Cap-snatching mechanism in yeast
L-A double-stranded RNA virus
Tsutomu Fujimura1and Rosa Esteban
Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Edificio Departamental, Avenida
del Campo Charro s/n, Salamanca 37007, Spain
Edited* by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved September 8, 2011 (received for review July 22, 2011)
The 5′ cap structure (m7GpppX-) is an essential feature of eukaryo-
tic mRNA required for mRNA stability and efficient translation.
Influenza virus furnishes its mRNA with this structure by a cap-
snatching mechanism, in which the viral polymerase cleaves host
mRNA endonucleolytically 10–13 nucleotides from the 5′ end and
utilizes the capped fragment as a primer to synthesize viral tran-
scripts. Here we report a unique cap-snatching mechanism by
which the yeast double-stranded RNA totivirus L-A furnishes its
transcript with a cap structure derived from mRNA. Unlike influen-
za virus, L-A transfers only m7Gp from the cap donor to the 5′ end
of the viral transcript, thus preserving the 5′ α- and β-phosphates
of the transcript in the triphosphate linkage of the final product.
This in vitro capping reaction requires His154 of the coat protein
Gag, a residue essential for decapping of host mRNA and known
to form m7Gp-His adduct. Furthermore, the synthesis of capped
viral transcripts in vivo and their expression were greatly compro-
mised by the Arg154 mutation, indicating the involvement of Gag
in the cap-snatching reaction. The overall reaction and the struc-
ture around the catalytic site in Gag resemble those of guanylyl-
transferase, a key enzyme of cellular mRNA capping, suggesting
convergent evolution. Given that Pol of L-A is confined inside
the virion and unable to access host mRNA in the cytoplasm, the
structural protein Gag rather than Pol catalyzing this unique
cap-snatching reaction exemplifies the versatility as well as the
adaptability of eukaryotic RNA viruses.
transcription ∣ Saccharomyces cerevisiae ∣ killer toxin
(1, 2). The cap structure is required for mRNA stability and effi-
cient translation. Capping of the RNA 5′ end is accomplished in
cells and for most viruses in three sequential catalytic reactions
(3, 4): removal of the 5′ γ-phosphate by RNA triphosphatase, ad-
dition of GMP from GTP by guanylyltransferase, and methylation
of the added GMP by methyltransferase. Influenza virus, how-
ever, employs a different mechanism (cap-snatching) to furnish
its mRNA with the structure (5–7). The trimeric viral polymerase
binds the cap of host mRNA, cleaves the RNA endonucleolyti-
cally 10–13 nucleotides downstream, and utilizes the capped frag-
ment as a primer to synthesize its transcript. So far, only negative
stranded RNA viruses and ambiviruses (the Orthomyxoviridae,
Bunyaviridae, and Arenaviridae families) have been known to use
this strategy to furnish their mRNAs.
The totivirus L-A, which infects the yeast Saccharomyces cer-
evisiae, has a nonsegmented dsRNA genome of 4.6 kb (8). Typical
of fungal viruses, L-A has no extracellular transmission pathway.
This virus is transmitted vertically from mother to daughter cells,
or horizontally through mating. The L-A genome contains two
overlapping genes, gag and pol, and the latter is expressed as a
Gag-Pol fusion protein by a −1 ribosomal frameshift (9, 10).
The genome is packed inside of a 39-nm icosahedral capsid con-
sisting of 60 asymmetric Gag dimers, in which one or two Gag
molecules are substituted by Gag-Pol. The N-terminal Pol region
is necessary for genome packaging, although Gag alone is suffi-
cient to form morphologically normal capsids (11). M1, a satellite
ukaryotic mRNA is capped at the 5′ end by a 7-methyl GMP
moiety via an inverted 5′-5′ triphosphate linkage (m7GpppX)
RNA of L-A, has a dsRNA genome (1.6–1.8 kb) that encodes a
protein toxin and immunity (12) but no proteins necessary for its
own replication. It requires L-A for encapsidation and
replication. Thus M1 can be maintained in the cell without the
helper virus provided that L-A proteins are expressed from a vec-
tor (13). Two decades ago Blanc et al. (14) found that the L-A
coat protein Gag covalently binds the cap structure of mRNA.
The reaction was inhibited by the cap analogue m7GpppG but
not by the nonmethylated GpppG. The subsequent study re-
vealed that m7Gp decapped from mRNA covalently attached
to His154 of Gag (15). Site-directed mutagenesis found His154
essential for decapping of mRNA, but its mutation (Arg154)
did not affect transcription, replication, and encapsidation of
viral RNA (15). L-A virions can synthesize positive strand tran-
scripts in vitro in a conservative manner (16). Recently we found
that L-A and M1 transcripts have diphosphate at their 5′ termini
(5′-ppGAAAAAU ...........; L-A and M1 share the same 7-nt
sequences at the 5′ ends) (17). More strikingly, when transcrip-
tion was primed with GTP or GMP, the transcripts again bore the
diphosphate at the 5′ ends. In the latter case, the 5′ β-phosphate
was derived from the γ-phosphate of ATP present in the reaction.
Therefore, L-A virus deliberately keeps its transcripts dipho-
sphorylated at the 5′ ends.
We speculated that, if m7Gp derived from host mRNA was
transferred to the diphosphorylated 5′ end of the viral tran-
script, it would produce an authentic cap structure in yeast. We
tested this hypothesis. Here we describe a unique cap-snatching
mechanism in the dsRNAvirus L-A. L-Aonly transfers the m7Gp
moiety derived from mRNA to its transcript. We also demon-
strate that this capping reaction is essential for efficient expres-
sion of the viral transcript.
Cap Transfer Reaction.A 5-nt cap donor,32p-labeled at the γ-phos-
phate of the triphosphate linkage, was incubated with L-A virions
in a transcription mixture containing the 4 NTPs. The full-length
L-A transcript incorporated the radioactivity (Fig. 1A, lane 1).
To further analyze the reaction, CTP was omitted and GTP was
replaced by GDP. Previously we observed that GDP is a better
substrate to prime transcription than GTP (17). Because the first
C appears at position 17 in the L-A transcript, the virions synthe-
size a 16-nt L-A fragment in the absence of CTP. L-A virions can
also incorporate a cap analogue (m7GpppG) as a primer and pro-
duced a 17-nt transcript (17). In the presence of the cap donor,
GDP-primed transcripts incorporated the label, which moved in a
polyacrylamide gel slightly slower than the 16-nt transcript but as
fast as a cap analogue (m7GpppG)-primed 17-nt transcript did
(Fig. 1B, lanes 4–6). The omission of GDP abrogated the incor-
Author contributions: T.F. designed research; T.F. and R.E. performed research; T.F. and R.E.
analyzed data; and T.F. and R.E. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1111900108 PNAS ∣ October 25, 2011 ∣ vol. 108 ∣ no. 43 ∣ 17667–17671
poration, indicating that the reaction is transcription-dependent
and that the cap donor (or part of it) was not used as a primer
(Fig. 1B, lane 3). The labeled product was isolated from the
gel and analyzed by TLC. Bacterial alkaline phosphatase (BAP)
treatment did not release radioactivity (Fig. 1C, lane 7), indi-
cating that the product was internally labeled. Nuclease S1
treatment digested the RNA body but could not work on the
triphosphate linkage, thus releasing the labeled cap analogue
(m7GpppG) (Fig. 1C, lane 5). Tobacco acid pyrophosphatase
(TAP) treatment released labeled m7Gp (Fig. 1C, lane 6). Be-
cause TAP can hydrolyze anhydrous bonds between α- and
β-, and also β- and γ-phosphates of the triphosphate linkage, it
indicates that the label was located at the γ-phosphate in the
capped product. These results demonstrate that, at least, the
m7Gp moiety of the cap donor was transferred to the 5′ end of
the viral transcript and formed a new cap structure on it. The
reaction required a high concentration of PEG 4000 (Fig. S1)
whereas a nonmethylated capped molecule made in the absence
of SAM did not function as cap donor (Fig. 1D, lane 4). PEG is
known to have bulk structure in aqueous solution that causes
crowding of macromolecules in the solution and has successfully
been used to reconstitute the transcriptase activity of L-A virus in
vitro (18). A 107-nt-capped molecule exhibited similar or better
donor activity than the 5-nt donor (Fig. S2).
Preservation of the 5′ α- and β-Phosphates of the Viral Transcripts in
the Triphosphate Linkage During Cap Transfer. The gel mobility of
the capped transcript (Fig. 1B) strongly suggests that only the
m7Gp moiety of the cap donor was transferred to the 5′ end of
the viral transcript. Here we confirm this. We found that the cap
analogue m7GpppG but not GpppG could be used as cap donor
and produced the 17-nt-capped product (Fig. 2A). This allowed
us to specifically label the 5′ end of the viral transcript (acceptor)
and examine its fate during cap transfer. In the absence of CTP,
(A) Labeled 5-nt cap donor was incubated in a transcription reaction mixture
with L-A virions (lane 1) or M1 virions with WT (lane 2) or mutant (Arg154,
lane 3) Gag protein and the full-sized transcripts were separated in an agar-
ose gel. Ethidium bromide staining and autoradiogram of the gel are shown.
λ, lambda-HindIII markers. (B) L-A virions were incubated with 5-nt cap donor
in a transcription mixture from which CTP and GTP were omitted. Transcrip-
tion was done in the presence of GDP (lane 4) or in its absence (lane 3), and
the products were analyzed on a 15% acrylamide gel. As mobility markers,
GTP (lane 1), cap donor (lane 2), 16-mer (lane 5), and 17-mer primed with
m7GpppG (lane 6) were run in parallel. The sequence of the cap donor
and the position of32P (indicated by the asterisk) are shown (Left). Under
the panel the 5′ end sequence of L-A transcript is shown. The first C appears
at position 17. (C) The labeled product (marked by the arrowhead in lane 4
of B) was isolated from the gel, treated with the enzymes as indicated,
and analyzed on polyethyleneimine-cellulose (PEI-cellulose) with 0.3 M
ðNH4Þ2SO4. Cap donor was also processed in parallel as control. The mobility
of nonlabeled nucleotides is indicated (Left). Deduced transfer reaction is
shown under the panel along with TAP and S1 cleavage sites. (D) 7-methy-
lated and nonmethylated cap donors were synthesized in the presence and in
the absence of S-adenosyl methionine (SAM), respectively, and incubated
with L-A virions as described in the legend to B. The product (17-mer) was
separated in a 8-M urea/15% acrylamide gel.
Transfer of m7Gp from cap donor to the 5′ end of viral transcript. Fig. 2.
linkage of the capped product during cap snatching. (A) L-A virions were in-
cubated in a CTP-omitted transcription mixture in the presence or absence of
methylated or nonmethylated cap analogue (0.5 mM). Transcription was
primed with [α-32P] GTP. BAP-treated or nontreated products were separated
in 8 M urea/15% acrylamide gel. The arrowheads indicate BAP-resistant
capped transcript (17-mer). (B) BAP-resistant 17-mer (capped) shown in A
was isolated from the gel, treated with the enzymes singularly or sequen-
tially as indicated, and analyzed by TLC using 0.3 M ðNH4Þ2SO4as solvent.
Noncapped transcript (16-mer) was also processed in parallel as control.
Deduced reaction scheme is drawn under the panel along with TAP and
S1 cleavage sites. (C) L-A virions were incubated in a GTP- and CTP-omitted
transcription mixture in the presence or absence of m7GpppG (0.5 mM). Tran-
scription was primed with GMP in the presence of [γ-32P] ATP. BAP-treated or
nontreated products were separated in an 8 M urea/15% acrylamide gel. The
arrowheads indicate BAP-resistant capped transcripts (17-mer). (D) BAP-resis-
tant 17-mer (capped) shown in C was isolated and processed as described in
the legend to Fig. 1C except that TLC was carried out with 1 M LiCl as solvent.
Noncapped transcript (16-mer) was also processed in parallel as control. RPP:
RNA 5′ polyphosphatase that hydrolyses β- and γ- phosphates at the 5′ ter-
minus of RNA. Lane 8: Pi standard. Deduced cap transfer reaction with some
enzymatic cleavage sites is drawn under the panel. The asterisks show the
positions of the32P label.
Preservation of 5′ diphosphate of L-A transcript in the triphosphate
www.pnas.org/cgi/doi/10.1073/pnas.1111900108 Fujimura and Esteban
L-A virion incorporates [α-32P] GTP at the 5′ end of the 16-nt
transcript with the label located at the α-position. In the presence
of m7GpppG, part of transcripts (ca. 20%) was converted to a
BAP-resistant 17-mer (Fig. 2A, lane 4). This species was gel-pur-
ified and analyzed by TLC. S1 treatment released m7GpppG,
thus confirming the formation of cap structure at the 5′ end
(Fig. 2B, lane 5). By contrast the 16-nt transcript released GDP
upon S1 digestion (Fig. 2B, lane 2) as observed previously (17).
After TAP treatment the capped product remained at the origin
(Fig. 2B, lane 6), indicating that the label was still associated with
the RNA body of 16 nt. A further treatment with S1, however,
converted it to GMP (Fig. 2B, lane 7). These results indicate that
during cap transfer the α-phosphate at the 5′ end of the acceptor
is preserved at the α-position in the triphosphate linkage of the
capped product. When transcription is primed with GMP, the
virion adds the β-phosphate at the 5′ end of the transcript at the
expense of ATP (17). Thus the 5′ β-phosphate of the acceptor can
be labeled specifically with [γ-32P] ATP, as shown in Fig. 2D. S1
treatment released GDP (Fig. 2D, lane 2), indicating that the
16-nt transcript had diphosphate at the 5′ end. Furthermore,
the transcript released labeled Pi upon RNA 5′ polyphosphatase
(RPP) treatment (Fig. 2D, lane 3). Because the enzyme does not
work on the 5′ α-phosphate, it indicates that the label was located
at the 5′ β-phosphate in the transcript. In the presence of
m7GpppG, part of 16-mer again was converted to a BAP-resistant
17-mer (Fig. 2C, lane 5). The formation of cap structure at the 5′
end of the 17-mer was confirmed by S1 digestion (Fig. 2D, lane 6).
This time, however, the 17-mer produced labeled Pi upon TAP
treatment (Fig. 2D, lane 7), thus confirming that the marker was
located at the β-position of the triphosphate linkage in the cap
structure. These results altogether depict the L-A virus’ cap-
snatching mechanism distinct from that of influenza virus: L-A
virus transfers only m7Gp from mRNA to the diphosphorylated
5′ terminus of its transcript. In addition, although influenza virus
requires a capped mRNA fragment as a primer for transcription
(19), L-A virus can synthesize its transcript in the absence of cap
Involvement of Gag in the Cap-Snatching Reaction. M1 requires L-A
for encapsidation and replication. M1 can be maintained in the
cell in the absence of the helper virus provided L-A proteins are
expressed from a vector (13). Like L-A, M1 virions formed with
wild-type L-A proteins have decapping activity and formed
m7Gp-Gag adduct when incubated with the 5-nt cap donor
(Fig. 3A, lanes 1 and 2). By contrast, M1 virions formed with a
mutant Gag (Arg154) failed to form the adduct as demonstrated
previously (15) (Fig. 3A, lane 3). This mutant Gag can support
M1; therefore, transcription, encapsidation, and replication of
M1 are not impaired by the mutation. When M1 virions were
incubated in a transcription mixture with the 5-nt cap donor, the
virions with the wild-type L-A proteins transferred m7Gp from
the donor to the transcript (Fig. 1A, lane 2), whereas the virions
with the mutant Gag failed to do so (Fig. 1A, lane 3). Because
mutant M1 virions synthesized transcripts with 5′ diphosphate
(Fig. S3) as wild-type virions did (17), these results indicate that
the Arg154 mutation specifically impaired the cap-snatching
reaction, thus signifying the involvement of the decapping reac-
tion in cap snatching.
Cap Snatching Is Essential for Efficient Expression of Viral Transcripts.
We have demonstrated that L-A and M1 virions can synthesize
capped transcripts in vitro. However, capped viral transcripts in
vivo have not been detected yet. We tried to prove their existence
in the cell. RNA was extracted from cells containing M1 virions
with wild-type or Arg154 mutant Gag protein. Capped RNA
was then isolated using a monoclonal anticap antibody. As shown
in Fig. 3B, Upper, lane 1, we could detect capped M1 transcripts
from virions with wild-type L-A protein, whereas capped RNA
from M1 virions with mutant Gag was barely detectable. Because
M1 in these strains was supported by plasmid-encoded L-A pro-
teins, capped L-A RNA transcribed by PolII was used as a posi-
tive control. There was no difference in the amount of capped
L-A RNA between these strains (Fig. 3B, Middle, lane 1). The
results therefore demonstrate that the Gag mutation Arg154 spe-
cifically affects capping of virion-derived transcripts. The strain
carrying M1 with wild-type L-A proteins produces protein toxin
and kills sensitive cells. By contrast the strain harboring the mu-
tant Gag barely produces the toxin as described (15) (Fig. 3C).
From these in vivo results we conclude that the cap-snatching
mechanism operates in the cell and does produce capped viral
transcripts, and that the capping reaction is essential for efficient
expression of the viral genome.
We have demonstrated a unique mechanism of cap snatching in
the yeast dsRNA virus L-A. The virus transfers the m7Gp moiety
from host mRNA to the diphosphorylated 5′ end of viral tran-
script, thus generating an authentic cap structure (referred to as
cap0) in the budding yeast. The mechanism is distinct from that of
influenza virus. Influenza virus endonucleolytically cleaves host
of Gag to form m7Gp-Gag adduct. L-A virions (lane 1) or M1 virions with WT
(lane 2) or mutant (Arg154, lane 3) Gag protein were incubated for decap-
ping with 5-nt cap donor labeled at the γ-phosphate with32P. Then proteins
were separated in an SDS gel and detected by Coomassie blue staining (Left)
or by autoradiography (Right). M; protein standards for mobility. (B) Detec-
tion of capped viral transcripts in vivo. RNA was extracted from cells contain-
ing M1 supported by L-A plasmid expressing WT or mutant Gag (Arg154) or
from L-A plasmid-cured cells (−) as control. Capped RNA was immunopreci-
pitated with (Anti-cap) or without (−) anti-cap antibody and hybridized with
M1 (+) strand-specific (Upper), L-A (+) strand-specific (Middle), or 25S rRNA-
specific (Lower) probe. As control, total RNA was processed in parallel (Total
RNA). (C) Killer assays.
Cap snatching is essential for viral expression. (A) Decapping activity
Fujimura and EstebanPNAS
October 25, 2011
mRNA 10–13 nt downstream from the 5′ end and utilizes the
resulting capped oligo fragment as a primer to synthesize its tran-
script. The L-A cap-snatching mechanism is essential for efficient
expression of viral transcripts and, strikingly, His154 of Gag is
involved in this process. In influenza virus the trimeric polymer-
ase performs cap snatching. It is surprising to find a unique cap-
snatching activity in the structural coat protein Gag. However,
considering that the Pol domain of the Gag-Pol fusion protein
is confined inside the virion and unable to access mRNA in the
cytoplasm, it is quite reasonable that Gag but not Pol performs
the reaction. Thus these findings exemplify the versatility and
adaptability of eukaryotic RNA viruses.
The cap-snatching reaction of L-A virus requires a high con-
centration of PEG 4000 and 7-methylation of cap is essential for
cap-donor activity. Interestingly L-A virions used m7GpppG in
vitro as a cap donor. Thus the virus can potentially utilize not
only host mRNA but also m7GpppG or capped oligo fragments
generated from the 3′-5′ mRNA degradation pathway carried
out by the exosome (20, 21), as cap donors. A substantial amount
(ca. 20%) of transcripts (16-mer) can acquire the cap structure
in vitro. The capped species preserved the α- and β-phosphates
of the transcript in the triphosphate linkage. Current data show
that 5′ monophosphorylated or triphosphorylated transcripts do
not function as cap acceptors in the reaction, thus suggesting that
the diphosphorylated 5′ end status is established at the very early
stage of transcription. His154 is essential for cap snatching in
vitro and in vivo. The model depicted in Fig. 4A postulates that
the m7Gp-Gag adduct generated by the decapping reaction is an
intermediate of m7Gp transfer reaction.
Crystallographic studies of the L-A virion have identified a
trench on the outer surface of Gag that includes His154 (22, 23).
L-A transcripts are made inside the virion and presumably
released to the cytoplasm through one of the pores located at
the icosahedral 5-fold symmetric axes (22). The trenches are
positioned in the Gag asymmetric dimer close to the 2-fold and
3-fold axes, far from the pores. It is likely that the high concen-
tration of PEG 4000 required in the reaction increases the local
concentration of the 5′ end of the viral transcript (or the host
mRNA cap donor) in the trench, presumably mimicking the
cytoplasmic environment overcrowded with macromolecules.
The physical separation of the cap-snatching site from the tran-
scription site may ensure that not all transcripts are capped. If
capped transcripts were destined for translation and the non-
capped ones for encapsidation, then the virus might actively
participate in this differentiation process by changing the ratio
of capping. Our current data suggest that only virions actively en-
gaging in polymerization have capping activity, implying that the
two sites are coordinated. Previously Masison et al. (24) failed to
detect the cap-snatching activity of L-A virus, presumably be-
cause they did not include PEG in the reaction. The authors
found that a ski1 mutation suppresses the effect of the mutation
of His154 on expression of M1 toxin and proposed an alternative
model, a decoy hypothesis in which L-A virus utilizes cellular
mRNAs decapped by the virus as a decoy to protect its non-
capped transcripts from degradation by the XRN1/SKI1 5′-3′ exo-
His154 is located at the tip of a loop (residues 144–163) that
is part of the upper rim of the trench. Upon ligand binding
(m7GDP) the rim moves inwardly and forms a closed conforma-
tion (23). Guanylyltransferase also contains a trench that can
adopt either open or closed conformations during the mRNA
capping reaction (25, 26). Furthermore, it has been noticed
that secondary structure elements around the trench of Gag
resemble those in guanylyltransferase (22). This enzyme forms
a Gp-enzyme intermediate with GTP and transfers Gp to the
diphosphorylated 5′ termini of PolII transcripts to generate a
nonmethylated cap structure. Thus, the cap-snatching reaction
of L-A (Fig. 4A) resembles that of guanylyltransferase. These
similarities suggest convergent evolution. In the standard capping
reaction, guanylyltransferase forms a covalent bond with Gp
through Lys but not His. However, it has been demonstrated that
in the capping reaction of vesicular stomatitis virus, L protein
covalently binds the 5′ monophosphorylated pre-mRNA through
His and transfers the bound pre-mRNA to GDP (27). Members
of the alphavirus-like superfamily methylate GTP first, then seem
to utilize His to form a phosphoamide bond with m7Gp and trans-
fer the m7Gp moiety to the 5′ diphosphate end of a viral tran-
script during the capping reaction (28–30).
In the trench, Tyr150, Asp152, Tyr452, and Tyr538 are located
close to the bound m7GDP, suggesting their involvement in cap
recognition (23). Mutagenesis studies indicate that these residues
are crucial for decapping activity. Tyr452 and Tyr538 sandwich
m7GDP by a potential stacking (23). Cap-binding proteins nor-
mally use two aromatic rings of Tyr, Phe, or Trp in cap recognition
to sandwich the m7G moiety (31, 32). Fungal totiviruses closely
related to L-A share these residues (or similar ones) as well as
His154 at comparative positions in their coat proteins (Fig. 4B).
Decapping activity has also been demonstrated in Gag of L-BC
virus (14). Therefore, it is likely that the cap-snatching me-
chanism of L-A virus is widespread among totiviruses of fungi.
Standard mRNA capping requires three sequential enzymatic
reactions. L-A virus, however, obtains these caps from host
mRNA, thus reducing the burden of encoding its own capping
machinery in a small dsRNA genome (4.6 kb). Typical of a fungal
virus, L-A has no extracellular transmission pathway. This further
eliminates the necessity to encode proteins for exit and reentry
to a new cell. These features have apparently contributed to re-
duce the viral genome to a single dsRNA segment. By contrast,
infectious dsRNA viruses (Reoviridae) found in higher eukaryotes
possess their own capping machinery (33) and have genomes with
10 or more segments.
Materials and Methods
Preparation of the 5-nt Cap Donor. Nonlabeled 106-nt RNA fragment contain-
ing the polylinker sequence from Bluescript KS+ vector was made by T7
dle virion, m7Gp-His154 adduct is depicted by the asterisk. It is not known
which Gag protein in the virion participates in the cap transfer reaction.
mRNA decapping and cap transfer may be coupled with transcription
(see text). (B) Comparison of selected L-A Gag sequences with those of other
fungal totiviruses. His154 and crucial residues for cap recognition are indi-
cated by the arrows. GenBank accession numbers are L-A (AAA50320.1),
TaV1 (ADQ54105.1), BRVF (YP 001497150.1), and L-BC (NP 042580.1).
(A) Schematic diagram of L-A cap-snatching mechanism. In the mid-
www.pnas.org/cgi/doi/10.1073/pnas.1111900108 Fujimura and Esteban
runoff transcription. The cap structure was installed on it using [α-32P] GTP
with the aid of mScript mRNA production system (Epicentre) following the
instructions provided by the manufacturer. The 5-nt cap donor was
generated by digesting it with RNase A (14) and purified through a 15%
Cap Transfer Reaction. The standard cap transfer reaction contained 50 mM
Tris HCl pH 7.5, 5 mM MgCl2, 0.1 mM EDTA, 20 mM NaCl, 5 mM KCl, 0.5 mM
each of ATP, CTP, GTP, and UTP, 20% PEG 4000, and labeled 5-nt cap donor
(2–5,000 cpm). The reaction was started by the addition of L-A or M1 virions
and kept at 30 °C for 1 h. Phenol-extracted products were analyzed on either
agarose or acrylamide gels. TLC using PEI-cellulose was done as described
(17). Decapping reaction was done as described (14).
Immunoprecipitation with Anticap Monoclonal Antibody. Freshly growing cells
(109cells) were harvested and broken by vortex mixing (15 s, 10 times) with
glass beads in a buffer containing 20 mM Tris HCl, pH 8.0 and 100 mM NaCl.
After removing cell debris, RNA was extracted with phenol from the super-
natant and precipitated with ethanol. RNA was incubated for 30 min at 4°C
with 30 μL of protein G-Sepharose (GE Healthcare) prebound to anti-
m3G∕m7G-cap monoclonal antibody (Synaptic Systems). RNA bound to the
Sepharose was extracted, blotted to a nylon membrane, and hybridized with
an L-A-, M1-, or 25S rRNA-specific probe as described previously (34). The L-A
and M1 probes recognize the positive strand sequences of L-A (nt 1,323–
1,786) and M1 (nt 14–500), respectively. The 25S rRNA probe recognizes nt
139–608 of 25S rRNA.
ACKNOWLEDGMENTS. We thank Reed B. Wickner (National Institutes of
Health, Bethesda, MD) for providing the L-A-free killer strains. This work
was supported by Grants BFU2007–60057 and BFU2010–15768 from the
Spanish Ministry of Education and Science.
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Fujimura and EstebanPNAS
October 25, 2011