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.

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.

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).

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).

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 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).

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.

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).

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 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 20–40 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 MVC 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