understanding how viral RNA
replication is initiated by the RNA-dependent
RNA polymerase (replicase) on the correct
template and nucleotide in an infected cell.
Alfalfa mosaic virus (AMV) and ilarviruses
are unusual positive-sense viruses, the ge-
nomic RNAs of which are replicated only in
the presence of the viral coat protein (CP)
(1, 2). These viruses are distinguished from
many other members of the virus family
Bromoviridae because they lack canonical
features of the tRNA-like structure (TLS)
common at the 3¶ termini of the viral RNA
genomes. The TLS is a necessary and suffi-
cient feature for recruitment of the bromo-
virus replicase (3, 4). CP-induced structural
organization of the AMV RNA 3¶ terminus
may create a functional homolog of the
tRNA tail and thereby permit recognition by
the RNA-dependent RNA polymerase.
CP binds specifically to the 3¶ untranslated
regions (3¶UTRs) found on all four RNAs of
the segmented AMV genome (5). The 180-
nucleotide 3¶UTR secondary structure likely
consists of six hairpins, most of which are
separated by single-stranded tetranucleotide
AUGC repeats (5–8). These repeats are
characteristic of AMV and ilarvirus RNA
sequences and are important for CP binding
(8–11). We previously identified a 39-
nucleotide minimal high affinity AMV CP-
binding site, consisting of the two terminal
hairpins and their flanking AUGC nucleo-
tides (nucleotides 843 to 881 in RNA4; i.e.,
AMV843–881) (8, 12, 13) (fig. S1A). This
fragment is competent to bind either full-
length CP or a 26–amino acid peptide (CP26,
fig. S1B) (13) representing the N-terminal
RNA binding domain (14). The CP N
terminus contains a Pro-Thr-x-Arg-Ser-x-x-
Tyr (PTxRSxxY) RNA binding domain
conserved among AMV and ilarvirus CPs
(14). The arginine at position 17 is critical
for both RNA binding and virus replication
(14–16). Circular dichroism experiments
suggest that the CP N terminus is unstruc-
tured in solution (17). Previous virus crys-
tallization attempts required proteolytic
cleavage of the AMV CP N terminus (18, 19).
CP26 in complex with 5-bromouridine–labeled
AMV843–881RNA were grown in hanging
drops by vapor diffusion. The structure was
1Department of Microbiology and Molecular Genet-
ics,2Department of Biological Chemistry and Molec-
ular Pharmacology, Harvard Medical School, Boston,
MA 02115, USA.
Sciences and Technology, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA.
3Harvard-MIT Division of Health
*To whom correspondence should be addressed.
E-mail: Lee_Gehrke@hms.harvard.edu (L.G.);
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17 DECEMBER 2004 VOL 306 SCIENCE www.sciencemag.org