JOURNAL OF VIROLOGY, Apr. 2010, p. 4330–4340
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 9
Achieving a Golden Mean: Mechanisms by Which Coronaviruses
Ensure Synthesis of the Correct Stoichiometric Ratios
of Viral Proteins?
Ewan P. Plant,1† Rasa Rakauskaite ˙,2Deborah R. Taylor,1and Jonathan D. Dinman2*
Laboratory of Hepatitis and Related Emerging Agents, Division of Emerging and Transfusion-Transmitted Diseases,
Office of Blood Research and Review, CBER, FDA, 8800 Rockville Pike, HFM310, Bethesda, Maryland 20892,1and
Department of Cell Biology and Molecular Genetics, Microbiology Building, Room 2135,
University of Maryland, College Park, Maryland 207422
Received 24 November 2009/Accepted 4 February 2010
In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed ?1 ribosomal
frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-
encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the
coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins,
the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and
replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of
frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open
reading frames affect virus propagation has not been explored before. Here, three different strategies were
employed to test the hypothesis that the ?1 PRF signals of coronaviruses have evolved to produce the correct
ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respi-
ratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency de-
creased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot
mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and
mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previ-
ously described frameshift attenuator element does not actually affect frameshifting per se but rather serves
to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a
“golden mean” model in which viruses use both programmed ribosomal frameshifting and translational
attenuation to control the relative ratios of their encoded proteins.
Viruses utilize programmed ribosomal frameshifting (PRF)
to posttranscriptionally regulate the expression of multiple
genes encoded on monocistronic viral mRNAs. In many RNA
viruses that utilize programmed ribosomal frameshifting (e.g.,
most retroviruses, totiviruses, and Ty elements), the mRNAs
transcribed from these viral templates contain two overlapping
open reading frames (ORFs). In these viruses, the ORF en-
coding the major viral nucleocapsid proteins (e.g., Gag) is
located at the 5? end of the mRNA, whereas ORFs encoding
proteins with enzymatic functions (typically Pro and Pol) are
located 3? of, and out of frame with, the Gag ORF. The
enzymatic proteins are translated only as a result of PRF
events that occur at frequencies of 1 to 40% depending on the
specific virus and assay system employed (reviewed in refer-
ence 6). Thus, the majority of translational events result in the
production of structural nucleocapsid proteins, while the inter-
vention of frameshifting results in a decreased yield of enzy-
matic products (23). The importance of maintaining precise
ratios of structural to enzymatic proteins on viral propagation
has been demonstrated using two endogenous viruses of the
yeast Saccharomyces cerevisiae and with two retroviruses (re-
viewed in reference 18). Small alterations in programmed
frameshifting efficiencies promote the rapid loss of the yeast
double-stranded RNA (dsRNA) L-A killer virus (13, 14, 17, 19,
38, 39, 40, 44, 49). Similarly, increasing or decreasing the effi-
ciency of the ?1 ribosomal frameshift in the Ty1 retrotrans-
posable element of yeast results in reduced retrotranspostion
frequencies (2, 17, 20, 27, 28, 33, 39). In L-A, Gag-pol dimer-
ization nucleates the formation of the virus particles (10–12,
22). Increasing the amount of Gag-pol protein synthesized may
cause too many particles to initiate nonproductively, while
producing too little may prevent efficient dimerization (19).
The proteolytic processing of the TyA-TyB (Gag-pol equiva-
lent) polyprotein of Ty1 is more akin to the situation observed
in retroviruses. In Ty1, increasing the amount of Gag-pol pro-
tein synthesized inhibited the proteolytic processing of the
polyprotein (33). As a consequence, the formation of the ma-
ture forms of RNase H, integrase, and reverse transcriptase is
blocked (33). Similarly, changing the ratio of Gag to Gag-pol
proteins in retroviruses like HIV or Moloney murine leukemia
virus interferes with virus particle formation (4, 24, 29, 32, 42,
53). In these cases, the overexpression of the Gag-pol protein
results in the inefficient processing of the polyprotein and the
inhibition of virus production. In sum, viral PRF efficiencies
* Corresponding author. Mailing address: Department of Cell Biol-
ogy and Molecular Genetics, Microbiology Bldg, Rm 2135, University
of Maryland, College Park, MD 20742. Phone: (301) 405-0918. Fax:
(301) 314-9489. E-mail: firstname.lastname@example.org.
† Present address: Division of Viral Products, Office of Vaccine
Research and Review, CBER, FDA, 8800 Rockville Pike, HFM445,
Bethesda, MD 20892.
?Published ahead of print on 17 February 2010.
at UNIV OF MARYLAND on April 6, 2010
have been fine-tuned to deliver the precise ratios of proteins
required for efficient viral particle assembly; too much or too
little frameshifting alters this ratio, with detrimental conse-
quences. Based on these studies, it has been proposed that ?1
PRF is a viable target for the prevention of viral propagation
(reviewed in 18).
Coronaviruses are positive-strand RNA viruses with large
genomes (?30,000 nucleotides [nt]) that also utilize ?1 PRF.
They can cause enteric and respiratory tract infections with
varying severity. For example, some genotypes affecting hu-
mans (HCoV-229E and HCoV-OC43) cause cold-like symp-
toms, while the coronavirus associated with severe acute respi-
ratory disease (SARS-CoV) is associated with a high mortality
rate. Similarly, the coronaviruses that affect other mammals
have assorted phenotypes: the mouse hepatitis virus (MHV)
enterotropic strains replicate initially in the intestinal epithe-
lium and tend not to disseminate, whereas the neurotrophic
MHV strains initially replicate in the respiratory tract and then
disseminate to the liver, brain, and lymph nodes. The latter
strains are used in models for acute and chronic central ner-
vous system infection (54). While the SARS-CoV and MHV
viruses have different pathologies, overall they are phylogeneti-
cally more similar to each other than SARS-CoV is to HCoV-
229E (21). The genomic organization of coronaviruses is dif-
ferent from that of retroviruses and totiviruses: the structural
proteins are encoded by subgenomic mRNAs, while the genes
regulated by ?1 PRF are involved in replicase/transcriptase
function (56, 59). The genomic organization of SARS-CoV is
shown in Fig. 1. The ORF1a-encoded polyprotein (pp1a) syn-
thesizes nonstructural proteins. The ?1 PRF signal is located
at the 3? end of ORF1a and redirects a fraction of the trans-
lating ribosomes into the ORF1b reading frame to synthesize
the larger pp1ab polyprotein. The enzymatic functions re-
quired for viral replication are derived from pp1ab (1, 5, 55).
Although frameshifting is an essential feature of the viral life
cycle per se because it is required for the production of most of
the replicase proteins, the consequences of changing ?1 PRF
efficiencies on the replication of this class of viruses have never
The cis-acting signals that promote frameshifting consist of a
heptameric slippery site and an strong mRNA structure sepa-
rated by a short spacer. In general, the slippery site can be
defined as N NNW WWH, where N is any three identical
bases, W is AAA or UUU, and H is A, C, or U (the frame of
the initiator AUG is indicated by the spacing) (8, 16). It ap-
pears that there is a preference within virus groups for certain
slippery sites, and these preferences likely reflect the differ-
ences in the host ribosomes (3, 45). The second element is
usually an mRNA pseudoknot that directs elongating ribo-
somes to pause with their A and P sites positioned over the
slippery site (34, 51). The initial demonstration that a
pseudoknot was required for efficient ?1 PRF was for the
avian infectious bronchitis coronavirus (IBV) (9). Subse-
quently, numerous pseudoknots have been described that fa-
cilitate frameshifting (reviewed in reference 25). Until re-
cently,allof the frameshifting
contained two stems. However, structural analyses revealed
that the SARS-CoV frameshift-stimulating pseudoknot con-
tains three stems (47, 52). In addition, another cis-acting ele-
ment affecting ?1 PRF located immediately upstream of the
SARS-CoV ?1 PRF signal was suggested to attenuate the
frameshifting efficiency of both the SARS-CoV and infectious
bronchitis virus (IBV) signals (52). The availability of se-
quences from several new coronaviruses now allows more in-
depth comparisons of regulatory sequences.
The current study begins by examining the question of the
importance of synthesizing the correct ratios of viral proteins
for SARS-CoV propagation and then addresses mechanisms
through which these ratios may be controlled. Initially, a series
of slippery-site mutants was introduced into an infectious clone
to test the hypothesis that correct levels of ?1 PRF are critical
for the propagation of this virus. The viable mutant viruses
produced less genomic RNA than subgenomic RNA. Further-
more, the infection of cells with equivalent amounts of wild-
type and mutant genomic RNAs revealed that the mutants
were significantly less infectious than the wild type, thus dem-
onstrating an important role for ?1 PRF in the viral life cycle.
pseudoknots have evolved in coronaviruses to promote frame-
shifting at specific levels so as to deliver the proper ratios of
ORF1a and ORF1ab products was tested using a series of
mutations that morphed the MHV ?1 PRF signal into that
from SARS-CoV. The results of this analysis reveal features of
the coronavirus pseudoknots that are important for stimulating
optimal levels of frameshifting. Lastly, the issue of an addi-
tional regulatory element, the so-called attenuator sequence
FIG. 1. Schematic of genomic and subgenomic RNAs. The open reading frames of the SARS-CoV gRNA are shown as open boxes. The
position of the frameshift signal where ORF1a and ORF1b overlap is indicated. The 5? leader sequence and 3? noncoding region common to the
gRNA and all sgRNAs are shown as filled boxes. The positions of the primers gF and gR used to detect gRNA and the primers sgF and sgR used
to dedect sgRNA are shown as arrows. Note that the detection of sgRNA requires a leader sequence proximal to the 3? ORF.
VOL. 84, 2010SHIFTING TOWARD A GOLDEN MEAN 4331
at UNIV OF MARYLAND on April 6, 2010
(52), was examined. Phylogenetic analyses reveal that while
there is little conservation of the sequence upstream of the
various coronavirus ?1 PRF signals, computational analyses
show that they all are predicted to fold into strong secondary
structures. Although prior findings suggested that the attenu-
ator element reduced ?1 PRF by ?40%, the experimental
design employed in that study did not preclude the hypothesis
that strong secondary mRNA structures simply cause ribo-
somes to dissociate from the mRNA prior to encountering the
frameshift signal, i.e., translational attenuation. Experiments
presented in the current study support this hypothesis, suggest-
ing that the function of the attenuator is to further help fine-
tune the ratios of ORF1a and ORF1b viral products by limiting
the number of ribosomes available to translate ORF1b. In sum,
the current study shows that the ratios of ORF1a- and ORF1b-
encoded proteins play a critical role for the coronaviruses, and
that both ?1 PRF and translational attenuation are employed
to guarantee the production of a “golden mean” of viral pro-
teins for optimal virus replication and viability.
MATERIALS AND METHODS
Sequence analysis. The GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) ac-
cession numbers for the sequences discussed in this paper are SARS-CoV
(NC_004718), Bt-CoV Rp3 (NC_009693), Bt-CoV HKU3 (NC_009694), Bt-CoV
Rf1 (NC_009695), Bt-CoV Rm1 (NC_009696), Bt-CoV HKU9-1 (NC_009021),
Bt-CoV HKU5-1 (NC_009020), Bt-CoV HKU9 (NC_009019), Bt-CoV 133/2005
(NC_008315), IBV (NC_001451), TCoV (NC_010800), MuCoV (NC_011550),
BuCoV (NC_011548), ThCoV (NC_011549), SW1-CoV (NC_010646), MHV-
A59 (NC_001846), MHV-JHM (NC_006852), HCoV OC43 (NC_005147),
BCoV (NC_003045), EqCoV (NC_010327), pigeon herpes encephalomyelitis
virus (NC_007732), HCoV HKU1 (NC_006577), Bt-CoV HKU2 (NC_009988),
feline infectious peritonitis virus (NC_007025), transmissible gastroenteritis virus
(TGEV) (NC_002306), HCoV 229E (NC_002645), HCoV NL63 (NC_005831),
porcine epidemic diarrhea virus (NC_003436), Bt-CoV 512/2005 (NC_009657),
Bt-CoV HKU8 (NC_010438), Bt-CoV 1a (NC_010437), and Bt-CoV 1b
(NC_010436). Sequences were aligned using ClustalW2 (35), and cladograms
were constructed on the EMBL website (http://www.ebi.ac.uk/). Pairwise align-
ments were performed using the default Clustal settings in the Lasergene soft-
ware (DNASTAR Inc., Madison, WI). RNA sequences were folded using mfold
on the web server at Rensselaer Polytechnic Institute (36, 60).
Strains and genetic methods. Escherichia coli strain DH5? was used to amplify
plasmids, and high-efficiency transformations were performed using the method
of Inoue et al. (30). Vero E6 cells were cultured at 37°C with 5% CO2in
Dulbecco’s modified eagle medium (Invitrogen, Carlsbad, CA) supplemented
with 10% fetal bovine serum (FBS; HyClone, Logan, UT). Cells were transfected
using FuGENE 6 (Roche, Indianapolis, IN) according to the manufacturer’s
Plasmid constructs. The parental plasmids pJD464 and pJD502, containing
the Renilla and firefly luciferase genes flanking the wild-type SARS-CoV frame-
shift, have been described previously (47). pJD502 is the test construct (T) for
measuring frameshifting efficiency, and pJD464 is a readthrough control plasmid
(C) to normalize against any defects in overall translation that the introduced
SARS sequence may cause. A PCR fragment corresponding to nucleotides 13057
to 14171 from MHV strain A59 (a kind donation from Paul Masters) was used
as a template for our MHV studies. The region from nucleotides 13545 to 13681,
which included 51 nucleotides upstream of the slippery site, the slippery site, and
the predicted pseudoknot, was amplified by PCR. The 51 nucleotides upstream
from the slippery site was the maximum amount that could be cloned while still
maintaining two open reading frames such that both test and control vectors
could be made. The primers (Table 1) included the restriction sites BamHI and
SacI to allow for cloning. The PCR amplicon was digested with these two
endonucleases and cloned into the similarly digested dual luciferase vector p2luci
(26) to create the readthrough control plasmid pJD768. This then was subcloned
as a BamHI/EcoRI fragment into the dual luciferase vector p2luc (26) to create
the test construct pJD769. Site-directed mutagenesis was used to introduce
mutations at various positions in the frameshift-stimulating pseudoknot on
pJD768. Mutagenesis was performed using Stratagene’s QuikChange II kit (La
Jolla, CA) and the primers listed in Table 1. The mutations were confirmed by
sequencing, and test constructs for each control were made by subcloning the
BamHI/EcoRI fragment into p2luc.
TABLE 1. Oligonucleotide primers used in this study
Oligonucleotide and functionSequence
4332 PLANT ET AL. J. VIROL.
at UNIV OF MARYLAND on April 6, 2010
SARS-CoV reverse genetics. Briefly, a full-length cDNA clone of the SARS-
CoV genome was constructed from six subclones (called SARS clones A through
F), and SP6 RNA polymerase and a GTP cap analog were used to generate
full-length infectious transcripts (58). The transfection of these into mammalian
cell lines results in a productive, lytic viral infection. RNA was prepared by in
vitro transcription, and the transcripts were transfected into Vero cells. The virus
was allowed to grow for 5 days at 37°C. Viral supernatants were plated on Vero
cells, and several clones were obtained by plaque purification. The plaque-
purified viruses were expanded on Vero cells. Viral assays were conducted in a
biosafety level 3 facility.
Quantitation of viral titer. The abundance of viral genomic and subgenomic
RNAs (gRNA and sgRNA, respectively) was determined by quantitative real-
time PCR using SYBR green chemistry. RNA was extracted from infected cells
using TRIzol (Invitrogen, Carlsbad CA) from which cDNA was produced using
the Applied Biosystems high-capacity cDNA reverse transcription kit (Foster
City, CA) according to the manufacturer’s instructions. Primers complementary
to nucleotides 13496 to 13516 and 13564 to 13542 (using the numbering of the
Urbani SARS-CoV strain; GenBank number AY278741) were used to detect
genomic transcripts, and primers complementary to nucleotides 30 to 50 and
28539 to 28521 were used to detect subgenomic RNA. Although the primers for
detecting the sgRNA can anneal to the gRNA, only the smallest sgRNA has the
5? leader and 3? sequence in close enough proximity to allow amplification to
proceed with normal PCR cycles (Fig. 1). To quantitate the ratio of subgenomic
to genomic equivalents, we measured the ratio of genomic to subgenomic RNA
from viral stocks, intracellular (unlysed cells) plus any secreted or membrane-
bound viral RNA. We believe that this ratio most fairly describes replication
events independent of viral infectivity.
For infection experiments, genomic RNA was quantitated in the viral stocks
for the purpose of determining the amount of viral stock to use as inoculum.
Equivalent amounts of genomic viral RNA were used to infect cells. Viral titers
were determined by observing infected Vero E6 monolayers in 96-well plates by
use of a 50% tissue culture infectious dose (TCID50) assay as previously de-
scribed (15). Briefly, 10-fold serial dilutions of viral samples were incubated at
37°C for 4 days and then examined for cytopathic effect (CPE) in infected cells.
The CPE of SARS-CoV-infected Vero E6 cells was determined by observing
rounded, detached cells in close association with each other. The first dilution of
viral sample was a 1:10 dilution, which set the limit of viral detection for this
assay at 1 log10TCID50. Error bars are the standard deviations from six mea-
surements. Where infectivity was at the lower level of detection for the assay,
error was not able to be calculated.
Translation assays. PCR primers were designed to amplify the frameshift
signal and the attenuator sequence when present from the dual luciferase plas-
mids. The forward primers included the T7 transcription promoter (Table 1).
Small amplicons were generated so that differences in the proteins from tran-
scripts with or without the attenuator sequence could be clearly resolved. Addi-
tionally, larger amplicons were generated that included the entire luciferase
cassette. PCR products were made using the Fermentas 2? PCR Master Mix
(Glen Burnie, MD). RNA was transcribed and translated from PCR amplicons
using the Ambion mMESSAGE mMACHINE T7, MEGAScript T7, and Retic
Lysate IVT kits (Austin, TX) or the Promega T7? coupled transcription/trans-
lation kit (Madison, WI). Products labeled with [26S]methionine were separated
by electrophoresis through Invitrogen 4 to 20% Tris-glycine gels (Carlsbad, CA),
and autoradiographs were produced.
Dual luciferase assays. Vero E6 cells were transfected with the dual luciferase
plasmids and grown overnight in DMEM supplemented with 10% FBS. Cells
were lysed using the passive lysis buffer (Promega, Madison, WI) per the man-
ufacturer’s instructions. Luminescence reactions were initiated by the addition of
10 to 20 ?l of cell lysates to 100 ?l of the Promega LAR II buffer and completed
by the addition of 100 ?l to the Stop’n’Glo reagent. Luminescence was measured
using a Turner Design TD20/20. At least three readings were taken for each
assay, and all assays were repeated (n ? 3 to 12) until the data were normally
distributed to enable statistical analyses both within and between experiments
RNA structure probing. The structures of the pseudoknots were probed using
the SHAPE procedure of Wilkinson et al. (57). Briefly, DNA was amplified from
each mutant plasmid using 2? PCR master mix (Fermentas, Glen Burnie, MD)
and the primers T7forSHAPE, 5?-TAATACGACTCACTATAGGGAAGA TG
CACCTGATGAAATGG-3?, and revSHAPE, 5?-GCCCATATCGTTTCATAG
CTTC-3?. These primers correspond to the 3? end of firefly luciferase and the 5?
end of Renilla luciferase coding sequences, respectively. RNA was transcribed
from the PCR products using the Ambion T7 MEGAscript kit per the manu-
facturer’s instructions. Two pmol of RNA in a total volume of 12 ?l H2O was
denatured at 95°C and cooled on ice. Six ?l of a buffer containing 333 mM
HEPES, pH 8.0, 20 mM MgCl2, 222 mM NaCl was added to each RNA sample,
and the mixture was incubated at 37°C for 20 min. The reaction mixtures were
split into two equal aliquots, one containing 1 ?l dimethylsulfoxide (DMSO) and
the other containing 1 ?l of 30 mM N-methylisatoic anhydride (NMIA) in
DMSO, and incubated at 37°C for 45 min. The RNAs were precipitated with 90
?l H2O, 4 ?l 5 M NaCl, 1 ?l 20 mg/ml glycogen, 2 ?l 100 mM EDTA, pH 8.0,
and 350 ?l ethanol overnight at ?80°C. After centrifugation, the RNA was
resuspended in 7 ?l of 0.5? Tris-EDTA. Fifty pmol of the oligonucleotide
5?-GCCGGGCCTTTCTTTATG-3? (Integrated DNA Technology, Coralville,
IA) was labeled with 30 ?Ci of [?-24P]ATP using T4 kinase (Roche) and purified
through a G-25 column (GE Healthcare; Piscataway, NJ). Seven ?l of RNA and
3 ?l of labeled oligonucleotide were annealed. Reverse transcription reactions
were performed using SuperScript III enzyme (Invitrogen, Carlsbad, CA) at 52°C
for 20 min. Products were separated through 8% polyacrylamide gels, dried, and
exposed to a phosphorimager cassette for analysis.
Frameshift efficiency plays a critical role in SARS-CoV
propagation. The composition of a slippery site can strongly
influence its ability to promote ?1 PRF (9, 19, 45). A series of
slippery-site mutants was constructed in a dual luciferase-
based ?1 PRF reporter plasmid (pJD502), and the effects on
?1 PRF efficiency were assayed as previously described (47).
The plasmids are based on those described by Grentzmann et
al. (26) and support transient expression. The activity of the
second luciferase is dependent on ?1 PRF and is normalized
to the activity of the first. As shown in Table 2, the different
slippery sites promoted a broad range of ?1 PRF efficiencies
from 14.4 to 0.15%. A reverse genetics system (58) then was
employed to assess the effects of three of the mutant slippery
sites (marked by an asterisk in Table 2) on SARS-CoV prop-
agation. The three mutant viruses (and wild-type control) were
constructed, recovered, and tested for infectivity using a tissue
culture infectious dose assay. Initial visual inspection revealed
that plaque numbers and diameters increased as ?1 PRF ef-
ficiencies approached wild-type levels. Consistent with the hy-
pothesis that ?1 PRF efficiency is critical for coronavirus prop-
TABLE 2. Slippery-site SARS-CoV sequencesa
U UUA AAC (WT)
U UUU UUC
A AAT TTA
U UUU UUU*
A AAA AAC
G GGA AAC
G GGU UUA
U UUA AAU
U UUA AAA
A AAA AAU*
C CCA AAC
G GGU UUC
U UUA AAG
G GGU UUU
U UUG AAA
G GGU UUG
U UUG AAC*
aSlippery-site SARS-CoV sequence mutants and incoming reading frames are
indicated by spaces. ?, slippery sites incorporated into infectious clones. ?1 PRF
efficiencies and standard deviations were measured in Vero cells as previously
described (60). The plaque sizes of infectious clones are indicated by the number
of plus signs. The TCID50(log10/10 viral genomes) is the average of six mea-
surements. The lower limit of the assay (1 log10) is 1.
VOL. 84, 2010SHIFTING TOWARD A GOLDEN MEAN4333
at UNIV OF MARYLAND on April 6, 2010
agation, the U UUG AAC mutant, which does not alter the
primary protein coding sequence of the slippery site but which
almost completely abrogated frameshifting, was not able to
support virus propagation. RNAs from the wild-type and re-
maining two mutant viruses were recovered and quantified
using real-time PCR. Interestingly, the abundance of genomic
RNA in infected cells from the mutant viruses was reduced by
approximately three orders of magnitude relative to wild-type
levels, while the subgenomic RNA levels only dropped by ap-
proximately one order of magnitude (Fig. 2). Equivalent
amounts of virus (based on the amount of genomic viral RNA)
subsequently were used to infect cells, after which TCID50
values were determined (Table 2). This analysis clearly shows
that as little as a 3-fold decrease in ?1 PRF efficiency essen-
tially abrogated SARS-CoV propagation. Consistent with ex-
perimental data from retroviruses and dsRNA viruses, this
demonstrates that maintaining the efficiency of frameshifting is
essential for the optimal production of infectious viral particles
for positive-sense single-stranded RNA (ssRNA) viruses as
Morphing the MHV frameshift-promoting pseudoknot
into the SARS-CoV pseudoknot supports the hypothesis
that viruses have evolved a golden mean for frameshift
efficiency. Although SARS-CoV and MHV belong to the same
group of coronaviruses, their predicted ?1 PRF-promoting
pseudoknots differ significantly. Comparing MHV with SARS
in Fig. 3A, the first stem of both pseudoknots is predicted to be
10 nucleotides long, i.e., one full helical turn. The second stem
of the SARS pseudoknot is 7 nucleotides long with a bulged
adenosine (47, 52), whereas the MHV stem is predicted to be
9 nucleotides long without a bulged residue. The loop joining
stem 3 with stem 2 also differs significantly between the two
pseudoknots: the loop in the MHV structure is predicted to be
8 nucleotides long, and that in the SARS structure is 2 nt.
Finally, each pseudoknot has a bulged adenosine in the third
stem but they differ slightly in placement, with the adenosine in
the SARS pseudoknot being closer to the loop of stem 3. We
hypothesized that the two viruses have similar functional re-
quirements with regard to ?1 PRF efficiency, and that the
different ?1 PRF signals have evolved over time toward a
functionally equivalent golden mean, i.e., different structures
have evolved to produce optimal levels of frameshifting for
The predicted structural differences between the SARS-
CoV and MHV frameshift-promoting pseudoknots provided
natural starting points to probe for how differences in structure
affect function. To this end, a series of four mutants was con-
structed that sequentially changed the MHV ?1 PRF promot-
ing pseudoknot into the SARS-CoV structure. Figure 3A
shows the entire series of six constructs, from wild-type MHV
to SARS-CoV, where the predicted secondary structures were
computationally determined using pknots (48). The circles de-
note bases sequentially mutagenized to morph the MHV
pseudoknot into the final SARS-CoV structure. To gain a
better sense of their solution structures, SHAPE analysis of T7
RNA polymerase primer extension reactions was employed
using NMIA (57). Representative autoradiograms of the
SHAPE reactions for each construct are shown in Fig. 3B, and
the results are color mapped onto the secondary structures in
Fig. 3A, where black denotes fully protected bases, green
means partial protection from modification, and red shows that
the sugars were fully available for modification by NMIA. Most
aspects of the predicted structures held true, with fully pro-
tected bases (black) mapping inside of the stems and partially
protected bases (green) mapping to stem junctions and in
loops. All of the stem 1 structures were highly stable. However,
this analysis did reveal some important differences from the
predicted structures. For example, although the MHV stem 2
is predicted to be 9 bp in length, the high A-U and G-U
content at its distal end rendered it quite unstable, decreasing
its actual length to a core of three to four G-C base pairs and
consequentially increasing the length of loop 1 from the pre-
dicted 2 nt to approximately 7 nt. The SARS-CoV stem 2 was
much more stable (5 to 6 bp plus the protected bulged A), and
its actual loop 1 was shorter than that of MHV (4 nt). In
contrast, while base pairing in the MHV stem 3 was very stable,
progressive mutations toward the SARS-CoV sequence led
base pairing in this region to become less stable. Further, while
the repositioning of the bulged A in stem 3 helped to stabilize
stem 2, this further destabilized stem 3. Similar patterns were
observed in the loop 2 and loop 3 regions, although we note
that loop changes simultaneous with moving the bulged A may
have influenced stem 3 stability. For example, while the larger
loop 2 of SARS-CoV was significantly more solvent accessible
than its shorter counterpart in MHV, the longer loop 3 of
MHV was more solvent accessible than the shorter loop 3 of
SARS-CoV. Interestingly, increasing the stability of loop 3 by
reducing its size progressively destabilized loop 2 (compare the
series ?2, ?2?3, ?2b?3 in Fig. 1A). In summary, while the
MHV and SARS-CoV ?1 PRF-promoting pseudoknots both
contain stable stem 1 structures, they each appear to have
exchanged solvent-accessible (stem 2/loop 3 for MHV and
stem 3/loop 2 for SARS-CoV) and solvent-inaccessible mod-
ules (stem 3/loop 2 for MHV and stem 2/loop 3 for SARS-
CoV). In contrast, the intermediate constructs appear to
progress to less-solvent-accessible, and thus more compacted
structures, culminating with ?2 and ?2?3, from which point
they sequentially become more solvent accessible and, thus,
FIG. 2. Relative abundance of genomic and subgenomic RNA in
viral stocks. Plaque-purified virus was used to infect Vero cells. Four
days postinfection CPE was observed. Media and detached cells were
removed and filtered. RNA was extracted from a 100-?l aliquot using
TRIzol. TaqMan analysis was used to determine the total number of
genomic and subgenomic RNA molecules compared to a reference
RNA transcribed from a SARS replicon (1). The number of copies per
ml of viral stock is shown with standard deviations.
4334PLANT ET AL.J. VIROL.
at UNIV OF MARYLAND on April 6, 2010
The effects of these constructs on ?1 PRF efficiency were
assayed as previously described (47). The wild-type MHV and
SARS-CoV sequences promoted roughly equivalent levels of
?1 PRF (SARS-CoV, ?18.4%; MHV, ?24.6%) (Fig. 3A).
Beginning with the MHV sequence, shortening loop 1 by one
codon (?2) increased the stability of stem 2 and increased
frameshifting efficiency to ?66%. Reducing the length of loop
3 further compacted the structure (?2?3), increasing frame-
shifting to ?90.8%. The insertion of a bulge into stem 2
(?2b?3) rendered loop 3 more solvent accessible (less com-
pacted) and lowered the frameshifting frequency to ?51.7%. A
final mutant that repositioned the bulge in stem 3 made the
loop more like that of the SARS-CoV pseudoknot (?2b?3b)
lowered frameshifting efficiency back to a level closer to that of
FIG. 3. Changing the MHV ?1 PRF-promoting mRNA pseudoknot to the SARS-CoV pseudoknot: structural and functional analysis. (A) The
predicted secondary structures of the MHV (left) and SARS-CoV (right) pseudoknots are shown, along with a series of mutants designed to
sequentially change the MHV sequence into that of SARS-CoV. S and L denote stem and loop elements, respectively. Circled bases show the
sequential mutations made to shorten stem 2 (?2) and loop 3 (?2?3) and to add and move the bulges in stems 2 (?2b?3) and 3 (?2b?3b).
Frameshifting efficiency (percent) with standard errors is shown below each construct. Color coding indicates the extent of protection from
modification by NMIA in the SHAPE reactions as indicated by the bar below. (B) Representative autoradiograms of SHAPE reactions.
Dideoxynucleotide sequencing reactions (GUAC) are included for each mutant. The minus sign indicates control samples, and the plus sign
represents NMIA-treated samples.
VOL. 84, 2010 SHIFTING TOWARD A GOLDEN MEAN4335
at UNIV OF MARYLAND on April 6, 2010
the wild-type SARS-CoV basal level (?26.8%). These results
demonstrate a relationship between pseukoknot stability/com-
pactedness and ?1 PRF efficiency and support the hypothesis
that the pseudoknots of SARS-CoV and MHV evolved to
promote the correct frequencies of ?1 PRF required for the
optimum synthesis of ORF1a- and ORF1b-encoded proteins.
The SARS-CoV attenuator sequence impedes ribosome pro-
cessivity rather than inhibiting frameshifting. A prior study
had suggested the presence of a frameshift attenuator element
located immediately 5? of the SARS-CoV ?1 PRF signal (52).
This conclusion was based on the observation that apparent
frameshifting efficiency was reduced by ?40% when an addi-
tional 150 nucleotides of sequence located 5? of the slippery
site of the SARS ?1 PRF signal was included in frameshift
reporter constructs. The inclusion of this sequence upstream of
the IBV frameshift signal resulted in a similar reduction in ?1
PRF, leading the authors to conclude that the function of this
element is to specifically attenuate frameshifting. An mfold
analysis revealed that this sequence may assume very stable
secondary structures, leading the authors to suggest that RNA-
RNA interactions between the attenuator sequence and the
?1 PRF signal promote decreased rates of ?1 PRF. An alter-
native interpretation is that the stable RNA structure assumed
by this sequence simply inhibits ribosome processivity, causing
a significant fraction of ribosomes to dissociate from the
mRNA before encountering the ?1 PRF signal. This would
result in an apparent, but not actual, change in ?1 PRF effi-
If such an element is functional, it should be conserved. In
an initial survey, windows of 150 nt upstream of the ?1 PRF
signals from all 32 coronaviruses sequenced to date were ex-
tracted and compared. Multiple-alignment analyses using
Clustal W2 (35) at both the peptide and nucleotide levels
revealed that although there was good conservation of se-
quences within the different subgroups of viruses, there was no
conservation of either primary nucleotide or peptide sequences
between coronavirus subgroups (data not shown). However,
since primary sequence information is not informative with
regard to potential secondary- or tertiary-structure interac-
tions, all of these sequences were folded in silico using mfold
(36, 60) to address this issue. This analysis revealed that they
all had the potential to form stable secondary structures, al-
though there was no predicted consensus structure tying the
different groups together. Figure 4 shows folding solutions
from six representative viruses. Thus, it is possible that coro-
naviruses have evolved strong RNA structures to modulate
To determine whether the effect of the SARS-CoV attenu-
ator element on ?1 PRF is direct or indirect, a series of dual
luciferase reporters was constructed either with or without the
attenuator element and/or the ?1 PRF signal (slippery site
plus pseudoknot) (Fig. 5A). The SARS-CoV attenuator se-
quence, slippery site, and pseudoknot (nucleotides 13224 to
13477) were cloned between the two Renilla and firefly lucif-
erase genes into p2luci to create the test plasmid (T?). The
insertion of an additional adenosine immediately 5? of the
slippery site created a readthrough control plasmid (C?), en-
abling the translation of the downstream firefly gene without
frameshifting. Test and control constructs lacking the attenu-
ator sequence and containing the ?1 PRF signal (pJD502 and
pJD464) were described previously (47) and were used to con-
trol for the absence of the attenuator sequence. These are
designated T? and C?. pLuci provided the readthrough con-
trol (RT) lacking both the attenuator and ?1 PRF signal.
The apparent level of frameshifting promoted by the atten-
uator-containing construct (T?/C?) was 12.1%, i.e., ?34%
less than the 18.4% frameshifting promoted from the construct
lacking this sequence (T?/C?) (Fig. 5B). This result compares
favorably with results of the prior study (52). However, the
comparison of firefly/Renilla luciferase ratios among the
readthrough controls revealed that both the attenuator and ?1
PRF signal inhibited ribosome processivity (Fig. 5C). Specifi-
cally, the addition of the slippery site plus pseudoknot reduced
the firefly/Renilla luciferase ratios to 92% of the control plas-
mid lacking any inserts (compare RT/RT to C?/RT), i.e., the
pseudoknot structure inhibited ribosome processivity by ?8%.
The addition of the attenuator reduced this ratio further to
62% of the no-insert readthrough control (C?/RT), i.e., the
FIG. 4. Coronavirus sequences upstream of the ?1 PRF signal are
predicted to fold into highly stable structures. mfold analyses of the
predicted secondary structures of sequences 5? of the slippery sites
from six distantly related coronaviruses are shown.
4336PLANT ET AL.J. VIROL.
at UNIV OF MARYLAND on April 6, 2010
combination of the attenuator and pseudoknot inhibited ribo-
some processivity by 38%. This number is nearly identical to
the apparent 34% reduction in ?1 PRF in the presence of the
attenuator. Thus, the attenuator element does not actually
inhibit ?1 PRF. Rather, we suggest that this element functions
to reduce the fraction of ribosomes that encounter the ?1 PRF
signal, from which point they may shift the reading frame and
translate ORF1b. In sum, we conclude that this element has
evolved as an additional means to control the stoichiometric
ratio of pp1a to pp1b.
If the attenuator functions to block elongating ribosomes,
then its presence should result in the accumulation of trun-
cated peptide products. To test this, PCR products containing
T7 RNA polymerase transcription promoters were synthe-
sized, T7 RNA polymerase runoff transcripts were synthesized
in vitro, [26S]methionine-labeled peptides were translated in
vitro, and the products were analyzed by sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (SDS-PAGE). The
frameshift-promoting (T? and T?2) template lacking the at-
tenuator primarily produced a product of 37 kDa (T? and
T?2 in Fig. 5D), which is consistent with the presence of a
0-frame termination codon. The addition of the attenuator
(T?) increased the size of this product to 43 kDa. Importantly,
the presence of the attenuator also resulted in the production
of a significant fraction of truncated peptides (indicated by an
asterisk in Fig. 5D). A similar range of truncated products was
produced from the readthrough reporter containing the atten-
uator (C?), and these were significantly less well represented
in the attenuatorless readthrough control (C?). In addition,
the presence of truncated products in the 37-kDa range in the
C? sample is consistent with pseudoknot-induced ribosome
dissociation. These results support the hypothesis that both the
attenuator and the frameshift-stimulating pseudoknot cause a
significant fraction of ribosomes to dissociate from the mRNA.
Maintaining the correct levels of coronavirus frameshifting
efficiency is essential for viral infectivity. The importance of
maintaining precise ratios of Gag to Gag-pol has been dem-
onstrated for the L-A dsRNA totivirus, the HIV and MMTV
retroviruses, and the Ty1 retrotransposable element (reviewed
in reference 18). In addition, the detrimental effects of altered
frameshifting efficiency has been shown for the positive-sense
ssRNA luteovirus barley yellow dwarf virus (43) and, more
recently, with regard to the neuroinvasiveness of the Kunjin
subtype of the positive-sense ssRNA flavivirus West Nile virus
(37). Although one of the earliest frameshift signals identified
was from a coronavirus (7), the importance of frameshifting
has not been established formally for this group. The ORFs in
FIG. 5. Apparent effect of the attenuator sequence on frameshifting is due to its affect on ribosome processivity. (A) pLuci (no-insert
readthrough, i.e., RT) was employed as the parent plasmid for all subsequent constructs. The SARS-CoV frameshift signal, including the slippery
site (ss) and pseudoknot (?k), were cloned between the two luciferase genes (test construct T?). A readthrough plasmid was created by the
addition of one nucleotide (n) upstream of the slippery site (control construct C?). Similar constructs containing the upstream attenuator sequence
(att) also were made (T? and C?). The reading frame of the firefly luciferase in each of the constructs is indicated. (B) Apparent frameshifting
efficiencies were assayed in Vero cells transfected with each of the indicated constructs. Firefly activity was normalized to Renilla activity, and
comparisons were made between the plasmids. The test constructs were compared to the control constructs to determine frameshifting efficiency.
(C) Firefly/Renilla luciferase ratios generated by the readthrough constructs containing either the pseudoknot alone (C?) or the pseudoknot and
the attenuator sequence (C?) were compared to those generated from pLuci (RT). (D) Full-length luciferase proteins produced using separate
transcription and translation reactions were separated by SDS-PAGE. Two different dilutions of T? (i.e., T? and T?2) are shown. An increase
in the abundance of smaller proteins from attenuator-containing transcripts is indicated with an asterisk. The expected sizes of the Renilla protein
with or without the attenuator at 43 and 37 kDa, respectively, are marked on the gel. The frameshift or readthrough products (Firefly/Renilla) with
or without the attenuator are predicted to be 101 and 107 kDa, respectively.
VOL. 84, 2010 SHIFTING TOWARD A GOLDEN MEAN 4337
at UNIV OF MARYLAND on April 6, 2010
which the coronavirus frameshift signals are found are very
large and encode many different proteins, many of whose func-
tions are uncharacterized. Altering the ORF1a/ORF1b protein
ratios by reducing ?1 PRF efficiency should result in fewer
enzymes encoded by ORF1b compared to the level of pro-
teases encoded by ORF1a.
The reduction of ?1 PRF to 0.15% completely abrogated
the production of infectious viral particles, whereas small
amounts of infectious virus were produced at ?1 PRF levels of
2.3%. These observations demonstrate that there is a lower
limit of frameshifting below which coronaviruses cannot repli-
cate to detectable levels. As anticipated, gRNA production was
greatly reduced in the two viable ?1 PRF mutants. After
normalizing for virus levels in infected cells, the infectivity
rates of the two viable slippery-site mutants remained at least
3.5 orders of magnitude less than that of the wild-type controls.
One possible explanation for this dramatic effect consequent to
mere 3- to 7-fold changes in ?1 PRF is that decreased ?1 PRF
results in the decreased synthesis of ORF1ab-encoded repli-
case proteins, which are required for the replication of the
virus. This species in turn serves as the enzyme for the synthe-
sis of gRNA and sgRNA. In positive-sense ssRNA viruses,
gRNA synthesis is greatly amplified from the negative strand,
and thus the observation that the mutants decreased infectivity
by ?3.5 orders of magnitude, and gRNA synthesis by ?3
orders of magnitude, may be accounted for by this amplifica-
tion process. Alternatively, the decreased viral amplification
may be due to a decrease in the abundance of the other nsp11
to nsp16 proteins relative to that of the nsp1 to nsp10 proteins.
It is not clear from our analysis whether the loss in infectivity
is directly the result of the altered RNA synthesis, protein
levels, or a subsequent defect in viral particle production. Fur-
ther study is needed to investigate these possibilities.
Interestingly, the slippery-site mutants had lesser effects on
the accumulation of sgRNA (Fig. 2). Viral RNA synthesis
occurs in the cytoplasm and requires only the ORF1a- and
ORF1ab-encoded proteins (1, 55). The RNA-dependent RNA
polymerase (RDRP) is responsible for the synthesis of both
gRNAs and sgRNAs, and the ratio of these two species nor-
mally remains constant throughout the infectious cycle (50).
The differences in this ratio observed between the wild-type
virus and frameshift mutants demonstrate that alterations in
the ?1 PRF signal impact the utilization of the template RNA
for the synthesis of these two RNA species. It has been shown
that additional nsp12 and nucleocapsid proteins each enhance
replication (41), as well as the presence of additional nsp3, a
papainlike cysteine protease (50), suggesting that the process-
ing of the polyprotein(s) is important for replication. However,
those studies did not distinguish between the production of
gRNA and sgRNA species. In the current study, reduced ?1
PRF is expected to result in less nsp11 to nsp16 relative to the
amount of nsp1 to nsp10 produced during infection. We ob-
served a change in the ratio of gRNA and sgRNA when RNA
from infected cells were analyzed by quantitative PCR. The
link between these two points is, at present, unclear but likely
is due to the inherent function of the nsp proteins. It is for-
mally possible, however, that the defect in gRNA synthesis
observed in the current study is due to changes in the RNA
slippery-site sequence itself.
Coronavirus frameshift-promoting mRNA pseudoknot struc-
tures have evolved to fine-tune ?1 PRF toward producing a
golden mean of ORF1a- and ORF1b-encoded peptides. While
the structural analyses presented here show that both the
SARS-CoV and MHV mRNA pseudoknots are three-
stemmed structures, their primary sequences and basic struc-
tural elements are significantly different from one another.
Specifically, although their stem 1 elements are highly stable,
the long loop 1, short stem 2, and long loop 3 of MHV are less
compact than those of their analogous SARS-CoV elements.
Conversely, reverse relationships apply to the stem 3 and loop
2 elements. Stabilizing/compacting any of these elements pro-
moted increased frameshifting efficiencies, which is consistent
with the notion that highly stable mRNA secondary structures
cause elongating ribosomes to pause longer over the slippery
site. The critical finding here is that, despite having signifi-
cantly different structures, both the MHV and SARS-CoV
pseudoknots promoted equivalent rates of ?1 PRF. Our re-
sults show that although a class of mRNA pseudoknots that
promote very high levels of frameshifting can exist, the fact
that all naturally occurring coronavirus frameshift signals as-
sayed to date promote ?1 PRF efficiencies in the 10 to 20%
range (46) provides indirect support for the notion that high
levels of frameshifting also are incompatible with coronavirus
replication. This is consistent with the golden mean model of
frameshifting, i.e., it appears that each virus has evolved the
right balance between more and less stable structural elements
to produce the optimum rates of ?1 PRF.
Coronaviruses also use translational attenuation to obtain
the correct ratios of upstream and downstream viral polypep-
tides. The description of a sequence with a novel function, the
attenuation of ?1 PRF by a novel cis-acting element (52),
warranted further inspection. Prior to that report, no other
cis-acting elements affecting ?1 PRF other than the slippery
site and downstream stimulatory element had been described,
and no concrete model for how the attenuator functioned was
proposed. The presence of a large number of previously un-
available coronavirus genomes allowed an initial phylogenetic
analysis. The alignment of these sequences revealed a large
degree of diversity among the analogous regions in both ho-
mology and length; the smallest coronaviral genomes con-
tained the shortest analogous sequences, and the most diverse
genomes showed the least homology. This indicated that no
specific attenuator sequence is conserved among coronavi-
ruses, suggesting that the element does not actually directly
affect the process of frameshifting. However, the computa-
tional prediction of highly stable secondary structures engen-
dered the hypothesis that the attenuator element impedes the
processivity of elongating ribosomes on the viral mRNA, caus-
ing them to dissociate prior to encountering the ?1 PRF sig-
nal, i.e., a translational attenuation model. As supported by the
experiments in the current study, the addition of the attenuator
sequence upstream of the dual luciferase assay confirmed that
its presence resulted in an apparent decrease in ?1 PRF.
However, the critical experiment, comparing the readthrough
controls, revealed that while the ?1 PRF signal itself pro-
moted a small but significant decrease in the production of the
full-length product (presumably due to the presence of the
mRNA pseudoknot), the addition of the attenuator decreased
this value by nearly 40%. Further, in vitro translation assays
4338PLANT ET AL.J. VIROL.
at UNIV OF MARYLAND on April 6, 2010
demonstrated that the attenuator sequence promoted the in-
creased synthesis of prematurely terminated peptide products.
Again, although indirect, this provides indirect support of the
golden mean hypothesis, in that they show that coronaviruses
have evolved a second cis-acting element to limit the fraction
of ribosomes able to eventually translate ORF1b. In sum, the
data presented here support a model in which a combination of
translational attenuation and limited ?1 PRF efficiency serve
to fine-tune the fraction of elongating ribosomes able to trans-
late the ORF1b mRNA sequence, thus ensuring that the pp1a
and pp1b ratios are optimized for coronavirus propagation.
We thank members of the Dinman laboratory for comments and
advice and give special thanks to Amy Sims and Ralph Baric for
construction of the infectious clones and to Paul Masters for the MHV
This work was supported by a grant from the National Institutes of
Health to J.D.D. (AI064307).
1. Almaza ´n, F., M. L. DeDiego, C. Galan, D. Escors, E. Alvarez, J. Ortego, I.
Sola, S. Zuniga, S. Alonso, J. L. Moreno, A. Nogales, C. Capiscol, and L.
Enjuanes. 2006. Construction of a severe acute respiratory syndrome coro-
navirus infectious cDNA clone and a replicon to study coronavirus RNA
synthesis. J. Virol. 80:10900–10906.
2. Balasundaram, D., J. D. Dinman, R. B. Wickner, C. W. Tabor, and H. Tabor.
1994. Spermidine deficiency increases ?1 ribosomal frameshifting efficiency
and inhibits Ty1 retrotransposition in Saccharomyces cerevisiae. Proc. Natl.
Acad. Sci. USA 91:172–176.
3. Barry, J. K., and W. A. Miller. 2002. A ?1 ribosomal frameshift element that
requires base pairing across four kilobases suggests a mechanism of regulat-
ing ribosome and replicase traffic on a viral RNA. Proc. Natl. Acad. Sci. USA
4. Biswas, P., X. Jiang, A. L. Pacchia, J. P. Dougherty, and S. W. Peltz. 2004.
The human immunodeficiency virus type 1 ribosomal frameshifting site is an
invariant sequence determinant and an important target for antiviral ther-
apy. J. Virol. 78:2082–2087.
5. Brian, D. A., and R. S. Baric. 2005. Coronavirus genome structure and
replication. Curr. Top. Microbiol. Immunol. 287:1–30.
6. Brierley, I. 1995. Ribosomal frameshifting viral RNAs. J. Gen. Virol. 76(Pt
7. Brierley, I., P. Digard, and S. C. Inglis. 1989. Characterization of an efficient
coronavirus ribosomal frameshifting signal: requirement for an RNA
pseudoknot. Cell 57:537–547.
8. Brierley, I., A. J. Jenner, and S. C. Inglis. 1992. Mutational analysis of the
“slippery-sequence” component of a coronavirus ribosomal frameshifting
signal. J. Mol. Biol. 227:463–479.
9. Brierley, I., N. J. Rolley, A. J. Jenner, and S. C. Inglis. 1991. Mutational
analysis of the RNA pseudoknot component of a coronavirus ribosomal
frameshifting signal. J. Mol. Biol. 220:889–902.
10. Bruenn, J. A. 1980. Virus-like particles of yeast. Annu. Rev. Microbiol.
11. Casto ´n, J. R., B. L. Trus, F. P. Booy, R. B. Wickner, J. S. Wall, and A. C.
Steven. 1997. Structure of L-A virus: a specialized compartment for the
transcription and replication of double-stranded RNA. J. Cell Biol. 138:975–
12. Cheng, R. H., J. R. Caston, G. J. Wang, F. Gu, T. J. Smith, T. S. Baker, R. F.
Bozarth, B. L. Trus, N. Cheng, R. B. Wickner, et al. 1994. Fungal virus
capsids, cytoplasmic compartments for the replication of double-stranded
RNA, formed as icosahedral shells of asymmetric Gag dimers. J. Mol. Biol.
13. Cui, Y., J. D. Dinman, T. G. Kinzy, and S. W. Peltz. 1998. The Mof2/Sui1
protein is a general monitor of translational accuracy. Mol. Cell. Biol. 18:
14. Cui, Y., J. D. Dinman, and S. W. Peltz. 1996. Mof4-1 is an allele of the
UPF1/IFS2 gene which affects both mRNA turnover and ?1 ribosomal
frameshifting efficiency. EMBO J. 15:5726–5736.
15. Darnell, M. E., E. P. Plant, H. Watanabe, R. Byrum, M. St Claire, J. M.
Ward, and D. R. Taylor. 2007. Severe acute respiratory syndrome coronavi-
rus infection in vaccinated ferrets. J. Infect. Dis. 196:1329–1338.
16. Dinman, J. D., T. Icho, and R. B. Wickner. 1991. A ?1 ribosomal frameshift
in a double-stranded RNA virus of yeast forms a gag-pol fusion protein.
Proc. Natl. Acad. Sci. USA 88:174–178.
17. Dinman, J. D., and T. G. Kinzy. 1997. Translational misreading: mutations in
translation elongation factor 1alpha differentially affect programmed ribo-
somal frameshifting and drug sensitivity. RNA 3:870–881.
18. Dinman, J. D., M. J. Ruiz-Echevarria, and S. W. Peltz. 1998. Translating old
drugs into new treatments: ribosomal frameshifting as a target for antiviral
agents. Trends Biotechnol. 16:190–196.
19. Dinman, J. D., and R. B. Wickner. 1992. Ribosomal frameshifting efficiency
and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus
propagation. J. Virol. 66:3669–3676.
20. Dinman, J. D., and R. B. Wickner. 1995. 5S rRNA is involved in fidelity of
translational reading frame. Genetics 141:95–105.
21. Escutenaire, S., N. Mohamed, M. Isaksson, P. Thoren, B. Klingeborn, S.
Belak, M. Berg, and J. Blomberg. 2007. SYBR green real-time reverse
transcription-polymerase chain reaction assay for the generic detection of
coronaviruses. Arch. Virol. 152:41–58.
22. Esteban, R., and R. B. Wickner. 1986. Three different M1 RNA-containing
viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-
stranded RNA synthesis. Mol. Cell. Biol. 6:1552–1561.
23. Farabaugh, P. J. 1996. Programmed translational frameshifting. Microbiol.
24. Felsenstein, K. M., and S. P. Goff. 1988. Expression of the gag-pol fusion
protein of Moloney murine leukemia virus without gag protein does not
induce virion formation or proteolytic processing. J. Virol. 62:2179–2182.
25. Giedroc, D. P., and P. V. Cornish. 2009. Frameshifting RNA pseudoknots:
structure and mechanism. Virus Res. 139:193–208.
26. Grentzmann, G., J. A. Ingram, P. J. Kelly, R. F. Gesteland, and J. F. Atkins.
1998. A dual-luciferase reporter system for studying recoding signals. RNA
27. Harger, J. W., A. Meskauskas, J. Nielsen, M. C. Justice, and J. D. Dinman.
2001. Ty1 retrotransposition and programmed ?1 ribosomal frameshifting
require the integrity of the protein synthetic translocation step. Virology
28. Hudak, K. A., A. B. Hammell, J. Yasenchak, N. E. Tumer, and J. D. Dinman.
2001. A C-terminal deletion mutant of pokeweed antiviral protein inhibits
programmed ?1 ribosomal frameshifting and Ty1 retrotransposition without
depurinating the sarcin/ricin loop of rRNA. Virology 279:292–301.
29. Hung, M., P. Patel, S. Davis, and S. R. Green. 1998. Importance of ribosomal
frameshifting for human immunodeficiency virus type 1 particle assembly
and replication. J. Virol. 72:4819–4824.
30. Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation
of Escherichia coli with plasmids. Gene 96:23–28.
31. Jacobs, J. L., and J. D. Dinman. 2004. Systematic analysis of bicistronic
reporter assay data. Nucleic Acids Res. 32:e160.
32. Karacostas, V., E. J. Wolffe, K. Nagashima, M. A. Gonda, and B. Moss. 1993.
Overexpression of the HIV-1 gag-pol polyprotein results in intracellular
activation of HIV-1 protease and inhibition of assembly and budding of
virus-like particles. Virology 193:661–671.
33. Kawakami, K., S. Pande, B. Faiola, D. P. Moore, J. D. Boeke, P. J.
Farabaugh, J. N. Strathern, Y. Nakamura, and D. J. Garfinkel. 1993. A rare
tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is es-
sential for Ty1 retrotransposition in Saccharomyces cerevisiae. Genetics
34. Kontos, H., S. Napthine, and I. Brierley. 2001. Ribosomal pausing at a
frameshifter RNA pseudoknot is sensitive to reading phase but shows little
correlation with frameshift efficiency. Mol. Cell. Biol. 21:8657–8670.
35. Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan,
H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thomp-
son, T. J. Gibson, and D. G. Higgins. 2007. Clustal W and Clustal X version
2.0. Bioinformatics 23:2947–2948.
36. Mathews, D. H., J. Sabina, M. Zuker, and D. H. Turner. 1999. Expanded
sequence dependence of thermodynamic parameters improves prediction of
RNA secondary structure. J. Mol. Biol. 288:911–940.
37. Melian, E. B., E. Hinzman, T. Nagasaki, A. E. Firth, N. M. Wills, A. S.
Nouwens, B. J. Blitvich, J. Leung, A. Funk, J. F. Atkins, R. Hall, and A. A.
Khromykh. 2009. NS1? of flaviviruses in the Japanese encephalitis serogroup
is a product of ribosomal frameshifting and plays a role in viral neuro-
invasiveness. J. Virol. 84:1641–1647.
38. Meskauskas, A., J. L. Baxter, E. A. Carr, J. Yasenchak, J. E. Gallagher, S. J.
Baserga, and J. D. Dinman. 2003. Delayed rRNA processing results in
significant ribosome biogenesis and functional defects. Mol. Cell. Biol. 23:
39. Meskauskas, A., and J. D. Dinman. 2001. Ribosomal protein L5 helps
anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae. RNA
40. Meskauskas, A., J. W. Harger, K. L. Jacobs, and J. D. Dinman. 2003.
Decreased peptidyltransferase activity correlates with increased pro-
grammed ?1 ribosomal frameshifting and viral maintenance defects in the
yeast Saccharomyces cerevisiae. RNA 9:982–992.
41. Pan, J., X. Peng, Y. Gao, Z. Li, X. Lu, Y. Chen, M. Ishaq, D. Liu, M. L.
DeDiego, L. Enjuanes, and D. Guo. 2008. Genome-wide analysis of protein-
protein interactions and involvement of viral proteins in SARS-CoV repli-
cation. PLoS One 3:e3299.
42. Park, J., and C. D. Morrow. 1991. Overexpression of the gag-pol precursor
VOL. 84, 2010 SHIFTING TOWARD A GOLDEN MEAN4339
at UNIV OF MARYLAND on April 6, 2010
from human immunodeficiency virus type 1 proviral genomes results in
efficient proteolytic processing in the absence of virion production. J. Virol.
43. Paul, C. P., J. K. Barry, S. P. Dinesh-Kumar, V. Brault, and W. A. Miller.
2001. A sequence required for ?1 ribosomal frameshifting located four
kilobases downstream of the frameshift site. J. Mol. Biol. 310:987–999.
44. Peltz, S. W., A. B. Hammell, Y. Cui, J. Yasenchak, L. Puljanowski, and J. D.
Dinman. 1999. Ribosomal protein L3 mutants alter translational fidelity and
promote rapid loss of the yeast killer virus. Mol. Cell. Biol. 19:384–391.
45. Plant, E. P., and J. D. Dinman. 2006. Comparative study of the effects of
heptameric slippery site composition on ?1 frameshifting among different
eukaryotic systems. RNA 12:666–673.
46. Plant, E. P., and J. D. Dinman. 2008. The role of programmed-1 ribosomal
frameshifting in coronavirus propagation. Front. Biosci. 13:4873–4881.
47. Plant, E. P., G. C. Perez-Alvarado, J. L. Jacobs, B. Mukhopadhyay, M.
Hennig, and J. D. Dinman. 2005. A three-stemmed mRNA pseudoknot in
the SARS coronavirus frameshift signal. PLoS Biol. 3:e172.
48. Rivas, E., and S. R. Eddy. 2000. The language of RNA: a formal grammar
that includes pseudoknots. Bioinformatics 16:334–340.
49. Ruiz-Echevarría, M. J., J. M. Yasenchak, X. Han, J. D. Dinman, and S. W.
Peltz. 1998. The upf3 protein is a component of the surveillance complex that
monitors both translation and mRNA turnover and affects viral propagation.
Proc. Natl. Acad. Sci. USA 95:8721–8726.
50. Sawicki, S. G., and D. L. Sawicki. 2005. Coronavirus transcription: a per-
spective. Curr. Top. Microbiol. Immunol. 287:31–55.
51. Somogyi, P., A. J. Jenner, I. Brierley, and S. C. Inglis. 1993. Ribosomal
pausing during translation of an RNA pseudoknot. Mol. Cell. Biol. 13:6931–
52. Su, M. C., C. T. Chang, C. H. Chu, C. H. Tsai, and K. Y. Chang. 2005. An
atypical RNA pseudoknot stimulator and an upstream attenuation signal for
?1 ribosomal frameshifting of SARS coronavirus. Nucleic Acids Res. 33:
53. Telenti, A., R. Martinez, M. Munoz, G. Bleiber, G. Greub, D. Sanglard, and
S. Peters. 2002. Analysis of natural variants of the human immunodeficiency
virus type 1 gag-pol frameshift stem-loop structure. J. Virol. 76:7868–7873.
54. Templeton, S. P., and S. Perlman. 2007. Pathogenesis of acute and chronic
central nervous system infection with variants of mouse hepatitis virus, strain
JHM. Immunol. Res. 39:160–172.
55. Thiel, V., J. Herold, B. Schelle, and S. G. Siddell. 2001. Viral replicase gene
products suffice for coronavirus discontinuous transcription. J. Virol. 75:
56. Thiel, V., K. A. Ivanov, A. Putics, T. Hertzig, B. Schelle, S. Bayer, B. Weiss-
brich, E. J. Snijder, H. Rabenau, H. W. Doerr, A. E. Gorbalenya, and J.
Ziebuhr. 2003. Mechanisms and enzymes involved in SARS coronavirus
genome expression. J. Gen. Virol. 84:2305–2315.
57. Wilkinson, K. A., E. J. Merino, and K. M. Weeks. 2006. Selective 2?-hydroxyl
acylation analyzed by primer extension (SHAPE): quantitative RNA struc-
ture analysis at single nucleotide resolution. Nat. Protoc. 1:1610–1616.
58. Yount, B., K. M. Curtis, E. A. Fritz, L. E. Hensley, P. B. Jahrling, E. Prentice,
M. R. Denison, T. W. Geisbert, and R. S. Baric. 2003. Reverse genetics with
a full-length infectious cDNA of severe acute respiratory syndrome corona-
virus. Proc. Natl. Acad. Sci. USA 100:12995–13000.
59. Ziebuhr, J. 2005. The coronavirus replicase. Curr. Top. Microbiol. Immunol.
60. Zuker, M. 2003. mfold web server for nucleic acid folding and hybridization
prediction. Nucleic Acids Res. 31:3406–3415.
4340 PLANT ET AL.J. VIROL.
at UNIV OF MARYLAND on April 6, 2010