Nidoviruses
Edited by S. Perlman, T. Gallagher, and E. J. Snijder
© 2008 ASM Press, Washington, DC
Chapter 3
Nidovirus Genome Organization and Expression Mechanisms
Paul Britton and Dave Cavanagh
The order Nidovirales comprises three families of
viruses with linear single-stranded RNA genomes of
positive polarity: the Coronaviridae, comprising the
two genera Coronavirus and Torovirus, together with
Arteriviridae and Roniviridae. The nidovirus mem-
bers have some common features with respect to
genome organization, replication, transcription, and
translation of gene products. The name Nidovirales,
derived from the Latin nidus for nest, reflects the nested-
set arrangement of the subgenomic mRNAs (sg mRNAs)
produced by this order of viruses during their replica-
tion cycle. The coronaviruses, toroviruses, and roni-
viruses possess the longest known RNA genomes,
ranging from 26 to 32 kb (7, 9, 27, 28, 49, 77, 79),
and are related by some common features to the arteri-
viruses, which have smaller genomes, ranging from 13
to 16 kb (37, 49) (Fig. 1). The coronaviruses are pres-
ently divided into three groups. The groupings were
based initially on cross-reactivity in neutralization and
immunofluorescence assays but more recently on phy-
logenetic relationships and genome organization. The
features of the various coronavirus subgroups are
summarized in greater detail in chapters 2, 12, and 15.
Nidoviruses replicate in the cytoplasm of an infected
cell, although one of the structural proteins, the
nucleocapsid (N) protein, for some viruses has been
found to be located within the nucleolar region of the
nucleus in virus infected cells (25, 56, 111, 113, 142,
153, 156). For general reviews, please see references
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42, 43, 77, 90, 125, 126, and 137.
This chapter covers the genome organization
and expression mechanisms of the nidoviruses as out-
lined in the five parts numbered in Fig. 2. Following
infection of a susceptible cell by a nidovirus and
uncoating of the RNA genome, the first step in a suc-
cessful replication cycle is the production of the repli-
case proteins. The nidovirus genomic RNA (gRNA)
initially acts as a eukaryotic mRNA for the translation
of the replicase proteins. Following synthesis of the
Paul Britton and Dave Cavanagh • Division of Microbiology, Institute for Animal Health, Compton, Newbury, United Kingdom.
29
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replicase proteins, the positive-sense gRNA is copied
into negative-sense counterparts which act as tem-
plates for the synthesis of new gRNAs. In addition to
a negative-sense gRNA, nidoviruses produce a series
of negative-sense counterparts of the sg mRNAs. The
synthesis of both full-length and subgenome-length
negative-sense RNAs is initiated at the 3
end of the
gRNA. Synthesis of negative-sense RNAs may termi-
nate at different points along the gRNA template,
yielding subgenome-length minus-strand RNAs.
Attenuation of minus-strand RNA synthesis occurs
at sequences, known as transcription regulatory
sequences (TRSs), which are well conserved in a virus
type but differ between groups, genera, and families
of viruses. Failure to terminate at any of the TRSs
results in the synthesis of a full-length negative-sense
RNA (or “antigenome”). The negative-sense sgRNAs
of coronaviruses and arteriviruses acquire a leader
sequence of 65 to 100 nucleotides for coronaviruses
but 150 to 210 nucleotides for arteriviruses, copied
from the 5
end of the gRNA, as part of the process
of discontinuous minus-strand RNA synthesis. The
negative-sense sgRNAs act as templates for the syn-
thesis of the positive-sense sgRNAs, which are usu-
ally generated in a large excess compared to their
negative-sense counterparts. The mechanism for the
synthesis of nidovirus sgRNAs is called discontinu-
ous extension of minus-strand RNA (115–118;
reviewed in chapter 8 and reference 119).
Toroviruses have two transcription strategies
involved in their production of the sg mRNAs. Only the
largest sg mRNA, RNA2, contains a leader sequence
that is derived from the 5
end of the gRNA and is
thought to be added by a mechanism involving discon-
tinuous RNA synthesis, according to the current
model, analogous to the method used for sgRNA syn-
thesis for corona- and arteriviruses (148). The other
three torovirus sg mRNAs are colinear with the 3
end
of the genome and do not contain a 5
leader sequence
 30 BRITTON AND CAVANAGH

Figure 1. Comparison of the genomic organizations of selected nidoviruses. A representative virus from each coronavirus
group is included. The genomes are drawn to scale to emphasize that the first two-thirds of most nidovirus genomes consist of
the replicase genes, ORF1a and ORF1b. The 1 frameshift site (RFS) for each virus is indicated. The drawings are derived
from complete genome sequences of the representative viruses: porcine TGEV, MHV, SARS-CoV, avian IBV, bovine torovirus
(BToV), EAV, and GAV. All the nonreplicase nonstructural protein genes are indicated with open boxes. The structural genes
are those encoding S, M, E, N, HE, and GP proteins; I is an internal ORF identified in the N protein genes of some group 2
coronaviruses. S is shown as a double-shaded box, with the GP116/GP64 region of GAV having some structural similarities to
an S gene. E is indicated as a dark gray box, M as a black box, and N as light gray box. The 5
end of the GAV GP116/GP64
is colored black, as this part of the gene product is predicted to have triple membrane-spanning motifs that are reminiscent of
the M protein. The arterivirus GP5 gene has a function equivalent to that of the S gene in coronaviruses and toroviruses.

derived from the gRNA (128). Analysis of RNA iso- and 12,700 to 15,700 nucleotides for arteriviruses.
lated from the lymphoid organ of prawns (Penaeus The genomes are capped at the 5
end and polyade-
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monodon) infected with the ronivirus gill-associated
virus (GAV), identified two sg mRNAs, neither of
which contained a leader sequence (29). The nidovirus
sg mRNAs, with or without a leader sequence, are
used for the expression of all viral genes with the
exception of the replicase gene products.
GENOME ORGANIZATION
All nidoviruses have a positive-sense single-
stranded RNA genome of 26,200 to 31,500 nucleo-
tides for roniviruses, toroviruses, and coronaviruses
nylated at the 3
end (49, 125). In addition, the
genomes contain untranslated regions (UTRs) at their
5
and 3
termini that play a role in both genome
replication and synthesis of sgRNAs.
The overall genomic organization of nidoviruses
is 5
-UTR-replicase open reading frame 1a (ORF1a)-
replicase ORF1b-nonreplicase genes-3
-UTR-poly(A).
However, as illustrated in Fig. 1, apart from the sizes
of the various regions, the major differences between
the nidovirus genomes involve the type and organi-
zation of the nonreplicase genes, with greater dif-
ferences occurring between the different families
of viruses. For example, the coronaviruses and

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 CHAPTER 3 • GENOME ORGANIZATION AND EXPRESSION MECHANISMS 31

Figure 2. Schematic diagram representing the replication cycle of a nidovirus following infection of a susceptible cell. The dia-
gram has five numbered regions that represent the topics discussed in this chapter: (1) gRNA, released from a virus particle
that has infected the cell, to highlight that it initially acts as an mRNA for the translation of the replicase proteins; (2) pro-
grammed 1 frameshifting event, common to all nidoviruses, for the translation of the replicase polyproteins, pp1a and
pp1ab, in differing amounts; (3) another common feature of nidoviruses, proteolytic cleavage of the replicase polyproteins by
virus-encoded proteinases; (4) another feature of nidoviruses, the generation of sg mRNAs (mainly polycistronic but function-
ally monocistronic) for the expression of the other virus-derived proteins; (5) expression strategies used for the translation of
the sg mRNAs into virus proteins. Only the sg mRNAs encoding the structural proteins are shown. The rest of the diagram
represents the interaction of the virus proteins for assembly and release of virus particles. ERGIC, endoplasmic reticulum-
Golgi intermediate compartment.

toroviruses have the general genome organization 5
-
UTR-ORF1a-ORF1b-S-E-M-N-3
-UTR-poly(A) (S is
spike protein, E is envelope protein, and M is integral
membrane protein); it should be noted that no
sequence for a protein equivalent to the coronavirus E
protein has been identified in a torovirus genome. As
can be seen from Fig. 1, the organization and type of
genes in the 3
-proximal region of arterivirus and
ronivirus genomes are very different.
Nidovirus genomes can be divided into two
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the features that distinguish this family of viruses
from other positive-sense RNA viruses. The two rep-
licase polyproteins, pp1a and pp1ab, are cleaved dur-
ing de novo synthesis by two types of virus-encoded
proteinases. There are common motifs within the
replicase gene that are shared between the different
members of the nidoviruses, characteristic of protein-
ase, polymerase, helicase, and endoribonuclease
activities that are located at specific regions of the
replicase gene. The ORF1b region of the replicase

regions, which vary within and between the different
virus families within the order Nidovirales. The first
region, gene 1, represents approximately two-thirds
of the genome (20 kb for coronaviruses, toroviruses,
and roniviruses) and encodes the replicase/transcrip-
tase functions of the viruses. Two large polyproteins
are expressed from gene 1, polyprotein 1a (pp1a) and
pp1ab; pp1ab is expressed in smaller amounts as a
C-terminal extension of pp1a following a 1 frame-
shift event (11–13), as discussed later in this chapter.
The second region of a nidovirus genome encodes the
structural and accessory proteins. The structure and
function of the nidovirus replicase genes are two of
encodes two proteins or domains that have not been
identified in other RNA virus families, a zinc-binding
domain (ZBD) (27, 50, 124, 130, 146) and a uridylate-
specific endoribonuclease (NendoU) (6, 60, 104,
129), which can be considered genetic markers for
nidoviruses (49).
5
and 3
UTRs
The 5
UTR is between 200 and 800 nucleotides
long for coronaviruses and toroviruses, 150 and 220
nucleotides for arteriviruses, and 34 nucleotides for
the ronivirus GAV. The very 5
end of the coronavirus

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 32 BRITTON AND CAVANAGH
and arterivirus UTR is the so-called leader sequence
that is attached to all coronavirus and arterivirus
sg mRNAs. The sg mRNAs from roniviruses do not con-
tain a leader sequence, possibly reflected in the fact
that roniviruses have very short 5
UTRs. The 3
UTR
is between 200 and 500 nucleotides long for coro-
viruses and toroviruses, 60 and 150 nucleotides for
arteriviruses, and 129 nucleotides for the ronivirus
GAV. The nidovirus 3
UTR occurs after the last func-
tional gene, mainly the N protein gene, except for
some group 1 coronaviruses, i.e., transmissible gas-
troenteritis virus (TGEV), canine coronavirus, and
feline coronavirus, which have one or two genes fol-
lowing the N protein gene, and the roniviruses that
have a predicted ORF (ORF4) as the last ORF pre-
ceding the poly(A) tract (Fig. 1). In addition, some
coronaviruses that may be similar to infectious bron-
chitis virus (IBV) have recently been isolated from a
variety of avian species (geese, mallards, and pigeons)
that also have one or two ORFs following the N pro-
tein gene (17, 65). The IBV 3
UTR can be divided
into two regions; the first is immediately downstream
of the N protein gene, is hypervariable among IBV
strains, and is deleted from some strains (e.g., M41),
whereas the second region is highly conserved (17).
A number of RNA secondary structures have
been either predicted or identified within the 5
and
3
UTRs of various nidoviruses and play a role in
both genome replication and synthesis of sgRNAs.
Coronavirus replication signals within the 5
and 3

ends of the genomes were initially investigated using
defective RNA (D-RNA) genomes; the minimal
lengths of these regions necessary for replication of
D-RNAs in the presence of helper virus are summa-
rized in Table 1 and include both the UTRs and 5

and 3
regions of the genomes containing parts of the
replicase and N protein genes, respectively. Three
stem-loop structures were predicted in the first 100
nucleotides of the 5
UTR of the group 3 corona-
virus IBV, of which the 5
-proximal stem-loop (I)
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Table 1. Minimal lengths of nidovirus terminal 5
and 3
regions involved in replication
Family Group Virus
Coronaviridae Coronavirus
1 TGEV
2 MHV
3 IBV
Torovirus EToV
Arteriviridae Arterivirus EAV
Roniviridae Okavirus GAV
a
ND, not determined.
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showed a high degree of covariance among different
IBV strains, providing phylogenetic support for this
structure (136). Similar stem-loop structures were
predicted in the first 106 nucleotides of the 5
UTR of
the group 2 coronavirus mouse hepatitis virus (MHV),
although analysis of a further 120 nucleotides identi-
fied two more stem-loop structures (151). Analysis of
the first 210 nucleotides of the 5
UTR of another
group 2 coronavirus, bovine coronavirus (BCoV),
predicted four stem-loop structures, of which stem-
loops III and IV showed phylogenetic conservation
and whose structures were supported by enzymatic
analysis of a BCoV D-RNA (109, 110). Analysis of
the 5
UTR of the arterivirus equine arteritis virus
(EAV) has also led to the prediction of secondary
RNA structures, most of which were supported by
phylogenetic conservation and by enzymatic and
chemical analyses (145).
The nidovirus 3
UTRs have also been predicted
to contain secondary and even tertiary RNA struc-
tures that have been implicated in RNA synthesis
during the nidovirus replication cycle. A 68-nucleo-
tide stem-loop was identified immediately down-
stream of the MHV N protein gene (59). A similar
structure has been found in the 3
UTR of the group
3 coronaviruses, IBV (Fig. 3) and turkey coronavirus
(TCoV). There is a high degree of covariance among
different IBV strains, providing phylogenetic support
for this structure (31). Interestingly, in most IBV
strains the stem-loop structure is found not adjacent
to the end of the N protein gene but at the junction of
the hypervariable and conserved regions of the IBV 3

UTR. However, in IBV strains lacking the hypervari-
able region, the stem-loop structure is adjacent to the
end of the N protein gene. It is possible that
the hypervariable region of the IBV 3
UTR may be
the remnants of ORFs that have been lost during evo-
lution of the virus. In addition to the 3
UTR stem-
loop structures, an RNA pseudoknot structure was
identified downstream of the stem-loop structure,
Minimum length (nucleotides)
5
end 3
end
649 492
466 436
544 338
607 242
296 354
a
ND ND
 CHAPTER 3 • GENOME ORGANIZATION AND EXPRESSION MECHANISMS
Figure 3. RNA structural elements in the 3
UTR of the avian coro-
navirus IBV. The IBV 3
UTR contains three predicted RNA struc-
tures, a stem-loop associated with a pseudoknot structure and a
second stem-loop structure, s2m. Similar stem-loop and associated
pseudoknot RNA structures have been identified in the 3
UTRs of
other coronaviruses. The s2m structure in the IBV 3
UTR also
occurs in the 3
UTRs of various astroviruses and a human rhino-
virus as well as in 3
UTR of TCoV, another group 3 coronavirus
related to IBV. Neither the s2m structure nor its composite
sequence was identified in the 3
UTR of any other non-group
3 coronavirus, until the isolation of SARS-CoV. The numbers rep-
resent the IBV genomic nucleotide positions.
initially in the 3
UTR of BCoV (152). The authors
postulated that the pseudoknot functions in the plus
strand as a regulatory control element in coronavirus
RNA replication. Similar structures were predicted to
be present in the 3
UTRs of other coronaviruses. An
example of a coronavirus 3
UTR stem-loop and
pseudoknot structure is shown in Fig. 3. Further
analysis of the MHV stem-loop/pseudoknot struc-
tures indicated that they may be involved in an RNA
switch governing a transition between different steps
of virus replication (47, 58). Analysis of the 3
UTR
of the severe acute respiratory syndrome coronavirus
(SARS-CoV) identified both a stem-loop and a pseu-
doknot structure that overlapped in a manner similar
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to that identified for MHV and BCoV, potentially
reflecting some shared common ancestry between
the viruses (47). Further work demonstrated that
the SARS-CoV 3
UTR could functionally replace the
MHV 3
UTR, whereas 3
UTRs from group 1 and 3
coronaviruses could not replace the MHV 3
UTR,
providing more evidence that SARS-CoV shares some
common ancestry with group 2 coronaviruses (48). A
second stem-loop structure, s2m (Fig. 3), was identi-
fied in the 3
UTR of all IBV strains to date, with
remarkable similarity in both sequence and structure
to stem-loop structures identified in the genomic 3

ends of astroviruses isolated from humans, pigs,
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33
sheep, and turkeys and of the picornavirus equine
rhinovirus serotype 2 (64). The sequence representing
the s2m stem-loop structure was also identified in the
3
UTR of the coronaviruses isolated from geese,
pigeons, and mallard ducks (65). Remarkably, an
almost identical sequence and predicted s2m struc-
ture was identified in the 3
UTR of SARS-CoV (89).
A 2.7-Å resolution three-dimensional crystal struc-
ture of the SARS-CoV s2m RNA element has been
determined (112). In addition to the RNA structures
identified in the 3
UTRs of coronaviruses, an octa-
nucleotide motif, 5
-GGAAGAGC-3
, of unknown
function has been identified 70 to 80 nucleotides
proximal to the poly(A) tract in all coronavirus
genomes examined to date.
Analysis of the nidovirus 3
UTR has been mainly
restricted to coronaviruses. However, a kissing-loop
interaction has been proposed for the arterivirus por-
cine reproductive and respiratory virus (PRRSV),
between the loops of stem-loop structures in the 3

UTR and N protein gene, to be crucial for minus-
strand RNA synthesis (149). A stem-loop structure in
the 3
UTR of the arterivirus simian hemorrhagic
fever virus (SHFV) was identified that contains bind-
ing sites for two cellular proteins (88). A stem-loop
structure that may act as a recognition sequence for
the initiation of minus-strand RNA synthesis has
been identified at the 3
-terminal end of the arterivi-
rus EAV 3
UTR (5). The identification of potential
RNA structures involved in RNA synthesis for toro-
virus or roniviruses has not been published.
Replicase Gene Region
The overall organization of the nidovirus repli-
pro
case gene motifs is TM1-TM2-3CL -TM3-RFS-
RdRp-ZBD-HEL-NendoU, in which TM represents
pro
hydrophobic transmembrane domains, 3CL is a
chymotrypsin-like proteinase (so called because it has
substrate specificities similar to those of picornavirus
3C proteinases), RFS is the ribosomal frameshift site
required for the expression of the ORF1b-encoded
proteins, RdRp is the RNA-dependent RNA poly-
merase, and HEL is a helicase (49). The three hydro-
phobic TMs have been postulated to be involved
in anchoring the replicase proteins to membrane
compartments, forming the so-called replication/
transcription complexes. In addition, other motifs,
representing other replicase gene functions but more
related to RNA processing, have been identified in
the replicase genes of coronaviruses, toroviruses, and
roniviruses, with one (NendoU) also occurring in
arteriviruses (129). Although the in vitro functions of
some of these gene products have been confirmed,
their roles in replication/transcription have yet to be
 34 BRITTON AND CAVANAGH
determined. The proteins include a 3
-to-5
exonucle-
ase, a 2
-O-ribose methyl transferase, and a cyclic
phosphodiesterase (CPD). The CPD motif (ns2;
Fig. 1) was initially identified in the group 2 corona-
virus MHV, not as a part of the replicase gene but as
ORF2a in sg mRNA2. The CPD motif is present within
the replicase gene of toroviruses. A domain corre-
sponding to an ADP-ribose 1-phosphatase (ADRP)
has been identified in coronaviruses (nsp3) (129) and
toroviruses (38). The activity of bacterially expressed
ADRP domains from SARS-CoV (114), human coro-
navirus 229E (HCoV-229E [106]), and TGEV (107)
has been verified in vitro, and the structure of the
SARS-CoV ADRP has been solved (114). The func-
tion of the nidovirus replicase proteins is discussed in
more detail in chapters 5 and 6.
The replicase polyproteins, pp1a and pp1ab, are
proteolytically processed during translation by two
types of virus-encoded proteinases, a papain-like pro-
pro
teinase (PL ) and a chymotrypsin-like viral main
pro
proteinase, 3CL , into 16 nonstructural proteins
(nsp1 to nsp16) for most coronaviruses, except the
avian coronavirus IBV, which lacks nsp1 and encodes
15 (nsp2 to nsp16) replicase gene-derived proteins.
pro
Most coronavirus genomes contain two PL
domains; however, IBV and SARS-CoV only encode
pro
one copy of PL . The arterivirus replicase polypro-
teins are cleaved by similar enzymes and may encode
pro
up to three PL domains, resulting in 13 or 14 prod-
ucts (nsp1 to nsp12), including the products of a
recently described internal cleavage of nsp7 (144).
Expression of the torovirus replicase gene has been
postulated to result in 12 proteins (128). The ronivi-
rus replicase gene appears to encode only one pro-
pro
teinase, equivalent to 3CL , and potentially gives
rise to at least four proteins (27, 49).
Structural and Accessory Protein Gene Regions
In contrast to the first (replicase) region of the
genome, the structural and accessory protein gene
products are expressed from a series of sg mRNAs. The
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sg mRNAs are produced as a “nested” set of RNAs
that have different 5
end regions but common 3

extensions, often referred to as a 3
-coterminal nested
set of sg mRNAs; synthesis of nidovirus sgRNAs is
discussed in chapter 8. The sg mRNAs encode the
virion structural proteins and, though only for coro-
naviruses, a set of small nonstructural proteins, of
unknown function, that are referred to as accessory
proteins. Synthesis of the coronavirus and arterivirus
sgRNAs is via a discontinuous process, as indicated
in the introduction. Negative-sense counterparts of
the sg mRNAs are synthesized from the full-length
gRNA as a result of being attenuated or terminated at
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conserved nucleotide sequences, the TRSs, upstream
of almost every virus gene. The consequence of the
polymerase terminating at TRSs is that the sgRNAs
can be produced in unequal amounts, in effect con-
trolling the amounts of the gene products. The fac-
tors and/or sequences that regulate whether or how
the polymerase terminates at a TRS have been an
area of considerable interest. Sequences flanking the
TRSs have been postulated to play a role in control-
ling the levels of sgRNA, as the lengths of identical
nucleotides in flanking regions of a TRS and those at
the leader junction vary (55). The roles of the flank-
ing regions have been investigated for both coronavi-
ruses (2, 55, 133, 158) and arteriviruses (35, 98–100,
147). These are discussed in chapter 8.
All of the sg mRNAs, except the smallest RNA,
are structurally polycistronic, but they are usually
functionally monocistronic. The coronaviruses syn-
thesize between five (IBV) and eight (SARS-CoV)
sg mRNAs encoding four structural proteins, S, E, M,
and N proteins, and various numbers of nonstruc-
tural accessory proteins (Fig. 1). The overall gene
arrangement of the coronavirus structural proteins is
highly conserved, following the pattern hemaggluti-
nin-esterase (HE)-S-E-M-N (Fig. 1). Some group 2
coronaviruses, BCoV, HCoV-OC43, and MHV,
encode a fifth structural protein, HE (67). Interest-
ingly, it has been demonstrated that rearrangement of
this gene order is not deleterious to MHV replication
(34). Coronaviruses differ with respect to both the
number and location of the accessory protein genes
(Fig. 1; reviewed in reference 77) and, in some cases,
in the mode of translation of the proteins, which is
discussed later. The toroviruses synthesize four
sg mRNAs and have the gene order S-M-HE-N (Fig. 1),
where the HE gene is intact in bovine torovirus but a
pseudogene in equine torovirus (37). The arterivi-
ruses EAV, PRRSV, and lactate dehydrogenase-elevat-
ing virus (LDV) synthesize six sg mRNAs all encoding
structural proteins, all of which, apart from the N
protein, are M proteins (Fig. 1). The arteriviruses
express proteins equivalent to the coronavirus E, M,
and N proteins, with either four extra membrane gly-
coproteins for EAV/PRRSV/LDV and possibly up to
seven extra M proteins for SHFV, of which three may
have arisen by a putative triple gene duplication
event. Only two viruses, GAV and yellow head virus,
which infect prawns, have been assigned to the family
Roniviridae, and on the basis of sequence analysis
their replicase genes have been shown to be related to
those of coronaviruses, toroviruses, and arteriviruses.
The roniviruses produce two sg mRNAs; both the gene
order and the format of the structural proteins are
very different from those identified in the genomes of
coronaviruses, toroviruses, and arteriviruses. A gene
 CHAPTER 3 • GENOME ORGANIZATION AND EXPRESSION MECHANISMS
equivalent to the N protein gene of other nidovi-
ruses is found immediately downstream of ORF1b
(Fig. 1).
In addition to the structural protein genes, the
coronavirus genomes contain a variety of accessory
protein genes, which are interspersed among the
structural protein genes and which in some group 1
and 3 coronaviruses are found downstream of the N
protein gene (Fig. 1). A number of laboratories have
shown that the accessory protein genes are not essen-
tial for virus replication in vitro, in vivo, or ex vivo
(21, 30, 33, 51, 57, 97, 132).
RIBOSOMAL FRAMESHIFTING
One of the main characteristic features of nido-
virus genomes is the large replicase gene region, of
which approximately two-thirds of the genome
encodes the proteins involved in replication and syn-
thesis of sgRNAs. The replicase polyproteins, pp1a
and pp1ab, are produced from the same initiation
codon; pp1ab is a C-terminally extended form of
pp1a as a result of a programmed 1 ribosomal
frameshifting event occurring in the region where
ORF1a and ORF1b briefly overlap. As a result, the
ORF1b-derived replicase proteins are produced in
smaller amounts (approximately 60 to 80% less)
than the ORF1a-derived replicase proteins. The
ORF1b-encoded proteins include proteins with key
replicative functions, like RdRp and HEL activities,
indicating that the conserved frameshift mechanism
is a way of down-regulating the expression of these
enzymes, which must be an important control system
in the nidovirus life cycle. Programmed 1 ribosomal
frameshifting has been reported to occur during the
translation of other viral genes, including those in the
animal virus families Retroviridae (61), Astroviridae
(63), Totiviridae (150), and Flaviviridae (26, 154)
and various plant viruses (68, 105).
Completion of the first coronavirus genome
sequence, for IBV (7), raised the question of the
Copyright © 2007. ASM Press. All rights reserved.
mechanism for expression of ORF1b. Sequence anal-
ysis showed that ORF1a terminated at genome nucle-
otide 12382 and that ORF1b, which overlapped
ORF1a by 42 nucleotides, was in a 1 reading frame
with respect to ORF1a (Fig. 4). No sg mRNA equiva-
lent to a size required for the expression of ORF1b
had been detected in IBV-infected cells, and no IBV
TRS, CUUAACAA, had been identified upstream of
ORF1b. Nucleotide comparisons of the IBV
ORF1a/1b junction with the gag-pol overlap region
of Rous sarcoma virus, a region suspected to elicit a
frameshifting event (62), showed significant sequence
identity. As a result of the conundrum of how ORF1b
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35
is expressed and the above observations, ribosomal
frameshifting, as a mechanism for translating ORF1b
of the nidovirus replicase genes, was confirmed for
IBV (10–12).
The potential for ribosomal frameshifting within
the IBV replicase gene was shown by the insertion of
the IBV ORF1a/1b overlap region into a reporter
gene and testing of the construct for any frameshift-
ing ability in vitro using a transcription and transla-
tion system (11). Results indicated that the region
mediated a 1 frameshift event, with about 30% of
the ribosomes changing frame within the ORF1a/1b
overlap region. Subsequently, similar experiments
were used to demonstrate ORF1a/1b frameshifting
for other coronaviruses (8, 15, 41, 54, 140), a toro-
virus (equine torovirus) (130), an arterivirus (EAV)
(36), and a ronivirus (GAV) (27), with putative frame-
shift sites identified in other sequenced nidovirus
genomes.
Following the demonstration that frameshifting
could occur in IBV, potential signals involved in
directing the frameshifting events were investigated
(12, 14, 16, 134). Potential signals were located
within the ORF1a/1b junction (Fig. 4) and comprised
two cis-acting RNA elements, the first representing
the heptameric slippery sequence, UUUAAAC, con-
forming to the predicted slippery sequence motif
XXXYYYN, the site of the actual 1 frameshift
(Fig. 4). In vitro experiments showed that 30% of the
ribosomes that entered the sequence in the ORF1a
frame, U-UUA-AAC, left in the ORF1b frame repre-
senting the codons UUU-AAA-C (Fig. 5). Interest-
ingly, all the coronavirus, torovirus, and arterivirus
genomes sequenced to date have the same UUUAAAC
slippery sequence, apart from EAV, which has the
sequence GUUAAAC (Fig. 6). The two ronivirus rep-
licase sequences available, those for GAV and yellow
head virus, have AAAUUUU as their slippery sequence.
Experiments showed that the slippery sequence was
not in itself sufficient to result in the ribosomes’
changing frame. A second element required for frame-
shifting to occur was identified as an RNA pseu-
doknot (Fig. 4 and 7), an RNA structure composed of
two base-paired regions stacked coaxially and con-
nected by two single-stranded loop regions (19, 103).
No particular nucleotide sequences were identified as
playing an essential role, although the overall shape
and predicted stabilities of the structure had to be
maintained for efficient frameshifting. The primary
role of the RNA pseudoknot is believed to involve
slowing down or stalling of the ribosome as it trans-
lates through the slippery sequence (Fig. 7), to allow
realignment of the decoding tRNA on the mRNA in a
new frame (Fig. 5). The RNA pseudoknot has to be
positioned within 5 to 7 nucleotides downstream of
 36 BRITTON AND CAVANAGH

Figure 4. Sequence of the IBV ribosome frameshifting site. The top drawing represents the IBV gRNA, as shown in Fig. 1
and 3. The sequence representing the junction of ORF1a and ORF1b, corresponding to the RFS site, is expanded below the
gRNA drawing. The numbers represent the IBV genomic nucleotide positions. The positions of the heptameric UUUAAC slip
site sequences (in bold) are shown; the nucleotides forming the stem and loop structures of the pseudoknot (shown in Fig. 6)
are underlined. The amino acid sequences corresponding to ORF1a, -1b, and -1ab are shown below the IBV genomic nucleo-
tide sequence. ORF1a terminates at nucleotide 12382. There is no initiation codon for ORF1b, but a contiguous amino acid-
encoding sequence starts at nucleotide 12342 and terminates at nucleotide 20417. The 1 frameshift site, highlighted in bold,
takes place within the coding context of the slip site in which an asparagine residue, encoded by ORF1a, is replaced by a lysine
residue, encoded by ORF1b, due to the ribosome moving the RNA back one nucleotide (shown in Fig. 5). As a result of the
1 frameshift event, ORF1a is extended by the ORF1b coding sequence and terminates as an ORF1ab fusion protein at
nucleotide 20415. Translation termination codons are marked with asterisks.

the slip site (Fig. 7) for efficient frameshifting (14).
The actual mechanism of frameshifting is thought to
occur by the simultaneous slippage, into the 1
frame, of two ribosome-bound tRNAs within the
aminoacyl and peptidyl sites of the ribosome during
the translation of the slippery sequence (44, 52, 86). For
Copyright © 2007. ASM Press. All rights reserved.
the tRNA accommodation step of elongation by
filling the entrance of the ribosomal mRNA tunnel
(40, 102). The restriction on the movement of the
RNA can be eased in two ways: (i) unwinding or melt-
ing of the pseudoknot, which allows the RNA to move
forward, or (ii) movement or slippage of the RNA one

the coronavirus IBV the incorporation of the amino
acid lysine, derived from the ORF1b sequence, into
pp1a, instead of the amino acid asparagine (Fig. 5),
results in the generation of pp1ab.
Although the pseudoknot structures cause the
ribosome to pause, resulting in the ribosomal amino-
acyl and peptidyl sites being placed over the slippery
sequence (70), the pausing is insufficient for 1 frame-
shifting to occur (143); the duration of the pausing
does not necessarily correlate with the level of frame-
shifting observed (70, 101). Crystallographic studies,
in concert with molecular studies, indicate that the
pseudoknot restricts movement of the RNA during
nucleotide backwards, resulting in the 1 frameshift
(reviewed in reference 52). Cryoelectron microscopy
of mammalian 80S ribosomes paused at a coronavirus
pseudoknot revealed an intermediate of frameshifting
in which it was possible to determine how the pseu-
doknot interacts with the ribosome (96).
REGULATION AND EXPRESSION STRATEGIES
The nidovirus gRNAs, like the genomes of other
positive-strand RNA viruses, such as the Flaviviridae
7
and Togaviridae, contain an m GpppN cap structure

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 CHAPTER 3 • GENOME ORGANIZATION AND EXPRESSION MECHANISMS 37

Figure 5. Schematic diagram representing the IBV 1 frameshift event for the synthesis of pp1ab. The top part shows the
progression of a ribosome along the IBV gRNA over the UUUAAAC slip site. Shown are the positions of the aminoacyl-tRNAs
decoding the ORF1a codons, elongation of the polypeptide chain, and decoding of the next codon, resulting in the synthesis
of pp1a. The lower part represents a 1 frameshift event as proposed by the simultaneous-slippage model, in which the
ribosome-bound aminoacyl-tRNAs are proposed to slip simultaneously one nucleotide to a 1 frame position from their ini-
tial frame. Frameshifting can only occur when the anticodons of the two aminoacyl-tRNAs associated with the ribosome and
mRNA can still form two base pairs with the RNA in the shifted 1 frame, indicated by the first and second anticodon-codon
Asn
base pairings, but with disruption of base pairing at the third position. Following the 1 slippage event, aminoacyl-tRNA
Lys
dissociates from the ribosome complex, before decoding the mRNA, allowing the next aminoacyl-tRNA, aminoacyl-tRNA ,
to move into the aminoacyl site of the ribosome, in which there is full complementarity between the anticodon of the
tRNA and codon on the mRNA in the ORF1b frame. The mRNA is then decoded at this position, allowing elongation and
movement of the ribosome to the next codon. The IBV replicase gene is now decoded in the ORF1b frame, resulting in transla-
tion of pp1ab.

at the 5
end. This essentially makes the gRNAs
into mRNAs, imparting a dual role in which the
gRNAs can act as a template for the synthesis of
negative-sense RNAs (replication) and for the trans-
lation of the replicase polyproteins. As nidovirus
gRNAs and sg mRNAs contain a 5
cap structure,
ribosomes are able to initiate translation in a cap-
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coronaviruses HCoV-229E and MHV (141), although
the presence of N protein was later shown to increase
the efficiency of rescue and replication of HCoV-229E
(120) and TGEV (1) replicons. This indicates that the
N protein has some effect on coronavirus replication,
possibly by protecting the viral RNAs, or involve-
ment in translation of the viral RNAs; however, N

dependent manner. protein is dispensable for synthesis of arterivirus

There is evidence from the analysis of some coro-
naviruses that translation can be regulated by both
viral and host cell factors acting in trans or by cis-
acting sequences within the virus 5
UTR (122). An
interaction of the MHV N protein with the leader
sequence has been proposed to stimulate translation
of viral mRNAs (138, 139). Interestingly, the IBV N
protein was found to be essential for the rescue of
IBV from intracellular T7 polymerase-generated
infectious RNA in IBV reverse genetics systems (22,
157). N protein was not required for the rescue of
infectious viruses in reverse genetics systems for the
RNA (95).
Nidovirus RNAs are very similar to host cell
mRNAs and therefore dependent on the availability
and activities of host cellular factors, although, as
indicated above, various viral RNA elements or virus-
encoded products (N protein) may increase the effi-
ciency of translation. Cellular initiation factors bind
7
to the m GpppN cap structure at the 5
end of the
viral RNAs for recruitment of the 43S ribosomal
complex and formation of the 48S ribosomal com-
plex, which scans the mRNA for AUG initiation
codons. On encountering the AUG codon, GTP is

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 38 BRITTON AND CAVANAGH
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Figure 6. Comparison of the nidovirus replicase gene frameshifting sites. (A) Independent alignment of the gRNA sequences,
from the slip site, over the pseudoknot sequences of various coronavirus, torovirus, arterivirus, and ronivirus sequences.
Nucleotides common within the grouped sequences are highlighted in black. The RFS slip site is underlined. (B) Phylogenetic
groupings of all the aligned nidovirus sequences from panel A to demonstrate that as well as falling within their genus group-
ings, the coronavirus-derived sequences also fall within their groups, indicating that the viruses are related through their RFSs.
FIPV, feline infectious peritonitis virus; PEDV, porcine epidemic diarrhea virus; BToV, bovine torovirus; YHV, yellow head
virus.

hydrolyzed, initiation factors are released, and the
60S ribosomal subunit binds to the preinitiation com-
plex for formation of the 80S ribosome for transla-
tion and elongation of the virus product (reviewed in
reference 39). One initiation factor, eukaryotic initia-
tion factor 4E (eIF-4E), the cap-binding protein
involved in binding of the initiation complex, can be
phosphorylated, which increases its binding to the
7
m GpppN cap structure at the 5
end of eukaryotic
mRNAs and to eIF-G4, usually enhancing translation
rates (46). The phosphorylation of eIF-4E plays a
regulatory role in translation initiation, and viruses

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 CHAPTER 3 • GENOME ORGANIZATION AND EXPRESSION MECHANISMS 39

Figure 7. Schematic diagram indicating the pausing of ribosomes by a pseudoknot structure. The top part represents ribosomes
encountering a stem-loop structure on an mRNA being decoded. The ribosomes may be slowed down by such a structure, but
they are able to melt the structure and decode the mRNA. The lower part represents ribosomes encountering a pseudoknot
structure with an upstream slip site. Provided the slip site and pseudoknot are separated by no more than 5 to 7 nucleotides,
the pausing effect of the pseudoknot can cause a 1 frameshift as illustrated in Fig. 6. The predicted RNA structure of the IBV
pseudoknot is shown as an example to highlight the nucleotides forming the slip site and the stem and loop structures.

have utilized this process for controlling host protein
synthesis (reviewed in reference 93). Increased phos-
phorylation of eIF-4E has been observed in cells
infected with the coronaviruses MHV (4) and SARS-
CoV (92) as a result of the activation of p38 mitogen-
activated protein kinase.
Regulation and Expression of the Replicase Gene
A complexity for the translation of RNAs from
viruses with a positive-sense RNA genome is associ-
ated with the gRNA, which can act either as an mRNA
or as a template for replication. The signals for replica-
tion can be in either the 5
or 3
UTR or both. Interac-
tions between the 5
and 3
ends of the gRNA of some
positive-sense RNA viruses have been shown to con-
trol the switch between translation and replication
(39). As discussed above, the 5
and 3
UTRs of the
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are required for synthesis of negative-strand RNA
(82). Evidence for interactions between the 5
and 3

ends of nidovirus genomes has not been documented.
As indicated in the introduction, the 5
UTRs of the
gRNA are between 200 and 800 nucleotides long for
coronaviruses and toroviruses and 150 and 220 nucle-
otides for arteriviruses. Although the 5
UTRs of coro-
naviruses and arteriviruses are relatively devoid of
initiation codons, a small ORF, the intraleader ORF,
coding for 3 to 11 amino acids, is found in the corona-
virus 5
UTRs. These include ORFs of 24 nucleotides,
beginning at position 99 in the 209-nucleotide 5
UTR
of MHV-A59 (80); 24 nucleotides, beginning at posi-
tion 101 in the 210-nucleotide 5
UTR of BCoV (109);
33 nucleotides, beginning at position 81 in the 293-
nucleotide 5
UTR of HCoV-229E (53); 9 nucleotides,
beginning at position 117 in the 313-nucleotide 5

UTR of TGEV (41); and 33 nucleotides, beginning at

nidovirus genomes contain a variety of RNA struc-
tures that may be involved in interactions with viral
and cellular proteins. Most studies have involved inter-
actions of proteins with the 3
UTR of group 2 coro-
naviruses. For example, the poly(A) tail of MHV and
BCoV was found to be an important cis-acting signal
for replication and was found to bind poly(A)-binding
protein (135). Studies using MHV D-RNAs indicated
that 466 nucleotides from the 5
end and 436 nucleo-
tides from the 3
end of the genome are required for
D-RNA replication (69, 81) (Table 1). In addition,
both ends of the MHV genome are required for posi-
tive-strand synthesis, but only the last 55 nucleotides
position 131 in the 528-nucleotide 5
UTR of IBV (7).
A slightly larger intraleader ORF, of 111 nucleotides,
is found beginning at position 14 in the 211-nucleotide
5
UTR of the arterivirus EAV (66). The intraleader
ORF initiation codon was identified as forming part of
the stem structure of stem-loop III of the group 2 coro-
navirus BCoV. The authors indicated that such ORFs
may be part of stem-loop structures identified in the 5

UTRs of other coronaviruses (109). The identification
of such ORFs led to the proposal that they may play a
role in regulation of translation of the replicase gene
from the gRNA (122). Recombinant EAVs in which
either the Kozak sequence of the intraleader ORF was

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 40 BRITTON AND CAVANAGH
optimized or the initiation codon was incapacitated
exhibited similar phenotypes but showed reduced rep-
lication kinetics, plaque size, and viral yields compared
to those of the parental virus (3). It should be noted
that inactivation of the EAV intraleader ORF by Molen-
kamp et al. (94) did not have an effect on virus growth,
indicating that the results obtained by Archambault et
al. (3) could be due to changes in RNA structure rather
than intraleader ORF expression.
Regulation and Expression of Genes Encoding
Structural and Accessory Proteins
The nidovirus nonreplicase proteins, the struc-
tural and accessory proteins, are not produced as poly-
proteins but are expressed from the sg mRNAs. In most
cases the cap-dependent mechanism directs translation
of the 5
-most ORF from an sg mRNA, which apart
from the smallest species are structurally polycistronic
but functionally monocistronic. The synthesis of the sg
mRNAs is not equimolar, although in most cases the
smallest sg mRNA, usually producing the N protein, is
produced in the largest amounts. Although the group 1
coronaviruses and some newly identified group 3 coro-
naviruses have ORFs downstream of the N protein
sequence, these smaller sg mRNAs are often produced
in much smaller amounts than other sg mRNAs. In
effect, a control on the amount of sg mRNA determines
the amount of protein produced. Nevertheless, some
coronaviruses have more than one ORF translated
from a particular sg mRNA. The majority of these are
bicistronic, though the group 3 coronavirus IBV and
the related TCoV and pheasant coronavirus (PhCoV)
express a tricistronic sg mRNA (23, 24, 83, 85).
All coronaviruses identified to date contain at
least one functionally bicistronic sg mRNA, some of
which are summarized in Table 2. The number and
position of the genes expressed from such sg mRNAs
vary with the different types and groups of coronavi-
ruses. The group 1 coronaviruses include viruses that
have only one functionally bicistronic sg mRNA, for
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Table 2. Coronavirus sg mRNAs expressing more than one product
Coronavirus Group No.
TGEV 1 1
HCoV-229E 1 1
FCoV 1 2
MHV 2a 2 5 and 8
SARS-CoV 2b 4 3, 7, 8, and 9
IBV 3 2
TCoV 3 2
PhCoV 3 2
a
It should be noted that the nidovirus gRNA is a functionally bicistronic mRNA for the replicase gene (gene 1), but
structurally a polycistronic mRNA.
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example, TGEV, in which the 3a and 3b genes are
expressed from sg mRNA3 (18), and HCoV-229E, in
which the 4a and 4b genes are expressed from
sg mRNA4 (108). Other group 1 coronaviruses have
more than one functionally bi- or tricistronic sg mRNA,
for example, feline coronavirus, in which sg mRNA3
has the capacity to express three genes (3a, 3b, and 3c
[51, 155]) and sg mRNA7 produces two genes (7a and
7b [32]). In general, the group 2 coronaviruses have
only one functionally bicistronic sg mRNA, e.g.,
sg mRNA5 of MHV, which expresses two genes, 5a
and 5b, the latter encoding the E protein (20, 127).
SARS-CoV yields three functionally bicistronic
sg mRNAs, sg mRNA3, -7, and -8, which produce the
3a and 3b, 7a and 7b, and 8a and 8b proteins, respec-
tively (140). In all the cases described above, the
functionally bi- or tricistronic sg mRNAs express two
or three ORFs from the 5
end of the mRNA. However,
some type 2 coronaviruses, MHV (45), BCoV (123),
and SARS-CoV (140), have a different type of func-
tionally bicistronic sg mRNA to express an I ORF
within the N protein gene. The I ORF is in a 1 read-
ing frame located downstream of the N protein gene
initiation codon. Recent studies of the structure of the
SARS-CoV I ORF (ORF9b) indicated that the protein
has a dimeric tent-like  structure with an amphipa-
thic surface and a central hydrophobic cavity that
binds lipid molecules (91). The authors indicated that
the hydrophobic cavity was likely to be involved in
membrane attachment and showed the ORF9b protein
to associate with intracellular vesicles.
The group 3 coronaviruses IBV, TCoV, and
PhCoV produce a functionally tricistronic sg mRNA3,
which encodes the 3a, 3b, and 3c proteins, where the
ORF3c product is the E protein (23, 24, 83, 85), and
a bicistronic sg mRNA5, which produces the 5a and
5b proteins (23, 24, 84). Arteriviruses also contain a
functional bicistronic sg mRNA; the EAV E and gly-
coprotein 2 (GP2) structural proteins are encoded
by two overlapping ORFs, ORF2a and ORF2b,
which are both expressed from sg mRNA2 (131).
a
sg mRNA Products
3 3a, 3b
4 4a, 4b
3 and 7 3a, 3b, 3c, 7a, 7b
5a, 5b (E), N, I
3a, 3b, 7a, 7b, 8a, 8b, N, I
3 and 5 3a, 3b, 3c (E), 5a, 5b
3 and 5 3a, 3b, 3c (E), 5a, 5b
3 and 5 3a, 3b, 3c (E), 5a, 5b
 CHAPTER 3 • GENOME ORGANIZATION AND EXPRESSION MECHANISMS
Homologous genes in other arteriviruses, PRRSV,
LDV, and SHFV, are also postulated to be expressed
from functionally bicistronic sg mRNAs.
The second and third ORFs expressed from the
functionally bicistronic and tricistronic sg mRNAs are
expressed as independent proteins. The simplest mech-
anism for the expression of a downstream ORF is
leaky scanning (71, 72, 74). The ribosome-scanning
model predicts that the efficiency with which a partic-
ular ORF is translated depends on its location in the
mRNA and the sequence context of the AUG initia-
tion codon (71, 73, 75, 76). In this mechanism, pre-
initiation complexes, on encountering the first AUG
initiation codon, either form an active 80S ribosome
for de novo synthesis of the associated protein or
bypass the first AUG codon and continue to scan down
the mRNA until they encounter another AUG codon.
For leaky scanning to occur, the first AUG codon ide-
ally should have an unfavorable sequence context,
allowing a proportion of ribosomes to bypass the first
initiation codon and have the opportunity to initiate
protein synthesis at a second or third AUG codon with
a more favorable sequence context (76). Most of the
coronavirus and arterivirus sg mRNAs that are capable
of expressing two gene products are believed to do so
as a result of leaky scanning. The sequence context of
the initiation codons may control the amounts of the
proteins produced. In some cases the second ORF may
play an important role in the replication cycle of the
virus; e.g., MHV ORF5b yields the E protein, a struc-
tural protein required for budding of virus particles.
The I ORF within the N protein gene of BCoV (121)
or MHV (45) is also thought to be produced as the
result of leaky scanning. Thus, expression of the MHV
E and I proteins as the second products of their
sg mRNAs indicates that bicistronic sg mRNAs con-
tribute to controlling the expression of these genes.
Another translation strategy for the control of
expression of virus gene products adopted by some
viruses, like picornaviruses, is the initiation of the
translation of an internal ORF using an internal ribo-
some entry site (IRES). An IRES element is usually
Copyright © 2007. ASM Press. All rights reserved.
part of a complex RNA structure for cap-indepen-
dent translation. Its use allows viruses the opportu-
nity to interfere with cap-dependent translation,
effectively inhibiting cellular protein synthesis with-
out affecting translation of virus-derived mRNAs.
However, as indicated above, coronaviruses, like all
members of the nidovirus order, employ the normal
eukaryotic cap-dependent strategy for the translation
of most virus-derived RNAs.
sg mRNA3 of the avian coronavirus IBV is a trcis-
tronic mRNA, for the 3a, 3b, and 3c proteins; 3c is
the E protein. Studies have shown that 3a and 3b are
in fact accessory proteins and not required for growth
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41
of IBV in cell culture (57). A series of experiments
using in vitro-generated RNA transcripts modifying
either the Kozak context sequences for the three gene
3 ORFs, expression in the absence of the 5
cap ana-
log 7-methyl-GTP, introduction of 5
structures to
prevent expression of ORF3a and ORF3b, or expres-
sion of the three ORFs downstream of the influenza
A virus N protein gene showed that ORF3c was still
expressed when the expression of ORF3a and ORF3b
had been prevented (85). These observations led the
authors to propose that the expression of 3c was
being directed by a cap-independent method involv-
ing internal initiation that is dependent on the 3ab
sequence and that the 3b protein was expressed as the
result of leaky scanning using cap-dependent transla-
tion. An RNA structure was predicted and proposed
to play a role in the internal entry of ribosomes for
the translation of ORF3c by Le et al. (78). The pro-
posed coronavirus IRES elements are very different
from those identified for picornaviruses; this is not
surprising, as the picornavirus IRES elements are for
cap-independent translation, whereas coronaviruses
must function in concert with cap-dependent transla-
tion. If proteins are produced from coronavirus IRES-
like elements, there remains the possibility that these
may also be expressed from other virus RNAs, gRNA
and larger sg mRNAs, which also contain these IRES-
like elements and downstream ORFs.
Most of the coronavirus products, apart from
the E proteins of MHV and IBV, expressed from the
functionally bicistronic or tricistronic sg mRNAs repre-
sent small nonstructural genes of unknown function
(for reviews, see references 19, 77, and 87). As out-
lined under “Structural and Accessory Protein Gene
Regions,” recent work has confirmed that these are
accessory proteins and are not required for replica-
tion per se. The observation that the synthesis of these
products involves a variety of expression “tricks”
raises the possibility that they play an important role
in the viral replication cycle.
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1. Almazan, F., C. Galan, and L. Enjuanes. 2004. The nucleopro-
tein is required for efficient coronavirus genome replication.
J. Virol. 78:12683–12688.
2. Alonso, S., A. Izeta, I. Sola, and L. Enjuanes. 2002.
Transcription regulatory sequences and mRNA expression lev-
els in the coronavirus transmissible gastroenteritis virus.
J. Virol. 76:1293–1308.
3. Archambault, D., A. Kheyar, A. A. de Vries, and P. J. Rottier.
2006. The intraleader AUG nucleotide sequence context is
important for equine arteritis virus replication. Virus Genes
33:59–68.
4. Banerjee, S., K. Narayanan, T. Mizutani, and S. Makino.
2002. Murine coronavirus replication-induced p38 mitogen-
activated protein kinase activation promotes interleukin-6
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production and virus replication in cultured cells. J. Virol.
76:5937–5948.
5. Beerens, N., and E. J. Snijder. 2006. RNA signals in the 3

terminus of the genome of equine arteritis virus are required
for viral RNA synthesis. J. Gen. Virol. 87:1977–1983.
6. Bhardwaj, K., L. Guarino, and C. C. Kao. 2004. The severe
acute respiratory syndrome coronavirus nsp15 protein is an
endoribonuclease that prefers manganese as a cofactor.
J. Virol. 78:12218–12224.
7. Boursnell, M. E. G., T. D. K. Brown, I. J. Foulds, P. F. Green,
F. M. Tomley, and M. M. Binns. 1987. Completion of the
sequence of the genome of the coronavirus avian infectious
bronchitis virus. J. Gen. Virol. 68:57–77.
8. Bredenbeek, P. J., C. J. Pachuk, A. F. H. Noten, J. Charite, W.
Luytjes, S. R. Weiss, and W. J. M. Spaan. 1990. The primary
structure and expression of the second open reading frame of
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