| Journal of General Virology |
| SUMMARY | INTRODUCTION | LIFE CYCLE | REVERSE GENETICS SYSTEMS | REPLICATION | VIRUS PROTEINS | GLYCOPROTEINS |
| ACCESSORY PROTEINS | VIRUS VECTORS | LIVE ATTENUATED VIRUS VACCINES | FUTURE PERSPECTIVES | REFS | ||
| First posted online 29 July 2002 | REVIEW ARTICLE |
| DOI: 10.1099/vir.0.18400-0 |
Gabriele Neumann,1 Michael A. Whitt2 and Yoshihiro Kawaoka1,3,4
1 Department of Pathobiological Sciences, School of
Veterinary Medicine, University of Wisconsin, 2015 Linden Drive
West, Madison, WI 53706, USA
2 Department of Molecular Sciences, University of
Tennessee Health Science Center, Memphis, TN, USA
3 Institute of Medical Science, University of Tokyo,
Tokyo, Japan
4 CREST, Japan Science and Technology Corporation, Japan
Since the first generation of a negative-sense RNA virus entirely from cloned cDNA in 1994, similar reverse genetics systems have been established for members of most genera of the Rhabdo- and Paramyxoviridae families, as well as for Ebola virus (Filoviridae). The generation of segmented negative-sense RNA viruses was technically more challenging and has lagged behind the recovery of nonsegmented viruses, primarily because of the difficulty of providing more than one genomic RNA segment. A member of the Bunyaviridae family (whose genome is composed of three RNA segments) was first generated from cloned cDNA in 1996, followed in 1999 by the production of influenza virus, which contains eight RNA segments. Thus, reverse genetics, or the de novo synthesis of negative-sense RNA viruses from cloned cDNA, has become a reliable laboratory method that can be used to study this large group of medically and economically important viruses. It provides a powerful tool for dissecting the virus life cycle, virus assembly, the role of viral proteins in pathogenicity and the interplay of viral proteins with components of the host cell immune response. Finally, reverse genetics has opened the way to develop live attenuated virus vaccines and vaccine vectors.
Introduction |
Negative-strand RNA viruses are classified into seven families (Rhabdo-, Paramyxo-, Filo-, Borna-, Orthomyxo-, Bunya- and Arenaviridae). The first four families are characterized by nonsegmented genomes, while the latter three have genomes comprising six-to-eight, three or two negative-sense RNA segments, respectively. The large group of negative-sense RNA viruses includes highly prevalent human pathogens, such as respiratory syncytial virus (RSV), parainfluenza viruses and influenza viruses, and two of the most deadly human pathogens (Ebola and Marburg viruses), as well as viruses with a major economic impact on the poultry and cattle industries [e.g. Newcastle disease virus (NDV) and rinderpest virus (RPV)].
Reverse genetics, as the term is used in molecular virology,
describes the generation of viruses possessing a genome derived
from cloned cDNAs. Reverse genetics systems have been established
to artificially produce members of the Rhabdo-, Paramyxo-, Filo-,
Bunya- and Orthomyxoviridae families (Table
1
An overview of the life cycle of negative-sense RNA viruses |
Negative-sense RNA viruses infect their host cells by binding to a
host cell receptor via a viral surface glycoprotein. Subsequent
fusion of the viral membrane with the plasma membrane
(pH-independent pathway) (Fig.
1A) or with endosomal membranes in the acidic
environment of late endosomes (pH-dependent pathway) (Fig. 1B) releases viral ribonucleoprotein
(RNP) complexes into the cytoplasm. RNP complexes are composed of
the viral RNA (vRNA), the nucleoprotein, which encapsidates the
vRNA, and the viral polymerase complex. Individual mRNAs are
synthesized during transcription, while replication leads to the
synthesis of full-length antigenomic RNAs, which, in turn, serve as
templates for genomic vRNA synthesis. Most of the negative-sense
RNA viruses replicate in the cytoplasm of infected cells, in
contrast to the nuclear replication of orthomyxoviruses and
bornaviruses. Thus, for these latter viruses, incoming RNP
complexes and newly synthesized NP and polymerase proteins have to
be imported into the nucleus, whereas newly assembled RNPs are
exported from the nucleus to the cytoplasm. Newly synthesized RNP
complexes are assembled with viral structural proteins at the
plasma membrane or at membranes of the Golgi apparatus, followed by
the release of newly synthesized viruses. For more detailed
information on the life cycles of negative-sense RNA viruses, the
reader is referred to the relevant chapters in Fields, Virology
(Knipe & Howley, 2001
), and to review articles on this topic
(Curran & Kolakofsky, 1999
; Buchmeier et
al., 2001
; de la Torre, 2001
; Lamb &
Kolakofsky, 2001a
; Lamb & Krug, 2001b
; Rose & Whitt,
2001
; Sanchez
et al., 2001
; Schmaljohn & Hooper, 2001
).
Fig. 1. Schematic diagram of virus entry. Negative-sense RNA viruses bind
to host cell receptors via viral surface glycoproteins. (A)
pH-independent pathway. Fusion of the viral membrane with the
plasma membrane releases RNP complexes into the cytoplasm. (B)
pH-dependent pathway. Viruses are internalized by receptor-mediated
endocytosis. The low pH in late endosomes triggers conformational
changes in the viral glycoproteins that lead to the fusion of the
viral and endosomal membranes and the subsequent release of RNP
complexes into the cytosol.
Reverse genetics systems |
The first reverse genetics systems were established with
positive-sense RNA viruses (Taniguchi et al., 1978
; Racaniello &
Baltimore, 1981
). Transfection of the full-length RNAs of
positive-sense RNA viruses into eukaryotic cells results in viral
protein expression which leads to virus replication. In contrast,
the genomes of negative-sense RNA viruses alone are noninfectious.
Initiation of virus replication and transcription requires
coexpression of the viral polymerase complex in addition to
vRNA(s); furthermore, the genome has to be complexed with the
nucleocapsid protein.
In 1989, Palese and colleagues established the first system for the
modification of a negative-sense RNA virus, influenza A virus
(Luytjes et al., 1989
; Enami et al., 1990
; reviewed by
Garcia-Sastre & Palese, 1993
; Garcia-Sastre et al., 1994b
; Palese, 1995
; Palese et
al., 1996
; Neumann & Kawaoka, 1999
). A cDNA encoding
the reporter protein chloramphenicol acetyltransferase (CAT) was
cloned in negative-sense orientation between the 5´ and
3´ noncoding viral sequences. A T7 RNA polymerase promoter
sequence and a recognition sequence for a restriction enzyme that
allowed the formation of authentic viral 3´ ends flanked this
construct. In vitro transcription yielded virus-like RNA
that was subsequently mixed with purified polymerase and NP
proteins to reconstitute RNP complexes. These artificially
generated RNPs were transfected into eukaryotic cells infected with
helper influenza virus. The viruses that were generated contained
the virus-like RNA encoding CAT in addition to the eight influenza
vRNAs (Luytjes et al., 1989
). This achievement was quickly followed by
the first alteration of a viral gene (Enami et al.,
1990
).
Although allowing site-directed mutagenesis of an influenza viral
gene for the first time, this system relies on helper-virus
infection and strong selection systems are necessary to distinguish
the modified virus from the (wild-type) helper virus.
For most negative-sense RNA viruses, the ability to generate
infectious negative-sense RNA virus entirely from cloned cDNA was
preceded by the establishment of minireplicon systems. In these
systems, T7 RNA polymerase was used to synthesize negative-sense
vRNA derived from cDNA clones of internally deleted viral genomes.
Upon transfection of the vRNA into eukaryotic cells that had been
infected with helper virus, the artificially generated vRNAs were
rescued into viruses (Collins et al., 1991
; Park et
al., 1991
). Pattnaik & Wertz (1991
) first established
a plasmid-based minireplicon system by providing all five vesicular
stomatitis virus (VSV) viral proteins from protein expression
plasmids. Experience with these different approaches demonstrated
that the RNA-dependent RNA polymerase L, the nucleoprotein N and
the phosphoprotein P are essential for the amplification of rhabdo-
and paramyxovirus genomes (Pattnaik & Wertz, 1990
, 1991
; Collins et
al., 1991
; Conzelmann et al., 1991
; Park et
al., 1991
; Calain et al., 1992
; Dimock &
Collins, 1993
; Sidhu et al., 1995
). For Marburg virus
(family Filoviridae), L, NP and VP35 (which is believed to
be the equivalent of the phosphoprotein) constitute the minimal
replication unit (Muhlberger et al., 1998
), while the VP30
protein is additionally required for replication of the closely
related Ebola virus (Muhlberger et al., 1999
). In contrast, two
proteins, L and NP, are sufficient for the transcription and
replication of bunyavirus (Dunn et al., 1995
; Lopez et
al., 1995
; Flick & Pettersson, 2001
) or arenavirus (Lee
et al., 2000
) minigenomes. For influenza viruses, the
nucleoprotein and the three polymerase subunits PB2, PB1 and PA are
necessary and sufficient for the amplification of vRNAs (Honda
et al., 1987
, 1988
, 1990
; Szewczyk et al., 1988
; Parvin et
al., 1989
).
In 1994, Conzelmann and colleagues (Schnell et al.,
1994
)
generated recombinant rabies virus (RV), demonstrating for the
first time the feasibility of producing a negative-sense RNA virus
entirely from cloned cDNA (Fig.
2A). Cells were cotransfected with protein
expression constructs for the L, P and N proteins and with a cDNA
construct encoding the full-length RV antigenome, all under control
of the T7 RNA polymerase promoter. Infection with recombinant
vaccinia virus (VV), which provided T7 RNA polymerase, was the
final step needed to produce infectious RV. The key element to this
success was the synthesis of a positive-sense antigenomic RNA from
cloned DNA. Positive-sense antigenomic RNA, in contrast to
negative-sense genomic RNA, cannot hybridize to positive-sense
mRNAs encoding the L, P and nucleoproteins and thus does not interfere
with virus generation. Moreover, the genomic RNAs of some
negative-sense RNA viruses contain stretches of uridine residues
followed by hairpin structures that resemble T7 RNA terminator
elements, which may cause premature abortion of T7 RNA polymerase
transcription (Whelan et al., 1995
). Since the initial
report by Schnell et al. (1994
), we have witnessed
the generation of an ever-growing number of rhabdo- and
paramyxoviruses by reverse genetics (Table
1) (reviewed by Conzelmann, 1996
, 1998
; Conzelmann &
Meyers, 1996
; Rose, 1996
; Roberts & Rose, 1998
, 1999
; Marriott &
Easton, 1999
; Nagai, 1999
; Nagai & Kato,
1999
; Munoz et
al., 2000
). Refinements of the original rescue
procedure included the expression of T7 RNA polymerase from stably
transfected cell lines (Radecke et al., 1995
), protein
expression plasmids (Lawson et al., 1995
) or heat shock
procedures to increase rescue efficiencies (Parks et al.,
1999
).
Recently, Ebola virus, a member of the family Filoviridae,
was also generated from cDNA (Volchkov et al., 2001
; Neumann et
al., 2002
). In contrast to these efforts with
positive-sense antigenomic RNA, a number of investigators have also
relied on negative-sense genomic RNA to produce Sendai virus (SeV)
(Kato et al., 1996
), human parainfluenza virus type 3 (hPIV3)
(Durbin et al., 1997a
) and Ebola virus (Neumann et al.,
2002
),
although with lower efficiencies.
Fig. 2. Systems
for the generation of negative-sense RNA viruses from cloned cDNA.
(A) Schematic diagram for the generation of nonsegmented
negative-sense RNA viruses. Cells are cotransfected with protein
expression plasmids for the N, P and L proteins and with a plasmid
containing a full-length viral cDNA, all under the control of the
T7 RNA polymerase promoter. Following infection with recombinant VV
encoding T7 RNA polymerase, vRNA is synthesized and the virus
replication cycle is initiated. (B) Schematic diagram for the
generation of influenza A virus. Cells are cotransfected with
plasmids that encode all eight vRNAs under the control of the RNA
polymerase I promoter. Cellular RNA polymerase I synthesizes vRNAs
that are replicated and transcribed by the viral polymerase and NP
proteins, all provided by protein expression plasmids.
Another breakthrough occurred in 1996 with the first generation of
a segmented negative-sense RNA virus from cloned cDNAs (Bridgen
& Elliott, 1996
). Following the approach outlined by
Schnell et al. (1994
), Bridgen & Elliott (1996
) created Bunyamwera
virus (family Bunyaviridae), thus demonstrating the
feasibility of artificially producing more than one vRNA.
The generation of influenza virus is far more complex than that of
nonsegmented negative-sense RNA viruses, as it requires eight vRNAs
as well as four proteins encoding the three polymerase
subunits and NP. Secondly, influenza virus replicates in the
nucleus of infected cells, so that the vRNAs and proteins have to
be delivered to this cellular compartment. A solution was provided
by Hobom and colleagues (Zobel et al., 1993
; Neumann et
al., 1994
), who established the RNA polymerase I
system for the intracellular synthesis of influenza vRNAs. RNA
polymerase I, a nucleolar enzyme, synthesizes ribosomal RNA, which,
like influenza virus RNA, does not contain 5´ cap or 3´
poly(A) structures. Hence, RNA polymerase I transcription of a cDNA
construct containing an influenza viral cDNA, flanked by RNA
polymerase I promoter and terminator sequences, results in
influenza vRNA synthesis. This system enabled the generation of
influenza virus from plasmids in 1999 (Neumann et al.,
1999
; Fodor
et al., 1999
; reviewed by Pekosz et al.,
1999
; Neumann
& Kawaoka, 2001
) (Fig.
2B). By transfecting eukaryotic cells with eight RNA
polymerase I plasmids encoding all vRNAs, together with protein
expression constructs for the polymerase and NP proteins (yielding
a total of 12 plasmids), one can now produce more than
108 infectious viruses per ml of supernatant derived 2
days after transfection. In a modified RNA polymerase I system,
both negative-sense vRNA and positive-sense mRNA can be synthesized
from the same template, thus reducing the number of plasmids
required (i.e. 8 instead of 12) (Hoffmann et al., 2000a
, b
; Hoffmann &
Webster, 2000
).
Thogoto virus, a second member of the family
Orthomyxoviridae, was recently generated from plasmids
(Wagner et al., 2001
). The genome of this tick-transmitted
orthomyxovirus consists of six segments of negative-sense RNA. The
RNA polymerase I system allowed the synthesis of all six vRNAs,
while the proteins required for transcription and replication were
expressed with the VV T7 RNA polymerase system.
Replication and transcription of nonsegmented negative-sense RNA viruses |
The genes of nonsegmented negative-sense RNA viruses are separated
by regulatory regions comprising a gene end signal (i.e.
transcription stop and polyadenylation), an intergenic region and a
gene start signal (i.e. transcription start signal) (Fig. 3). These signals are
both sufficient and necessary for gene transcription (Kuo et
al., 1996b
; Schnell et al., 1996b
). The gene
transcription units are flanked by so-called leader and trailer
regions, which contain the viral promoters for replication and
transcription.
Fig. 3. Genome
organization of nonsegmented negative-sense RNA viruses, as
exemplified by VSV. The coding regions for the nucleoprotein (N),
phosphoprotein (P), matrix protein (M), glycoprotein (G) and
polymerase protein (L) are separated by regulatory sequences that
contain a transcription stop (gene end) signal, a polyadenylation
signal, a nontranscribed intergenic region and a transcription
start (gene start) signal. Transcription units are flanked by a
leader (Le) and trailer (Tr) region that contain the genomic and
antigenomic viral promoters, respectively.
Gene end signals
In VSV, the gene end signal is highly conserved among all genes.
Alteration of the conserved tetranucleotide transcription stop
signal reduced the ability to terminate RNA transcription (Barr
et al., 1997a
; Hwang et al., 1998
) and affected
polyadenylation (Barr & Wertz, 2001
). Furthermore, the
tetranucleotide is functional only when juxtaposed to the uridine
stretch that functions as a polyadenylation signal (Barr et
al., 1997a
) and when positioned at a minimal distance
from the gene start sequence (Whelan et al., 2000
). In addition to
the tetranucleotide, the uridine stretch constituting the
polyadenylation signal is critical for termination, since its
deletion, shortening or interruption resulted in read-through
transcripts (Barr et al., 1997a
; Hwang et
al., 1998
). In contrast to VSV, the polyuridine
tracts of simian virus type 5 (SV5) are of variable lengths and
nucleotide insertions or deletions did not affect termination but
did affect reinitiation at the downstream gene (Rassa et
al., 2000
).
Intergenic region
The intergenic regions of nonsegmented negative-sense RNA viruses
are highly variable, consisting of a conserved dinucleotide (VSV),
trinucleotide (morbilli- and respiroviruses) or regions of up to
143 nt (filoviruses). For VSV, alterations of the conserved
dinucleotide affected the efficiency of termination of the upstream
mRNA as well as the mRNA levels of the downstream gene (Barr et
al., 1997b
; Stillman & Whitt, 1997
, 1998
). The various
lengths of the intergenic regions of RV correlate with
transcriptional attenuation (Finke et al., 2000
), whereas the
diverse intergenic regions of RSV do not modulate gene expression
(Kuo et al., 1996a
; Bukreyev et al., 2000a
). Similarly,
nucleotide insertions into the middle of the SV5 MF
intergenic region did not affect transcription termination and/or
initiation; however, replacement of the entire MF
intergenic region with nonviral sequences abolished termination of
M gene transcription (Rassa & Parks, 1999
).
Gene start signals
The transcription start signals are identical for all VSV genes.
Mutational analyses revealed that the first three nucleotides of
the 5´ start sequence are critical for gene expression
(Stillman & Whitt, 1997
). Interestingly, mutations in this
sequence did not abolish the initiation of transcription per se but
affected polymerase processivity as well as capping and/or
methylation of mRNA transcripts (Stillman & Whitt, 1999
). Further insights
into transcription initiation came from RSV minigenomes containing
a nucleotide replacement at position 1 of the gene start signal.
mRNAs were synthesized with the predicted nucleotide at the 5´
end; however, a subpopulation of mRNAs contained the (nontemplated)
parental wild-type nucleotide at the 5´ end (Kuo et
al., 1997
). Thus, the polymerase complex may have a
preference for the wild-type nucleotide at the start position. For
SeV, the start sequence of the F gene was significantly weaker than
the P, M and HN start signals (Kato et al., 1999
). A recombinant
SeV, whose F gene start signal was replaced with the strong P, M or
HN gene start signal replicated faster and was more virulent than
wild-type virus in mice (Kato et al., 1999
).
Replication
The switch from transcription to replication was once thought to be
triggered by increasing amounts of soluble N protein, which is
required to encapsidate replicating RNA. However, increasing levels
of RSV N did not alter the ratio between transcription and
replication (Fearns et al., 1997
). Mutational
analysis of the genomic and antigenomic promoter elements has
resulted in the generation of minireplicons that are replicated
poorly but are transcribed well, and vice versa. Rather than
switching between transcription and replication, a balance between
these two processes may be controlled by the different activities
of the genomic and antigenomic promoters (Li & Pattnaik,
1999
; Whelan
& Wertz, 1999
; Hoffman & Banerjee, 2000b
). This idea is
supported by experiments analysing a copy-back ambisense RV
minigenome (Finke & Conzelmann, 1999
).
For rhabdo- and paramyxoviruses, the minimal promoter sequences
have been mapped to the 1244 3´-terminal nucleotides
of VSV (Li & Pattnaik, 1997
, 1999
; Whelan & Wertz, 1999
), SeV (Tapparel
& Roux, 1996
), hPIV3 (Hoffman & Banerjee,
2000b
) or RSV
(Kuo et al., 1996b
; Fearns et al., 2000
) genomes or
antigenomes. For VSV, the extent of complementarity between the
3´ and 5´ ends can affect the level of replication (Wertz
et al., 1994
; Whelan & Wertz, 1999
). However, other
reports indicate that the base pairing potential between the
3´ and 5´ ends of the vRNAs does not affect replication
efficiency; rather, the primary sequence of the promoter elements
determines their strength (Tapparel & Roux, 1996
; Hoffman &
Banerjee, 2000a
). In contrast to rhabdoviruses, the
genomic and antigenomic promoters of paramyxoviruses (with the
possible exception of RSV) contain second promoter elements (Pelet
et al., 1996
; Murphy et al., 1998
; Tapparel et
al., 1998
; Murphy & Parks, 1999
; Hoffman &
Banerjee, 2000b
). These elements are composed of three
hexamers that contain a repeated motif (Tapparel et al.,
1998
; Murphy
& Parks, 1999
; Hoffman & Banerjee, 2000b
). Nucleotide
deletions or insertions between the two promoter elements were
detrimental for their activity, indicating that their relative
spacing is critical (Murphy et al., 1998
; Tapparel et
al., 1998
). In structural models, the two elements
are positioned on the same face of the RNP helix (Murphy et
al., 1998
; Tapparel et al., 1998
). Thus, upon
alteration of their spacing, the interaction of the polymerase
complex with the two promoter elements may be abrogated. Recent
studies also identified a third promoter element for SV5 (located
between the first and the second element) that is required for
optimal replication (Keller et al., 2001
).
The genomic promoter localizes to the 3´ end of the
negative-sense vRNA and hence serves as a promoter for both
replication and transcription. Distinct elements have been
identified in the genomic promoter that are required for
transcription but not for replication, or vice versa (Li &
Pattnaik, 1999
; Whelan & Wertz, 1999
; Peeples &
Collins, 2000
). In contrast, the antigenomic promoter
that localizes to the 3´ end of the positive-sense antigenome
functions in replication only.
'Rule of six'
Efficient replication for most paramyxoviruses was observed only
when the total number of nucleotides was divisible by six (dubbed
the 'rule of six') (reviewed by Kolakofsky et
al., 1998
). The nucleoprotein interacts with exactly
6 nt, and the rule of six seems to depend on the recognition of
nucleotides positioned in the proper N-phase context
(Vulliemoz & Roux, 2001
). Extensive mutagenesis revealed that the
rule of six seems to follow taxonomic grouping, being apparently
specific to the rubula- (Murphy & Parks, 1997
; He & Lamb,
1999
; Peeters
et al., 2000
; Kawano et al., 2001
), respiro- (Calain
& Roux, 1993
, 1995
; Harty & Palese, 1995
; Hausmann et
al., 1996
; Durbin et al., 1997b
) and morbillivirus
(Radecke et al., 1995
; Sidhu et al., 1995
) genera of the
family Paramyxoviridae, although with different stringency.
The rule of six is not followed by RSV (Samal & Collins,
1996
) nor
does it apply to VSV (Pattnaik et al., 1995
).
Replication and transcription of influenza viruses |
Each RNA segment of the segmented negative-sense RNA viruses
constitutes a separate transcription unit. The 5´ 13-terminal
and the 3´ 12-terminal nucleotides are highly conserved among
the eight viral segments of influenza A viruses and form the basic
promoter for transcription and replication (Parvin et al.,
1989
;
Yamanaka et al., 1991
; Li & Palese, 1992
; Seong &
Brownlee, 1992a
, b
; Piccone et al., 1993
). Extensive
mutational analyses revealed the contributions of individual
nucleotides to in vitro and in vivo promoter activity
(Li & Palese, 1992
; Piccone et al., 1993
; Fodor et
al., 1994
, 1995
; Neumann et al., 1994
; Neumann &
Hobom, 1995
; Pritlove et al., 1995
; Flick et
al., 1996
; Kim et al., 1997
). In contrast to
the promoter elements of nonsegmented negative-sense RNA viruses,
which consist of specific sequence elements at the 3´ ends of
the vRNA or cRNA, the genomic and antigenomic promoters of
influenza viruses are composed of both the 5´ and the 3´
ends of the vRNA or complementary RNA (cRNA), respectively (Fodor
et al., 1993
, 1994
; Tiley et al., 1994
; Neumann &
Hobom, 1995
; Pritlove et al., 1995
). The influenza
viral promoters can be divided into two elements: a terminal
element (nt 19 at the 3´ and 5´ ends of the
vRNA or cRNA) and a distal element (nt 1015 and
1116 at the 3´ and 5´ ends of the vRNA), which
are connected by a flexible joint formed by an unpaired nucleotide
(Fodor et al., 1995
; Neumann & Hobom, 1995
; Flick et
al., 1996
). Within the terminal element, the nature
of the nucleotide sequence is critical, whereas in the distal
element, base-pairing between the 3´ and 5´ ends of the
vRNA or cRNA is crucial (Fodor et al., 1994
, 1995
; Neumann &
Hobom, 1995
; Flick et al., 1996
; Kim et al.,
1997
). Three
promoter models, the 'panhandle' model (Hsu et
al., 1987
), the 'fork' model (Fodor
et al., 1994
, 1995
; Kim et al., 1997
) and the
'corkscrew' model (Flick et al., 1996
; Flick & Hobom,
1999
), have
been proposed. The panhandle model suggests formation of a
partially double-stranded structure in the entire promoter region,
while the fork model proposes double-strand formation for the
distal promoter element only. Recent data favour the corkscrew
model, which predicts base-pairing between nucleotides at the
5´ or 3´ end rather than base-pairing between the 5´
and 3´ end (Flick & Hobom, 1999
; Pritlove et
al., 1999
; Leahy et al., 2001a
, b
). The influenza B
virus promoter has structural features similar to its influenza A
virus counterpart (Lee & Seong, 1996
).
During transcription, the polymerase complex proceeds until it
encounters the polyadenylation signal, which is formed by a uridine
stretch located adjacent to the promoter element. At the uridine
stretch, the polymerase complex 'stutters', resulting
in polyadenylation (Zheng et al., 1999
; Poon et
al., 2000
). Efficient polyadenylation requires that
an uninterrupted uridine stretch be located 1522 nt from
the 5´ end of the vRNA (Luo et al., 1991
; Li & Palese,
1994
).
The terminal promoter elements contain the information sufficient
for replication and transcription; however, signals that modulate
these processes seem to be located in the noncoding regions that
lie between the promoter and the start or stop codon, respectively.
Recombinant viruses with deletions, insertions or mutations in
these regions have been generated (Garcia-Sastre et al.,
1994b
; Barclay
& Palese, 1995
; Bergmann & Muster, 1996
; Zheng et
al., 1996
). Although these viruses were viable, some
had altered amounts of vRNA (Bergmann & Muster, 1996
; Zheng et
al., 1996
).
Phosphoprotein |
Although the RNA-dependent RNA polymerase L is believed to contain
all catalytic activities, phosphoprotein P is essential for vRNA
synthesis. Its interaction with the N protein keeps the latter in a
soluble form and imparts specificity to the N protein to
encapsidate viral, but not cellular, RNAs. Moreover, binding of P
to L allows efficient interaction of L with the NRNA template
(Horikami et al., 1992
). Both the amino- and the carboxy-terminal
domains of P are critical to execute these functions (De et
al., 2000
). For VSV P, replacement of single amino
acids in the carboxy-terminal region resulted in P mutants that
were defective in transcription but supported replication,
suggesting that the transcriptase and replicase complexes may not
be identical (Das et al., 1997
, 1999
).
Reverse genetics has also allowed researchers to address the
biological significance of P protein phosphorylation in the virus
life cycle. The hPIV3 P protein is phosphorylated by the cellular
protein kinase C isoform
(PKC-
) and growth of hPIV3 was
abrogated in the presence of a PKC-
inhibitor (De et al.,
1995
). The
VSV P protein is phosphorylated at several sites in both the amino-
and the carboxy-terminal domains. Replacement of the amino-terminal
phosphorylation sites affected transcription (Spadafora et
al., 1996
; Pattnaik et al., 1997
), while the
carboxy-terminal phosphorylation sites are necessary for optimal
replication (Hwang et al., 1999
). In contrast,
replacement of the SeV P phosphorylation site did not affect virus
replication in cell culture nor did it affect virus pathogenicity
in mice (Hu et al., 1999
).
Matrix proteins |
Matrix proteins constitute the major structural component of the
virion shell. Both the VSV and influenza A virus matrix proteins
are essential for virion formation and have the ability to induce
particle formation (Sakaguchi et al., 1999
; Gomez-Puertas
et al., 2000
; Jayakar et al., 2000
; Latham &
Galarza, 2001
). In contrast, recombinant MV and RV
lacking the M gene (
M) have been generated (Cathomen et
al., 1998a
; Spielhofer et al., 1998
; Mebatsion et
al., 1999
). MV
M was more efficient in inducing cell
fusion than its wild-type counterpart (Cathomen et al.,
1998a
).
Interestingly, MVs isolated from patients with subacute sclerosing
panencephalitis are often defective in M gene expression (Baczko
et al., 1984
).
The SeV M protein is phosphorylated in infected cells but not in
mature virions; therefore, M phosphorylation was thought to
regulate processes such as virion assembly or budding. However, a
recombinant SeV containing a mutation in M that abrogated
phosphorylation was indistinguishable from wild-type virus both
in vitro and in vivo (Sakaguchi et al.,
1997
).
Influenza A virus M2 protein |
The influenza virus M2 protein is expressed from a spliced mRNA
encoded by segment 7. It is an integral membrane protein that
executes functions early and late in the virus replication cycle.
In late endosomes, the M2 ion channel activity allows proton influx
into the virions, thus causing a pH shift that leads to the
dissociation of the M1 protein from RNP complexes. Late in
infection, proton influx through the M2 ion channel raises the pH
in the trans-Golgi network, thereby preventing acid-induced
conformational changes in intracellularly cleaved haemagglutinin
(HA). Reverse genetics experiments indicated that the M2
cytoplasmic tail is critical for virus replication, most likely
through its interaction with other viral proteins (Castrucci &
Kawaoka, 1995
); in contrast, replacement of either the
serine residue at position 64 (the primary M2 phosphorylation site)
or the conserved cysteine residues did not compromise the
recombinant viruses in vitro or in vivo (Castrucci
et al., 1997
; Thomas et al., 1998
).
M2 ion channel activity was long considered essential for virus
replication. However, an influenza virus with a deletion in the M2
transmembrane domain was growth-impaired but still replicated in
cell culture (Takeda et al., 2002
). Another
recombinant virus whose M2 transmembrane and cytoplasmic domains
were deleted grew in cell culture but not in mice (Watanabe et
al., 2001
). Thus, M2 ion channel activity is not
essential for virus replication in vitro but it is required
for in vivo virus replication.
Influenza A virus NS2 protein |
The influenza A virus NS2 protein is translated from a spliced mRNA
encoded by segment 8. It contains a classical nuclear export signal
(NES) in its amino-terminal region and mediates nuclear export when
fused to a reporter construct (O'Neill et al.,
1998
).
Moreover, NS2 interacted with the cellular nuclear export factor
CRM1, which mediates export of proteins with classical NESs
(Neumann et al., 2000a
). In cells infected with virus-like
particles (VLPs) that lacked NS2 or encoded an NS2 protein with an
altered NES, viral RNP complexes were retained in the nucleus, thus
supporting the role of NS2 as the virus nuclear export factor of
viral RNP complexes (Neumann et al., 2000a
).
Viral glycoproteins |
Rhabdoviruses possess a single glycoprotein (G), which is involved in cell attachment and pH-dependent fusion of the viral and cellular membranes. For orthomyxoviruses, the HA protein binds to cellular receptors and mediates the pH-dependent fusion event, while the sialidase activity of the neuraminidase (NA) protein is required to prevent self-aggregation and to prevent viruses from rebinding to cells from which they were released. In contrast to orthomyxoviruses, different proteins encode the attachment and fusion activities for paramyxoviruses. The G (rhabdo- and pneumoviruses), H (morbilliviruses) or HN (respiro- and rubulaviruses) glycoprotein brings about binding to cellular receptors. The fusion (F) protein is involved in the fusion of the viral membrane with the endosomal or plasma membrane, resulting in the release of RNP complexes into the cytoplasm of infected cells. However, for most paramyxoviruses, coexpression of both F and HN is required for fusion activity, indicating a fusion-promoting activity of the HN protein.
Deletion of glycoproteins
A human RSV (hRSV) strain passaged at progressively lower
temperatures contains a deletion of the G gene (as well as of the
SH gene), demonstrating that these proteins are not essential for
virus replication (Karron et al., 1997a
); however, a
recombinant hRSV lacking G was attenuated in its replication
(Techaarpornkul et al., 2001
). Similarly, reverse genetics allowed the
generation of bovine RSV (bRSV) that lacked the G and/or SH genes
(Karger et al., 2001
). The artificially generated viruses did
not display an altered morphology and grew to titres similar to
wild-type virus in cell culture (Karger et al., 2001
). Thus, for hRSV
and bRSV, the F protein seems to be able to function as an
attachment protein, in addition to its critical role in cell fusion
(Kahn et al., 1999
); however, the G protein is needed for
efficient growth of hRSV in mice (Teng et al., 2001
).
Proteolytic cleavage of viral glycoproteins determines pathogenicity
Proteolytic cleavage of the viral fusion protein is one of the key
factors determining virus pathogenicity. Many of the fusion
proteins are synthesized as inactive precursor proteins that are
cleaved by host cell proteases to generate two disulfide-linked
subunits. It is this activated form that initiates the fusion of
the viral and cellular membranes. In avian influenza virus and NDV,
fusion proteins (HA for the former virus) possess multiple basic
amino acids at their cleavage site that are recognized by
ubiquitous cellular proteases, such as furin (Stieneke-Grober et
al., 1992
). In contrast, the fusion proteins of
avirulent viruses contain single basic residues at this site that
are not recognized by the ubiquitous proteases, resulting in local
infections. For avian influenza A viruses, researchers established
a direct link between HA cleavability and virus pathogenicity and
demonstrated that two features were critical for HA cleavage: the
length and composition of the basic amino acid stretch as well as
the presence or absence of a nearby carbohydrate side chain
(Kawaoka & Webster, 1988
, 1989
; Horimoto & Kawaoka, 1994
; Klenk &
Garten, 1994
). The importance of multiple basic amino
acids at the F cleavage site has also been demonstrated for NDV
(Peeters et al., 1999
). For MV, alteration of the multibasic
furin cleavage site of the F protein resulted in recombinant virus
that did not induce neural disease, in contrast to wild-type virus
(Maisner et al., 2000
). Reverse genetics also allowed
researchers to replace the multibasic furin-recognition motif of
the Ebola virus GP protein with nonbasic amino acids.
Interestingly, the recovered virus grew to titres similar to
wild-type virus in cell culture (Neumann et al., 2002
). Hence,
furin-mediated cleavage of the Ebola virus GP protein is not
critical for virus replication in cell culture; however, it may be
required for virus propagation in animals.
Cross et al. (2001
) determined the significance of the large
hydrophobic amino acids of the influenza A virus fusion peptide,
which become exposed after HA cleavage by host cell proteases.
Replacement of hydrophobic amino acids with alanine but not with
glycine yielded viruses capable of replication. Moreover, cell
culture propagation of viruses containing alanine substitutions in
the fusion peptide resulted in pseudoreversion to valine,
indicating a preference for hydrophobic amino acids with a large
side chain in this region (Cross et al., 2001
).
Viral glycoproteins determine host cell tropism
Viral glycoproteins are a major factor in determining host cell
tropism. To study MV cell tropism, two groups of investigators
generated recombinant viruses that contained the H and/or F
proteins derived from a lymphotropic wild-type strain (WTF) of MV
in the background of the tissue culture-adapted Edmonston vaccine
strain (Johnston et al., 1999
; Ohgimoto et al., 2001
). These studies
demonstrated that the WTF H protein can confer the ability to
viruses to replicate in secondary lymphoid tissues in cotton rats
(Ohgimoto et al., 2001
). Furthermore, replacement of the
Edmonston strain H gene with that of the wild-type IC-B strain, or
vice versa, altered the host cell specificity of the resulting
recombinant viruses (Takeuchi et al., 2002
).
MV has the potential to cause neurological complications.
Substitution of the H gene of the Edmonston B vaccine strain with
its counterpart derived from a rodent brain-adapted strain resulted
in a neurovirulent, recombinant virus (Duprex et al.,
1999
).
However, infection progressed more slowly in mice infected with
recombinant virus compared to the rodent brain-adapted parental
strain. Thus, the H protein is an important (although not the only)
factor required for neurovirulence.
Two studies addressed the significance of the RV G protein for
neurovirulence by introducing G protein derived from neurovirulent
strains into the genetic background of less neurotropic strains.
While G is the major determinant of neurotropism, other factors
seem to contribute to pathogenicity (Morimoto et al.,
2000
; Ito
et al., 2001
). Similarly, reciprocal exchange of the
canine distemper virus and MV H proteins demonstrated that they are
important determinants of the growth characteristics and the cell
tropism of the recombinant viruses (von Messling et al.,
2001
).
Cytoplasmic tails of glycoproteins
The cytoplasmic tails of the attachment and fusion proteins are
thought to be critical for virus assembly, most likely through
their interaction(s) with internal viral proteins. The influenza A
virus HA protein contains conserved, palmytilated cysteine residues
in both its cytoplasmic tail and its transmembrane domain.
Replacements of these amino acids yielded conflicting results.
While two groups reported the generation of influenza viruses
lacking the conserved cysteine residues (Jin et al.,
1996
; Lin
et al., 1997
), another group failed to generate such
mutants (Zurcher et al., 1994
). However, these findings may simply
reflect the fact that the different HA subtypes used for these
studies differ in their abilities to tolerate amino acid
replacements. Analysis of recombinant influenza A viruses whose NA
tail was replaced with that of influenza B virus, or whose NA
and/or HA cytoplasmic tails were deleted, demonstrated that the HA
and NA cytoplasmic tails are not absolutely required for virus
propagation (Bilsel et al., 1993
; Jin et al.,
1994
,
1997
;
Garcia-Sastre & Palese, 1995
; Mitnaul et al., 1996
). However, deletion
of the cytoplasmic tails affected the particle shape, reduced the
vRNA:protein ratio and attenuated the recombinant viruses in mice,
suggesting that the HA and NA cytoplasmic tails affect virion
formation and confer a growth advantage to influenza virus
(Garcia-Sastre & Palese, 1995
; Mitnaul et al., 1996
; Jin et al.,
1997
; Zhang
et al., 2000
). Similarly, truncation of the cytoplasmic
tails of the SV5 HN (Schmitt et al., 1999
) or the SeV HN or F
proteins (Fouillot-Coriou & Roux, 2000
) resulted in
recombinant viruses that were impaired in their growth. Recombinant
SV5 or MV with alterations in their HN, F and/or H cytoplasmic
tails displayed enhanced cell-to-cell fusion (Cathomen et
al., 1998b
; Schmitt et al., 1999
). In addition,
basolateral targeting signals have been identified in the
cytoplasmic tails of the MV H and F proteins (Moll et al.,
2001
). For
VSV, replacement of the transmembrane and cytoplasmic domains of G
with those of human CD4 protein did not affect virion budding
(Schnell et al., 1998
). Further analysis demonstrated that a
short cytoplasmic tail without specific sequence requirements is
sufficient to promote efficient budding (Schnell et al.,
1998
). In
addition, a short region in the extracellular stem of G was
identified that confers efficient virus assembly (Robison &
Whitt, 2000
).
Influenza A virus NA protein
The influenza A virus NA protein is composed of a stalk of variable
length and amino acid composition that connects the transmembrane
domain with the globular head, which contains the enzymatic centre.
Recombinant viruses with altered stalks demonstrated that viruses
with longer stalks replicated to higher titres in eggs (Castrucci
et al., 1992
, 1994
; Castrucci & Kawaoka, 1993
; Luo et al.,
1993
).
Stalk-less viruses were not restricted in their growth in tissue
culture but failed to grow in eggs and displayed attenuated
phenotypes in mice (Castrucci & Kawaoka, 1993
). These findings
indicate that the NA stalk is not essential for virus replication
but modulates the host range, probably by affecting the enzymatic
activity of NA.
The NA protein of the neurovirulent influenza A/WSN/33 virus strain
differs from all other influenza A virus NAs by virtue of the lack
of a glycosylation site at amino acid position 130. To assess the
contribution of this glycosylation site for neurovirulence, Li
et al. (1993c
) generated a recombinant virus containing
the conserved glycosylation site. The recombinant virus, unlike its
parent, did not replicate in mouse brain, demonstrating that this
NA glycosylation site is critical for neurovirulence. Further
studies demonstrated that influenza A/WSN/33 virus NA binds
plasminogen, which, after being activated to plasmin, cleaves HA
(Goto & Kawaoka, 1998
). In vitro and in vivo
studies revealed that two NA structural features, a
carboxy-terminal lysine residue and the lack of the glycosylation
site at position 130, were critical for plasminogen binding (Goto
& Kawaoka, 1998
; Goto et al., 2001
). Recombinant
viruses in which either one of these features was altered did not
replicate in the brain of infected mice, in contrast to wild-type
influenza A/WSN/33 virus (Goto et al., 2001
).
Ebola virus glycoprotein GP
Expression of the complete, membrane-anchored form of the Ebola
virus glycoprotein is achieved through editing of its mRNA, while
the unedited mRNA is translated to the secreted glycoprotein (sGP).
About 80 % of the GP mRNAs remain unedited, resulting in high
amounts of sGP in patient sera (Volchkov et al., 1995
; Sanchez et
al., 1996
). In contrast, the closely related Marburg
virus translates its GP from an unedited mRNA and does not express
sGP. To address the role of transcriptional editing and sGP
synthesis in Ebola virus replication, Volchkov et al.
(2001
)
generated a virus that expressed GP from an unedited mRNA. The
mutant virus expressed higher levels of GP and was more cytotoxic
than its wild-type counterpart. This finding suggested that
transcriptional editing might control the expression levels of GP
and hence the cytotoxic effects of this protein.
Accessory proteins |
Accessory proteins are not essential for basic levels of virus replication and/or transcription but regulate or modulate a variety of steps in the virus life cycle. For VSV and members of the Paramyxovirinae subfamily, accessory proteins are encoded by the P gene and expressed through RNA editing and/or they are expressed from a second reading frame overlapping the P gene. In contrast, accessory proteins of pneumoviruses are expressed from separate, additional transcription units, involving the use of overlapping reading frames.
Influenza A virus NS1 protein
Among negative-sense RNA viruses, the NS1 protein was the first
protein shown to counteract the cellular interferon (IFN) response
(reviewed by Garcia-Sastre, 2001). In 1998, Garcia-Sastre et
al. (1998
) generated a
NS1 influenza A virus that
was restricted in its replication in MDCK cells and mice (hence, in
systems with a functional IFN response). In contrast,
NS1
influenza A virus replicated to titres comparable to wild-type
virus in IFN-compromised systems, such as Vero cells or
STAT-1/ mice (Garcia-Sastre et
al., 1998
; Talon et al., 2000b
). NS1 interferes
with several cellular pathways to combat the antiviral IFN
response. Double-stranded RNA-activated protein kinase (PKR)
indirectly stimulates type I IFN, thus playing a critical role in
antiviral responses. NS1 binds to RNA (Yoshida et al.,
1981
),
thereby suppressing PKR (Hatada et al., 1999
; Bergmann et
al., 2000
). In line with these findings,
NS1
influenza A virus, but not wild-type virus, stimulated the
expression of a reporter gene under the control of an
IFN-responsive promoter (Garcia-Sastre et al., 1998
). Moreover,
NS1
influenza A virus activated NF-
B (Wang et al., 2000
), which
transactivates IFN-
-regulated promoters and IFN regulatory factor
3 (Talon et al., 2000a
), which forms a transcription complex with
signal transducer and transcriptional activator-1 (STAT-1) and
STAT-2.
In a study to assess the contribution of NS1 to the extreme
pathogenicity of the 'Spanish Flu' virus, which killed
2040 million people in 1918 and 1919, recombinant viruses
were generated that contained the NS1 or both the NS1 and the NS2
encoding regions of the Spanish Flu virus in an influenza A/WSN/33
virus genetic background (Basler et al., 2001
). The recombinant
viruses did not kill mice, in contrast to the parental influenza
A/WSN/33 virus, and the results were considered inconclusive
because of gene constellation effects.
C proteins
C proteins (reviewed by Nagai, 1999
; Nagai & Kato, 1999
) are expressed by
VSV, morbilli- and respiroviruses from a second reading frame that
overlaps the 5´ region of the P gene in the +1 open reading
frame (ORF). Morbilliviruses and hPIV3 encode one C protein. For
VSV and SeV, ribosomal choice results in the use of different
initiation codons (all in the same reading frame), yielding a
nested set of two C proteins (designated C' and C) for VSV or
a nested set of four proteins (C´, C, Y1 and Y2) for SeV
(Latorre et al., 1998b
). C proteins share a common
carboxy-terminal region but differ in their amino termini.
The relative importance of C proteins for the virus life cycle
differs among rhabdoviruses and paramyxoviruses. VSV defective in C
protein expression is indistinguishable from wild-type virus in
cell culture (Kretzschmar et al., 1996
), while alteration
of the C proteins of hPIV3 (Durbin et al., 1999
) and RPV (Baron
& Barrett, 2000
) restricted virus replication in cell
culture and/or experimental animals. The growth characteristics of
MV
C depended on the cell line tested (Radecke & Billeter,
1996
;
Escoffier et al., 1999
) and MV deficient in C protein expression
was attenuated in mice (Patterson et al., 2000
). For SeV, C´
protein-deficient virus was as virulent as wild-type virus in mice,
while C protein-deficient virus was highly attenuated in these
animals (Latorre et al., 1998a
); this phenotype
was attributed to a single amino acid substitution in the SeV C
protein (Garcin et al., 1997
). Recombinant SeV deficient in the
expression of all four C proteins was severely attenuated and grew
to titres of 4 log units lower than those observed for the
wild-type counterpart (Kurotani et al., 1998
). Most importantly,
SeV employs all four C proteins to diminish the induction of
IFN-stimulated gene expression (Didcock et al., 1999a
; Garcin et
al., 1999
, 2000
; Kato et al., 2001
), as demonstrated
by the experimental requirement to ablate the entire complement of
C proteins to fully suppress IFN-
-stimulated gene expression
(Gotoh et al., 1999
). While all four C proteins interfered
with STAT-1 phosphorylation, only C and C´ proteins induced
its instability (Garcin et al., 2001
).
The various C proteins have both positive and negative effects on
virus replication and seem to execute their function(s) in a highly
coordinated manner (Cadd et al., 1996
; Garcin et
al., 1997
; Tapparel et al., 1997
; Kurotani et
al., 1998
; Latorre et al., 1998a
; Baron &
Barrett, 2000![]()