Journal of General Virology (2002),
83, 2635-2662.
© 2002 Society for General Microbiology
A decade after the generation of a negative-sense RNA virus from cloned cDNA – what have we learned?
Gabriele Neumann1,
Michael A. Whitt2 and
Yoshihiro Kawaoka1,3,4
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706, USA1
Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN, USA2
Institute of Medical Science, University of Tokyo, Tokyo, Japan3
CREST, Japan Science and Technology Corporation, Japan4
Author for correspondence: Yoshihiro Kawaoka. Fax +1 608 265 5622. e-mail kawaokay{at}svm.vetmed.wisc.edu
 |
Abstract
|
|---|
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
) and have revolutionized research on these viruses. In this review, we will describe these experimental systems and discuss their contributions to our understanding of virus replication, the pathogenicity of negative-sense RNA viruses and the development of live attenuated virus vaccines.
|
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 ).
View larger version (53K):
[in this window]
[in a new window]
|
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.
View larger version (61K):
[in this window]
[in a new window]
|
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.
View larger version (21K):
[in this window]
[in a new window]
|
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 M–F intergenic region did not affect transcription termination and/or initiation; however, replacement of the entire M–F 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 12–44 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 1–9 at the 3' and 5' ends of the vRNA or cRNA) and a distal element (nt 10–15 and 11–16 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 15–22 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 N–RNA 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 ). MVM 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
TOP
Abstract
Introduction
