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Development of mouse hepatitis virus and SARS-CoV infectious cDNA constructs

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The genomes of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) have been generated with a novel construction strategy that allows for the assembly of very large RNA and DNA genomes from a panel of contiguous cDNA subclones. Recombinant viruses generated from these methods contained the appropriate marker mutations and replicated as efficiently as wild-type virus. The MHV cloning strategy can also be used to generate recombinant viruses that contain foreign genes or mutations at virtually any given nucleotide. MHV molecular viruses were engineered to express green fluorescent protein (GFP), demonstrating the feasibility of the systematic assembly approach to create recombinant viruses expressing foreign genes. The systematic assembly approach was used to develop an infectious clone of the newly identified human coronavirus, the serve acute respiratory syndrome virus (SARS-CoV). Our cloning and assembly strategy generated an infectious clone within 2 months of identification of the causative agent of SARS, providing a critical tool to study coronavirus pathogenesis and replication. The availability of coronavirus infectious cDNAs heralds a new era in coronavirus genetics and genomic applications, especially within the replicase proteins whose functions in replication and pathogenesis are virtually unknown.
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CTMI (2005) 287:229--252
 Springer-Verlag 2005
Development of Mouse Hepatitis Virus
and SARS-CoV Infectious cDNA Constructs
R. S. Baric ()) · A. C. Sims
Department of Epidemiology, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599–7400, USA
rbaric@email.unc.edu
1 Introduction ................................. 230
2 The Coronavirus Genome .......................... 230
3 Systematic Approaches to Assembling Coronavirus cDNAs
from a Panel of Contiguous Subclones ................... 231
4 Assembling MHV Infectious cDNAs .................... 235
4.1 Applications in Genomics .......................... 240
4.2 Engineering MHV Genomes ......................... 241
5 SARS-CoV Infectious Clone ......................... 245
6 Future Applications ............................. 246
References....................................... 248
Abstract The genomes of transmissible gastroenteritis virus (TGEV) and mouse hep-
atitis virus (MHV) have been generated with a novel construction strategy that al-
lows for the assembly of very large RNA and DNA genomes from a panel of contigu-
ous cDNA subclones. Recombinant viruses generated from these methods contained
the appropriate marker mutations and replicated as efficiently as wild-type virus.
The MHV cloning strategy can also be used to generate recombinant viruses that
contain foreign genes or mutations at virtually any given nucleotide. MHV molecular
viruses were engineered to express green fluorescent protein (GFP), demonstrating
the feasibility of the systematic assembly approach to create recombinant viruses ex-
pressing foreign genes. The systematic assembly approach was used to develop an
infectious clone of the newly identified human coronavirus, the serve acute respira-
tory syndrome virus (SARS-CoV). Our cloning and assembly strategy generated an
infectious clone within 2 months of identification of the causative agent of SARS,
providing a critical tool to study coronavirus pathogenesis and replication. The
availability of coronavirus infectious cDNAs heralds a new era in coronavirus genet-
ics and genomic applications, especially within the replicase proteins whose func-
tions in replication and pathogenesis are virtually unknown.
1
Introduction
Molecular analysis of the structure and function of RNA virus genomes
has been profoundly advanced by the availability of full-length cDNA
clones, the source of infectious RNA transcripts that replicate efficiently
when introduced into permissive cell lines (Boyer and Haenni 1994).
Coronaviruses contain the largest single-stranded, positive-polarity
RNA genome of about 30 kb (Cavanagh et al. 1997; de Vries et al. 1997;
Eleouet et al. 1995). Until recently, coronavirus genetic analysis has been
limited to analysis of temperature-sensitive (ts) mutants (Fu and Baric
1992, 1994; Lai and Cavanagh 1997; Schaad and Baric 1994; Stalcup et
al. 1998), defective interfering (DI) RNAs (Izeta et al. 1999; Narayanan
and Makino 2001; Repass and Makino 1998; Williams et al. 1999), and
recombinant viruses generated by targeted recombination (Fischer et al.
1997; Hsue and Masters 1999; Kuo et al. 2000). Among these, targeted
recombination is the seminal approach developed to systematically as-
sess the function of individual mutations in the 30-most ~10 kb of the
MHV genome. Methods to assemble an MHV full-length infectious con-
struct have been hampered by the large size of the genome, the regions
of chromosomal instability, and the inability to synthesize full-length
transcripts (Almazn et al. 2000; Masters 1999; Yount et al. 2000). This is
especially problematic within the group 2 coronavirus replicase, where
several regions of chromosomal toxicity and instability have hampered
the development of infectious cDNAs. Full-length infectious constructs
will allow for the systematic dissection of the structure and function of
each viral gene, the phenotypic consequences of gene rearrangement on
virus replication and pathogenesis, the development of coronavirus het-
erologous gene expression systems, and a clearer understanding of the
transcription and replication strategy of the Coronaviridae. In this re-
port, we review strategies for building coronavirus infectious cDNAs by
using mouse hepatitis virus strain A59 as a model.
2
The Coronavirus Genome
The coronavirus genome, a single-stranded RNA, is the largest viral
RNA genome known to exist in nature (27.6–31.3 kb). Genomic RNAs
have a 50terminal cap and a 30terminal poly (A) tail. In addition, a lead-
er sequence of 65–98 nucleotides and a 200- to 400-base pair untranslat-
ed region are located at the 50terminus, whereas a 200- to 500-base pair
230 R.S. Baric · A.C. Sims
untranslated region is located at the 30terminus. The 50most two-thirds
of the genome encodes the replicase gene in two open reading frames
(ORFs), 1a and 1b, the latter of which is expressed by ribosomal
frameshifting (Almazn et al. 2000; Eleouet et al. 1995). Like many other
positive-sense RNA viruses, the coronavirus replicase is translated as a
large precursor polyprotein that is processed by viral proteinases, giving
rise to ~15 replicase proteins. The functions of most of the coronavirus
replicase proteins are unknown. However, based on nucleotide sequence
homology and empirical studies, identifiable functions include two pa-
painlike cysteine proteases, a chymotrypsin-like 3C protease, a cysteine-
rich growth factor-related protein, an RNA-dependent RNA polymerase,
a nucleoside triphosphate (NTP)-binding/helicase domain, and a zinc-
finger nucleic acid-binding domain (Enjuanes et al. 2000a; Penzes et al.
2001; Siddell 1995). Most of the replicase gene products colocalize with
replication complexes at sites of RNA synthesis on internal membranes.
However, a spectrum of genetically informative mutations have not been
systematically targeted to any of these replicase proteins, so we have lit-
tle insight into the organization of the replicase complex and the loca-
tion of functional motifs, which regulate transcription, replication, and
RNA recombination. Because of the extremely rich milieu of molecular
reagents that are available against the replicase proteins, the availability
of a molecular clone of MHVallows for the first time a systematic genet-
ic analysis of gene 1 function in coronavirus replication.
3
Systematic Approaches to Assembling Coronavirus cDNAs
from a Panel of Contiguous Subclones
Coronavirologists have seized on several different strategies to build in-
fectious cDNA clones. However, all were primarily designed to circum-
vent problems associated with the large size of the coronavirus genome,
regions of chromosomal instability, and other problems associated with
the production of full-length infectious transcripts (Almazn et al. 2000;
Masters 1999; Yount et al. 2000). Our solution was to assemble infectious
cDNAs from a panel of contiguous subclones that spanned the entire
length of the TGEV and MHV genomes. Each subclone was flanked by
unique restriction sites with characteristics that allow for the systematic
and precise assembly of a full-length cDNA with in vitro ligation. For
this strategy to be efficient, restricted subclone fragments had to be in-
capable of self-concatemer formation and not spuriously assemble with
other noncontiguous subclones.
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 231
Conventional class II restriction enzymes, such as EcoRI, leave identi-
cal sticky ends that assemble with similarly cut DNA in the presence of
DNA ligase (Pingoud and Jeltsch 2001; Sambrook et al. 1989). Because
these enzymes leave identical compatible ends, digested fragments ran-
domly self-assemble into large concatamers and, therefore, they are poor
choices for assembling large intact genomes or chromosomes. However,
a second group of class II restriction enzymes (i.e., BglI, BstXI, SfII) also
recognize a symmetrical sequence but leave random sticky ends 1–4 nu-
cleotides in length, and consequently, restrict assembly cascades along
specific pathways (Table 1). For example, the type II restriction enzyme,
BglI, recognizes the symmetrical sequence GCCNNNN
#
NGGC and
cleaves a random DNA sequence on average every ~4,096 base pairs. Be-
cause 64 different 3-nucleotide overhangs can be generated, DNA frag-
Table 1. Selected restriction enzymes used in assembly of recombinant full-length gen-
omes
Restric-
tion
enzyme
a
Recognition site No. of
variable
sticky end
Average
cutting
frequency
b
Actual frequency
of compatible
ends
b
BglI GCCNNNN#NGGC 3 nt/64
potential ends
~4,096 nt ~261,344 nt
CGGN"NNNNCCG
BstXI CCANNNNN#NTGG 4 nt/256
potential ends
~4,096 nt ~1,045,376 nt
GGTN"NNNNNACC
SfII GGCCNNNN#NGGCC 3 nt/64
potential ends
~65,536 nt ~4,194,304 nt
CCGGN"NNNNCCGG
SapI GCTCTTCN#NNNN 3 nt/64
potential ends
~16,385 nt (in
either strand)
~1,048,640 nt*
CGAGAAGNNNNN"
AarI CACCTGCNNNN#NNNN 4 nt/256
potential ends
~16,385 nt (in
either strand)
~4,194,304 nt*
GTGGACGNNNNNNNN"
Esp3I
(BsmBI)
CGTCTCN#NNNN 4 nt/256
potential ends
~4,096 nt (in
either strand)
~1,048,576 nt*
GCAGAGNNNNN"
a
Other enzymes leaving many different overhangs: BsmFI, EclHkI, FokI, MboII,
TthIIII, AhdI, DrdI, BspMI, BsmAI, BcgI, BmRI, BpmI, BsaI, BseI, EarI, PfiMI, BstV2,
VpaK32I, AbeI, PpiI.
b
Assuming a totally random DNA sequence; *asymmetric cutters like SapI, AarI
and Esp3I can have recognition sites in either strand of DNA so actual site frequen-
cy is ~1/2 of indicated values and can be engineered as “no-see-um” (Yount et al.
2002).
232 R.S. Baric · A.C. Sims
ments will only assemble with the appropriate 3-nucleotide complemen-
tary overhang generated at an identical BglI restriction site. As a result,
identical ends are generated every ~264,000 base pairs, providing a pow-
erful means for the construction of very large DNA and RNA genomes.
Consonant with these findings, the type IIS restriction enzyme, Esp3I,
recognizes an asymmetric sequence and makes a staggered cut 1 and
5 nucleotides downstream of the recognition sequence, leaving 256,
mostly asymmetrical, 4-nucleotide overhangs (GCTCTCN
#
NNNN). As
identical Esp3I sites are generated every ~1,000,000 base pairs or so in a
random DNA sequence, most restricted fragments usually do not self-as-
semble (Yount et al. 2002). Rather, specific recursive assembly pathways
can be designed that hypothetically allow assembly of >1 million base
pair DNA genomes (~2
256
fragments) (Table 1). We took advantage of
several unique properties inherent in type II restriction enzymes to
build coronavirus infectious cDNAs.
Initially, we isolated five cDNA subclones spanning the entire TGEV
genome (designated TGEVA, B, C, D/E, and F) by RT-PCR using primers
that introduced unique BglI restriction sites at the 50and 30ends of each
fragment without altering the amino acid coding sequences of the virus
(Table 2). The TGEVA, C, DE, and F clones were stable in plasmid DNAs
in Escherichia coli. The B fragment, however, was unstable, containing
deletions or insertions in the wild-type sequence at a region of instabili-
ty in the TGEV genome noted by other investigators (Almazn et al.
2000; Eleouet et al. 1995). To prevent fragment instability, we used prim-
er-mediated mutagenesis to bisect the B fragment at the unstable site
with an adjoining BstXI (CCATTCAC
#
TTGG) site, resulting in TGEV B1
and TGEV B2 amplicons (Fig. 1; Table 2). It is likely that sequences
Table 2. Design of TGEV junction sequences
Restriction site junction Location Junction
50-GCCTGTT
#
TGGC-30BglI, nt 6,159 A-B1
30-CGGA
"
CAAACCG-50
50-CCATTCAC
#
TTGG-30BstXI, nt 9,949 B1-B2
30-GGTA
"
AGTGAACC-50
50-GCCGCAT
#
TGGC-30BglI, nt 11,355 B2-C
30-CGGC
"
GTAGCCG-50
50-GCCTTCT
#
TGGC-30BglI, nt 16,595 C-D/E1
30-CGGA
"
AGAACCG-50
50-GCCGTGC
#
AGGC-30BglI, nt 23,487 D/E1-F
30-CGGC
"
ACGTCCG-50
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 233
234 R.S. Baric · A.C. Sims
(9600–9950) in and around the TGEV 3C like protease (3CL
pro
) motif
are either bactericidal or unstable in microbial vectors (Almazn et al.
2000; Yount et al. 2000). The resulting 6 fragments, TGEV A, B1, B2, C,
D/E, and F, were ligated in vitro to generate a full-length cDNA of the
TGEV genome (Fig. 1). Molecularly cloned viruses were indistinguish-
able from wild type and contained the marker mutations and unique
BglI and BstXI junction sequences used in the assembly of the infectious
construct (Yount et al. 2000).
4
Assembling MHV Infectious cDNAs
One potential problem with the original approach was that several “si-
lent” mutations were inserted to introduce the unique BglI sites into the
TGEV component clones. To circumvent this problem, a variation of the
systematic assembly approach was used to build the group II coronavi-
rus, mouse hepatitis virus (MHV) infectious cDNA (Yount et al. 2002).
The enzyme Esp3I recognizes an asymmetrical site and cleaves external
to the recognition sequence, allowing for traditional and “no-see-um”
cloning applications (Fig. 2, Table 1). With traditional approaches, Esp3I
sites can be oriented to reform the recognition site after ligation of two
MHV cDNAs, leaving the restriction site within the genomes of recombi-
nant viruses. However, the Esp3I recognition site is asymmetrical, so a
simple reverse orientation allows for the insertion of an Esp3I recogni-
tion sequence on the ends of two adjacent clones with the cleavage site
derived from virtually any 4-nucleotide sequence combination dictated
by the virus sequence. On cleavage and ligation with the adjoining frag-
ment, the Esp3I sites are lost from the final ligation products, leaving a
Fig. 1. Strategy for the systematic assembly of TGEV full-length cDNA. The TGEV
genome is a positive-sense, single-stranded RNA of about 28.5 kb. Six independent
subclones (A,B1,B2,C,DE, and F) that span the entire length of the genome were
isolated by RT-PCR using primer pairs that introduced unique NotI, BglI, and/or
BstXI restriction sites at each end. On ligation, the intact viral genome is generated
as a cDNA. A unique T7 start site and a 25 poly(T) tail allow for in vitro transcrip-
tion of full-length, capped, polyadenylated transcripts (Yount et al. 2000). PL, pa-
painlike protease; 3CL
pro
, 3CL protease; GFL, growth factor like; pol, polymerase mo-
tif; MIB, metal binding motif; hel, helicase motif; VD/CD, variable or conserved do-
mains
t
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 235
seamless junction compiled from the exact MHV-A59 sequence. Because
of this property, unique junctions can be inserted at virtually any posi-
tion between two component clones without mutating the viral genome
sequence. Additionally, a large number of other restriction enzymes
share this property (e.g., SapI, AarI), expanding the utility of the “no-
see-um” technology (Table 1).
During the isolation of the MHV component clones, it was also neces-
sary to remove three preexisting Esp3I sites located throughout the
MHV ORF1 sequence (Bonilla et al. 1994). Mutations inserted to ablate
these sites were used as marker mutations to distinguish molecularly
Fig. 2. Use of Esp3I in the traditional and “no-see-um” approaches. The traditional
approach to the use of Esp3I involves the ligation of two fragments containing iden-
tical Esp3I restriction sites, resulting in a ligation product with an intact Esp3I site
remaining. In the “no-see-um” approach, a simple reverse orientation of the restric-
tion sites allows for the specific removal of the Esp3I site from the two fragments,
resulting in a ligation product lacking the engineered restriction site. The use of the
“no-see-um” technology allows for the assembly of large DNAs from smaller sub-
clones without the incorporation of unique restriction sites into the genome. (Yount
et al. 2002)
236 R.S. Baric · A.C. Sims
cloned and wild-type virus. We then isolated seven consensus cDNAs
that spanned the entire length of the MHV-A59 genome in the same
manner as the TGEV infectious construct (Fig. 3). This was necessary
because the MHV-A59 genome contains several major regions of se-
quence toxicity in microbial cloning vectors, most of which map be-
tween ~10 and 15 kb in the MHV ORF 1a/ORF 1b polyprotein and an
unstable region mapping ~5.0 kb in ORF 1a. As described for the TGEV
B fragment, cDNAs were isolated after intersecting the toxic domains
and separating them into independent subclones. However, many sub-
clones were still unstable in traditional PUC-based cloning vectors (e.g.,
pGem, TopoII) even when maintained at low temperature. Consequently,
we used pSMART cloning vectors (Lucigen), which lack a promoter and
indicator gene and contain transcriptional and translational terminators
surrounding the cloning site. Instability appears to be associated with
expression, as this entire domain (nucleotides 9,555–15,754) is also sta-
ble in yeast vectors (pYES2.1 Topo TA Cloning Kit from Invitrogen) that
maintain tight regulation over foreign gene expression (Yount et al., un-
published results). Full-length MHV-A59 cDNA was systematically as-
sembled through the simultaneous in vitro ligation of a series of seven
subgenomic cDNAs (Yount et al. 2002). In the future, it may be possible
to construct larger subgenomic fragments spanning the entire genome
by using the pSMART cloning vectors, thereby simplifying the assembly
strategy, although we have not tested this directly.
The TGEV and MHV A fragments contain a T7 promoter, whereas the
TGEV F and MHV G fragments terminate in a poly(T) tract at the 30
end, allowing for in vitro T7 transcription of infectious capped, poly-
adenylated transcripts. The poly(A) tails generated from these tran-
scripts are 25 nucleotides in length, which appears sufficient for tran-
script infectivity. At this time, we do not know the minimal number of
30poly(A) residues necessary for transcript infectivity or whether a 50
methylated cap is essential. Electroporation of the genomic-length RNAs
resulted in the production of recombinant MHV virus with growth char-
acteristics identical to those of the wild-type viruses (Yount et al. 2000,
2002). Importantly, the molecularly cloned viruses contained marker
mutations engineered into the component clones. Inclusion of nuclocap-
sid(N)-encoding transcripts enhanced the infectivity of full-length MHV
and TGEV transcripts. In MHV, N transcripts enhanced the infectivity of
full-length MHV-A59 transcripts by 10- to 15-fold as evidenced by in-
creased viral antigen expression and virus titers at 25 h postinfection
(Yount et al. 2002). It is unclear whether MHV N transcripts, N protein,
or both are essential for increased virus yields after electroporation, or
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 237
238 R.S. Baric · A.C. Sims
whether this effect would be observed with transcripts encoding unrelat-
ed genes. Coronaviruses have been demonstrated to package low con-
centrations of subgenomic mRNAs, especially N transcripts, and several
studies have suggested that N may function in transcription and replica-
tion and are tightly associated with the replication complex. With IBV,
but not TGEV or HCoV-229E, N transcripts are absolutely essential for
full-length transcript infectivity (Casais et al. 2001). With HCoV-229E,
other groups have shown that the N gene is not required for subgenomic
transcription (Thiel et al. 2001). Clearly, additional studies are needed to
evaluate the role of N protein in RNA transcript infectivity.
The MHV cDNA cassettes can be ligated systematically as described
for TGEV or simultaneously. Although numerous incomplete assembly
intermediates were evident, our demonstration that simultaneous liga-
tion of seven cDNAs will result in full-length cDNA will simplify the
complexity of the assembly strategy. At this time, there is no evidence to
indicate that this approach might introduce spurious mutations or ge-
nome rearrangements from aberrant assembly cascades. However, it is
possible that such variants might arise after RNA transfection, as a con-
sequence of high-frequency MHV RNA recombination between incom-
plete and genome-length transcripts. It is likely that such variants would
be replication impaired and rapidly out-competed by wild-type virus. A
second limitation is that the yield of full-length cDNA product is re-
duced, resulting in less robust transfection efficiencies compared with
the more traditional systematic assembly method. At this time, the
MHV approach suffers from the large number of component clones (sev-
en), which increase the complexity of the system and reduce the yield of
full-length cDNA product after in vitro ligation. If the large number of
toxic domains in the MHV genome is duplicated in other group II coro-
naviruses, this will likely interfere with the development of other infec-
Fig. 3. Systematic assembly strategy for the construction of MHV-A59 full-length
cDNA. The MHV genome is a positive-sense, single-stranded RNA of ~31.5 kb. Sev-
en independent subclones (A,B,C,D,E,F, and G) that span the entire MHV genome
were isolated by RT-PCR. Unique BglI and Esp3I restriction sites, located at the 50
and 30ends of each subclone, were used to assemble a full-length cDNA. A unique
T7 start site was inserted at the 50end of the MHV A fragment and a 25 poly(T) tail
was inserted at the 30end of the MHV F fragment, allowing for in vitro transcription
of full-length, capped, poly-adenylated transcripts. Note: Esp3I sites are lost in the
assembly process. (Yount et al. 2002)
t
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 239
tious cDNAs as well. Topics of future research include: (1) Can group II
coronavirus cDNAs be stabilized as full-length constructs in bacterial ar-
tificial chromosomes or poxvirus vectors as has been reported with
TGEV, IBV, and HCoV 229E? (2) How does N function to enhance infec-
tivity of full-length transcripts? (3) How can we enhance yields or the in-
fectivity of coronavirus infectious cDNAs and transcripts and allow for
critical review of the consequences of lethal mutations? (4) Can we re-
duce the number of component clones needed to assemble group II co-
ronavirus infectious cDNAs?
4.1
Applications in Genomics
Our assembly strategy for coronavirus infectious constructs is simple
and straightforward, although the synthesis of full-length transcripts is
technically challenging. In contrast to infectious clones of other posi-
tive-strand viruses, our TGEV and MHV constructs must be assembled
de novo and do not exist intact in bacterial or viral vectors. This does
not restrict the methods applicability for reverse genetic applications.
Rather, it allows for rapid genetic manipulation of independent sub-
clones, which minimizes the introduction of spurious mutations else-
where in the genome during recombinant DNA manipulation. Theoreti-
cal limits of our method may exceed several million base pairs of DNA
and will likely surmount the cloning capacity of bacterial (BAC) and eu-
karyotic artificial chromosome vectors (Grimes and Cooke 1998). Our
systematic assembly method should also be appropriate for constructing
full-length infectious clones of other large RNA viruses, including coron-
aviruses (27–32 kb), toroviruses (24–27 kb), and filoviruses like Mar-
burg (19 kb) (de Vries et al. 1997; Peters et al. 1996). Viral genomes that
are unstable in prokaryotic vectors can also be cloned by these methods
(Boyer and Haenni 1994; Rice et al. 1989). Moreover, the technique
should allow the systematic assembly of full-length infectious dsDNA
genomes of adenoviruses, herpesviruses, and perhaps other large DNA
viruses that promise to be powerful tools in vaccination, gene transfer,
and gene therapy (Smith and Enquist 2000; van Zijl et al. 1988). Recent-
ly, genome sequences from a large number of prokaryotic and eukaryot-
ic organisms have been obtained, providing significant insight into gene
organization, structure, and function (Cho et al. 1999; Hutchison et al.
1999) (TIGR homepage http://www.tigr.org). Using this strategy, it may
be possible to reconstruct a minimal microbial genome from the bottom
up. However, problems associated with isolating large DNA fragments
240 R.S. Baric · A.C. Sims
and the introduction of large DNA genomes into environments that per-
mit replication will likely be significant hurdles. Nevertheless, our as-
sembly strategy may provide a means to analyze the function of large
blocks of DNA, such as pathogenesis islands, or to engineer chromo-
somes that contain large gene cassettes of interest (Cho et al. 1999).
4.2
Engineering MHV Genomes
Coronaviruses provide a unique system for the incorporation and ex-
pression of one or more foreign genes (Enjuanes and Van der Zeijst
1995). Coronavirus genes rarely overlap, simplifying the design and ex-
pression of foreign genes from downstream intergenic sequences (IS)
start sites. Integration of the coronavirus RNA genome into the host cell
chromosome is unlikely (Lai and Cavanagh 1997). Additionally, recom-
binant viruses or replicon particles could be readily targeted to other
mucosal surfaces in swine or to other species by simple replacements in
the S glycoprotein gene, which has been shown to determine tissue- and
species tropism (Ballesteros et al. 1997; Delmas et al. 1992; Kuo et al.
2000; Leparc-Goffart et al. 1998; Snchez et al. 1999; Tresnan et al. 1996).
Furthermore, coronaviruses infect a number of different species, includ-
ing human, porcine, bovine, canine, and feline, and are available for the
development of expression systems (Snchez et al. 1992). Additionally,
the coronavirus helical ribonucleocapsid structure may further relax the
packaging constraints of the virus, as compared to icosahedral struc-
tures (Enjuanes and Van der Zeijst 1995; Lai and Cavanagh 1997; Risco
et al. 1996). Selected questions that remain unanswered include: (1)
What is the coding capacity of coronavirus based expression systems?
(2) What is the minimal genome required for efficient replication? (3)
Can high-titer coronavirus replicon particles be obtained for vaccine ap-
plications? (4) What are the minimal sequence requirements for subge-
nomic transcription? (5) How many foreign genes can be coordinately
regulated without impeding virus replication or immunogenicity? (6)
What are the efficacy, stability, and safety of the recombinant coron-
aviruses in natural settings? Clearly, these vaccine-related topics will
provide fruitful avenues of investigation over the next decade and will
greatly enhance our understanding of the mechanics of coronavirus
transcription, replication, assembly and release, and pathogenesis.
The future development of vaccines and expression vectors are partic-
ularly intriguing applications of our TGEV and MHV infectious clones.
Importantly, at least two TGEV downstream ORFs encode luxury func-
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 241
Fig. 4. Rapid mutagenesis of the MHV infectious cDNA with Class IIS restriction en-
donucleases. Seamless insertion of foreign genes into the coronavirus genome can
be accomplished with Class IIS restriction enzymes. In this case, a target gene is sys-
tematically removed and replaced by a new gene (new insert). Using a primer with
overlaps a unique upstream (Site A) restriction site, the upstream arm amplicon is
242 R.S. Baric · A.C. Sims
tions (ORF 3a and 3b) that may be deleted from the viral genome
without impacting infectivity (Curtis et al. 2002; Laude et al. 1990;
McGoldrick et al. 1999; Wesley et al. 1991). We have developed a rapid
approach that allows seamless insertion of foreign sequences into virtu-
ally any nucleotide position in the MHV genome, based on class IIS re-
striction endonucleases (Fig. 4). In this approach, flanking sequences
around the target domain are amplified as separate arms linked by un-
ique class IIS restriction site oriented as described in Fig. 3. A third am-
plicon encoding the payload sequence of interest is isolated and flanked
by similar class IIS sites. After restriction digestion and ligation, the for-
eign sequences are inserted into the backbone sequence at any given nu-
cleotide, leaving no evidence of the restriction sites that were used to
“sew” the new sequences into the MHV backbone. We have successfully
expressed GFP from the ORF 3a locus of TGEV (Curtis et al. 2002) and
ORF 4 of MHV (Fig. 5) (manuscript in preparation), demonstrating the
feasibility of the method and the use of TGEV and MHV as expression
vectors. In the case with TGEV, GFP expression was stable for at least
10 passages. In addition, we have removed the ORF 3a and replaced it
with GP5 of PRRSV to create icTGEV PRRSV GP5 recombinant viruses
(Curtis KM and Baric RS, unpublished data). Recombinant viruses ex-
pressed the PRRSV GP5 glycoprotein as evidenced by indirect immuno-
fluorescence assay (IFA) and RT-PCR using primer pairs within the
TGEV leader and PRRSV GP5 gene (data not shown). Recently, expres-
sion of the reporter gene b-glucuronidase (GUS) and PRRSV ORF 5
from a TGEV-derived minigenome was demonstrated (Alonso et al.
2002). Importantly, strong humoral immune responses against GUS and
PRRSV ORF5 were generated in swine with these vectors, demonstrating
the feasibility of coronavirus-based vectors for future vaccine develop-
ment.
amplified with a second primer (Site B) containing a Esp3I recognition at the 50end
of the nonsense strand of DNA by PCR. A similar approach is used to amplify the
downstream arm (Site C and Dprimers). The insert DNA is amplified with primer
pairs containing compatible C and D Esp3I sites. After PCR amplification and re-
striction digestion, the new insert can be inserted into the viral genome without evi-
dence of the restriction sites used in the assembly cascade. A large number of class
IIS restriction enzymes greatly enhances the plasticity of the approach
t
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 243
Fig. 5a, b. Recombinant MHV-A59 expressing GFP. With standard molecular tech-
niques, ORF 4 was removed and the gene encoding GFP inserted downstream of the
ORF 4 IS (a). DBT cells were infec ted with wild-type MHV-A59 (Aand C) or icMHV-
A59 GFP (Band D) and subsequently analyzed for CPE by light microscopy (Aand
B) and GFP expression by fluorescent microscopy (Cand D)(b)
244 R.S. Baric · A.C. Sims
5
SARS-CoV Infectious Clone
Rapid response and control of exigent emerging pathogens require an
approach to quickly generate full-length cDNAs from which molecularly
cloned viruses are rescued, allowing for genetic manipulation of the ge-
nome. Identification of the first human coronavirus to cause consider-
able morbidity and mortality worldwide provided the first template to
test the rapidity of our systematic assembly strategy (Drosten et al. 2003;
Ksiazek et al. 2003). Development of novel vaccine candidates and thera-
peutics requires a better understanding of viral pathogenesis, a process
greatly facilitated by the availability of an infectious clone. A systematic
assembly strategy based on the TGEV infectious clone was employed to
create an infectious construct of the SARS-CoV, within ~2 months of the
identification and isolation of genomic SARS-CoV RNA (Yount et al.
2003). Consensus clones were assembled from sibling clones of each
SARS-CoV fragment by taking advantage of the special properties of
asymmetric type IIS restriction enzymes. Within 9 weeks, infectious
clone SARS-CoV was isolated that was phenotypically indistinguishable
from wild-type SARS-CoV strains.
The SARS-CoV genome was cloned as six contiguous subclones that
could be systematically linked by unique BglI restriction endonuclease
sites (Fig. 6). Two BglI junctions were derived from sites encoded within
the SARS-CoV genome at nt 4,373 (A/B junction) and nt 12,065 (C/D
junction). A third BglI site at nt 1,577 was removed, and new BglI sites
were inserted by the introduction of silent mutations into the SARS-CoV
sequence at nt 8,700 (B/C junction), nt 18,916 (D/E junction) and nt
24,040 (E/F junction). The resulting cDNAs include SARS A (nt 1–4,436),
SARS B (nt 4,344–8,712), SARS C (nt 8,695–12,070), SARS D (nt 12,055
18,924), SARS E (nt 18,907–24,051), and SARS F (nt 24,030–29,736) sub-
clones. The SARS A subclone also contains a T7 promoter, and the SARS
F subclone terminates in 21Ts, allowing synthesis of capped, polyadenyl-
ated transcripts. SARS-CoV infectious clone virus was assembled, tran-
scribed and transfected as described previously, and recombinant viruses
contained the marker mutations inserted into the infectious clone. Re-
combinant viruses produced a mild pneumonia on x-ray in macaques
similar to wild-type viruses and replicated to similar titers in the mouse
model (unpublished observation). These data suggest that recombinant
viruses recapitulated the pathogenesis of wild type in animal models, al-
lowing for the identification of pathogenesis determinants and develop-
ing attenuated viruses as candidate live and killed vaccines.
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 245
6
Future Applications
The availability of infectious cDNA clones will undoubtedly have a pro-
found effect on the field of coronavirology. These new tools will facilitate
basic studies and allow for more precise analyses of the molecular mech-
anisms of viral replication, including the definition of RNA elements im-
portant for RNA replication, subgenomic RNA transcription, and ge-
Fig. 6. Systematic assembly strategy for the SARS-CoV infectious clone. The SARS-
CoV genome is about 30 kb in length and contains ~14 open reading frames (ORFs).
The predicted functions of the group specific ORFs (ORF 3a/b,ORF 6,ORF 7a/b,
ORF 8a/b,ORF 9b) are unknown. Dark gray squares indicate highly conserved con-
sensus sequence sites that function in subgenomic RNA synthesis. Six independent
subclones (A,B,C,D,E, and F) that span the entire SARS-CoV genome were isolated
by RT-PCR (genome fragments are not shown to scale). The A fragment spans nt 1–
4436, the B fragment nt 4344–8712, the C fragment nt 8695–12,070, the D fragment
nt 12,055–18,924, the E fragment 18,907–24,051, and the F fragment nt 24,030–
29,736. Unique BglI restriction sites located at the 50and 30ends of each subclone
were used to assemble a full-length cDNA. A unique T7 start site was inserted at the
50end of the SARS-CoV A fragment, and a 21 poly(T) tail was inserted at the 30end
of the SARS-CoV G fragment, allowing for in vitro transcription of full-length,
capped, polyadenylated transcripts
246 R.S. Baric · A.C. Sims
nomic RNA packaging. In addition, studies of gene function will be en-
hanced by the availability of infectious cDNA clones by allowing for the
construction of recombinant viruses and/or replicons containing muta-
tions and the analysis of their effects on viral replication and assembly.
MHV has long been used as a premiere model to study coronavirus as-
sembly and release, replication, transcription, entry, and pathogenesis.
The availability of MHV and SARS-CoV infectious cDNA clones will
complement the existing targeted recombination approaches by provid-
ing a tool for the mutagenesis of the replicase gene, which encode a large
number of cleavage products that have not been fully characterized. The
structure and function of the ~20-kb MHV replicase domain will likely
remain a fertile area of research for the next decade and reveal novel
protein functions that participate and regulate discontinuous transcrip-
tion and high-frequency RNA recombination. Although large panels of
reagents are available for analyzing replicase protein expression, pro-
cessing, and subcellular localization, a spectrum of genetically informa-
tive mutations have not been systematically targeted to any of these
replicase proteins. Given the complexity and size of the coronavirus
replicase gene, the number of potential mutants that can be generated is
enormous and will likely require bioinformatic approaches for building
and testing specific hypotheses. For example, the ORF1a C-terminal
MHV p15 protein is highly conserved among group I through III coron-
aviruses and contains a large number of conserved cysteine residues and
predicted phosphorylation, myristylation, and glycosylation sites (pro-
site, unpublished) (Fig. 7). The original sequence report of p15 also sug-
gested possible similarities to growth factor-like proteins (Lee et al.
1991). Recent studies with an IBV homolog suggest that p15 exists as a
dimer and accumulates on stimulation with epidermal growth factor,
providing some evidence that the protein might be involved in the
growth factor signaling pathway (Ng and Liu 2002). A single amino acid
mutation has been identified in p15 of the temperature sensitive mutant,
LA6, an MHV-A59 mutant with a defect in RNA synthesis at nonpermis-
sive temperature (Siddell et al. 2001). The availability of infectious cD-
NAs allows, for the first time, a systematic mutagenesis approach for
studying the function of specific structural features within this and oth-
er replicase proteins.
Coupled with the capacity to isolate large panels of mutants in each
of the replicase proteins, selected questions include: (1) Are each of the
PL1
pro
, PL2
pro
, and 3CL
pro
cleavage sites necessary for MHV replication?
(2) Are the PL1
pro
, PL2
pro
, or 3CL
pro
proteases essential for replication?
(3) Are any replicase proteins nonessential? (4) Is replicase gene order
Development of Mouse Hepatitis Virus and SARS-CoV Infectious 247
critical? (5) Are replicase proteins interchangeable between the group 1
and/or group 2 coronaviruses? (6) How do replication complexes form
on membranes? (7) What replicase complexes regulate discontinuous
transcription and synthesis of genome-length and subgenomic-length
mRNAs and negative-strand RNAs? (8) What are the cis-acting sequence
elements required for genomic RNA packaging and replication? (9)
What are the structure-function relationships within and between vari-
ous replicase proteins and/or RNAs? (10) What are the functions of the
group-specific ORFs, and how do they influence pathogenesis? The next
decade of research may well be defined as the golden age of coronavirus
genetics.
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Coronaviruses were recognized as a group of enveloped, RNA viruses in 1968 and accepted by the International Committee on the Taxonomy of Viruses as a separate family, the Coronaviridae, in 1975. By 1978, it had become evident that the coronavirus genomic RNA was infectious (i. e. , positive strand), and by 1983, at least the framework of the coronavirus replication strategy had been per­ ceived. Subsequently, with the application of recombinant DNA techniques, there have been remarkable advances in our understanding of the molecular biology of coronaviruses, and a mass of structural data concerning coronavirus genomes, mRNAs, and pro teins now exists. More recently, attention has been focused on the role of essential and accessory gene products in the coronavirus replication cyde and a molecular analysis of the structure-function relation­ ships of coronavirus proteins. Nevertheless, there are still large gaps in our knowledge, for instance, in areas such as the genesis of coronavirus subgenomic mRNAs or the function of the coronavirus RNA-dependent RNA polymerase. The diseases caused by coronaviruses have been known for much longer than the agents themselves. Possibly the first coronavirus-related disease to be recorded was feline infectious peritonitis, as early as 1912. The diseases associ­ ated with infectious bronchitis virus, transmissible gastroenteritis virus, and murine hepatitis virus were all well known before 1950.
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