The interferon (IFN) system is the first line of defense against viral infection in mammals. This system is designed to block the spread of virus infection in the body. Detection of virus infection by intracellular receptors (sensors) results in activation of multiple pathways, leading to the production of IFN-α/β, which activates the Jak/STAT signaling pathway and stimulates transcription of a variety of antiviral genes in neighboring cells. Many of the pathogenic viruses, however, have evolved mechanisms to evade the IFN system by blocking IFN synthesis, the signaling pathway or both. In the case of paramyxoviruses, accessory proteins encoded by the P/V/C gene mediate antagonism of innate immunity. Mutant viruses unable to express the accessory proteins are attenuated in vivo, indicating the critical role of the accessory proteins in viral growth and pathogenicity. Although most of the paramyxoviruses are able to antagonize IFN activity, the mechanism and proteins responsible for the antagonism differs among the viruses. This minireview describes a variety of anti-innate activities mediated by accessory proteins of viruses belonging to the Paramyxoviridae subfamily, with the exception of henipavirus, which will be covered in a different section of this issue.

Family Paramyxoviridae, in the order Mononegavirales, is composed of a wide range of “classic” and “emerging” viruses of medical or veterinary significance. The family is categorized into two subfamilies, Paramyxovirinae and Pneumovirinae, according to several criteria, such as morphology, genomic organization, role of the encoded viral proteins and the sequence relationship between these proteins. Subfamily Paramxyxovirinae, which is the focus of this review, consists of five genera: Respirovirus, Morbillivirus, Rubulavirus, Avulavirus and Henipavirus. The well-studied viruses belonging to this subfamily include Sendai virus (SeV) and human parainfluenza virus types 1 and 3 (hPIV1 and 3)(Respirovirus), parainfluenza virus 5 (PIV5, formerly SV5) and mumps virus (MuV)(Rubulavirus), measles virus (MeV)(Morbillivirus), Newcastle disease virus (NDV)(Avulavirus) and Hendra virus (HeV) and Nipah virus (NiV) (Henipavirus).

Paramyxoviruses contain nonsegmented negative-strand RNA genomes. Their genomes are 15-19 kB in length, and the genomes contain six to ten tandemly linked genes. Each gene is flanked by conserved transcriptional control sequences, which are linked by an intergenic sequence. The viral mRNAs are transcribed monocistronically, resulting in one protein being expressed from a single mRNA, except the P gene, as described below. Viral RNA is encapsidated with nucleocapsid (N) protein, which is associated with a polymerase complex composed of phospho- (P) and large (L) proteins. The virion contains a lipid bilayer envelope that is derived from the plasma membrane of the host cell. Inserted into the viral envelope are two surface glycoproteins responsible for virus attachment and membrane fusion. Residing between the envelope and the core is the viral matrix protein that is important in virion assembly and incorporation of nucleocapsid into progeny virions. Paramyxovirus replication takes place entirely in the cytoplasm, and progeny virions are formed at the plasma membrane of infected cells [1].

Most mRNAs of Paramyxovirinae are translated into one protein, with the exception of transcripts from the P gene, which express accessory proteins, in addition to the P protein. Non-rubulavirus Paramyxovirinae P proteins are expressed from a faithful transcript of the P gene, and all the viruses in this group express at least one accessory protein (Figure 1). They are expressed by two different strategies: one by RNA editing for V/W/D expression, and the other by overlapping open reading frame (ORF) for C expression. All the viruses of Paramyxovirinae encode a characteristic editing site in the P gene except hPIV1, and the number of inserted G residues, as well as the frequency of G nucleotide insertion is determined by sequences surrounding and within the editing site [2-4]. All the V proteins are expressed from edited RNA containing a single G residue inserted at the editing site, except Rubulavirus that produces V protein from intact unedited mRNA, and edited RNA with 2 inserted G residues translate the P protein. The V protein is a ~25- to ~30-kDa polypeptide that shares an N-terminal domain with the P protein but has a distinct C-terminal domain as a result of frame shift due to inserted G nucleotide(s). The C-terminal V-specific domain is highly conserved among related paramyxoviruses, with spaced histidine and cysteine residues forming a zinc-binding domain, and indeed binds two atoms of zinc molecules per V protein [5-7]. Respiroviruses, Morbilliviruses, Avulaviruses and Henipaviruses express W/D proteins from mRNAs with two inserted G residues. The relative abundance of these proteins is low compared to the total P mRNA [8]. The function of the W and D proteins is not clear, however, disruption of both V and D proteins of hPIV3 was shown to affect in vivo hPIV3 replication [9], while disruption of the W protein had little effect on NDV growth in cultured cells [10].

In addition to RNA editing, Respiro-, Morbilli- and Henipaviruses use alternative translation initiation codons to yield the C proteins (Figure 1). The number of expressed C proteins differs among paramyxoviruses, ranging from 1 to 4. SeV expresses a nested set of C proteins, C’, C, Y1 and Y2, that range in size from 175 to 215 residues. The ORFs are located upstream of the editing site and overlap the P ORF in the +1 frame. The initiation codons of C’, C, Y1, and Y2 are ACG⁸¹, AUG¹¹⁴, AUG¹⁸³ and AUG²⁰¹, respectively. Translation of each of the C’, C, Y1, and Y2 ORFs is terminated at the same downstream stop codon; therefore, these proteins share a common C-terminal region. The hPIV1, which is highly homologous with SeV expresses only C’ and C. The hPIV3 and bPIV3 encode a single species of C protein composed of 199 and 201 residues, respectively. MeV also expresses a single species of C protein with 186 amino acids [11]. HeV and NiV express C proteins composed of 166 residues, and share 83% identity between the viruses [12].

C proteins are small basic polypeptides that contribute multiple functions to viral growth [1]. The most prominent roles of C proteins are inhibition of intracellular Jak/STAT IFN signaling pathway and downregulation of viral RNA synthesis [13,14]. Details of the anti-innate activities are described below. Earlier studies showed that SeV C protein binds L protein and inhibits viral mRNA synthesis [15-17]. Also, SeV C proteins have been shown to make significant contributions to viral budding from the plasma membrane [18], possibly by escorting ESCRT to the site of virion formation [19], which is required for pinching off from budding plasma membrane patches. Recombinant SeV that does not express C proteins can be recovered, indicating that C proteins are categorically nonessential gene products [20]. However, replication of this knockout virus was greatly impaired even in tissue culture cells, possibly due to the role of C protein in viral transcription, genome replication, and assembly. As expected, because of the functions of C protein in viral replication and anti-IFN activities, growth of recombinant SeV, hPIV1 and hPIV3 with disrupted C protein expression are severely attenuated in vivo [9,21,22]. In the case of MeV, C-deficient virus still grows well in certain culture cells but is less virulent in vivo [23]. Changes in pathogenesis of C mutant viruses are likely to be related to the ability of C proteins to inhibit IFN responses, which is discussed in further detail below.

V protein also plays a number of important roles in the virus replication cycle, as evidenced by many studies using recombinant viruses that have been engineered to disrupt expression of the whole V protein or its conserved cys-rich domain. In many cases, these mutant viruses grow well in tissue culture cell lines [24-26], but are severely attenuated for growth in vivo or cleared rapidly from infected animals [9,23,25,27], likely due to its capacity as an IFN antagonist.

The innate immune response is a non-adaptive host defense that forms early barriers to infectious disease, and acts within minutes of infection to eradicate a pathogen or avoid the spread of infection until the threat can be eliminated by the adaptive immune response [28]. Once the immune system has sensed viral infection, multiple signal transduction cascades are initiated resulting in expression of innate cytokines, including IFNs [29]. IFNs are categorized as type I (IFN-α/β), type II (IFN-γ) or type III (IFN-λ) based on sequence homology and the receptor complex used for signaling. Type I IFNs are secreted in direct response to virus infection and consist of the products of the IFN-α multigene family, predominantly synthesized by hematopoietic cells and the product of the single IFN-β gene, which is synthesized mainly by fibroblasts. The released IFNs bind to cell surface receptors and upregulate the expression of more than 300 cellular proteins. The expression of these proteins confers an antiviral state on the cells, providing the cells with an early defense against viral infections [30].

Like most other viruses, paramyxoviruses have evolved mechanisms that allow them to at least partially overcome the IFN response in order to establish a productive infection. They achieve this by reducing the production of IFN from infected cells, blocking IFN signaling or both. Interestingly, accessory proteins that are responsible for anti-IFN activities vary among the viruses, as well as their strategies to achieve these tasks (Table 1 and Table 2).

When infected with a virus, the host attempts to suppress virus replication in infected cells and viral spread to neighboring cells by various means including induction of apoptosis. Because viruses are intracellular parasites, control over the cell’s death machinery is crucial for viral replication and pathogenicity [104]. Apoptosis is a process in which cells activate intracellular death pathways to terminate themselves in a systematic way in response to a wide variety of stimuli. Apoptosis results from a collapse of cellular infrastructure through regulated internal proteolytic digestion, which leads to cytoskeletal disintegration, metabolic derangement and genomic fragmentation. Many viruses manipulate the apoptotic machinery to their advantage, and in the case of paramyxoviruses, accessory proteins expressed from the P/V/C gene play a role in blocking apoptotic cell death.

Many paramyxoviruses lacking V or C protein expression show enhanced apoptosis in infected cells [21,105-108]. One example is a recombinant PIV5 lacking the C-terminus of the V protein that induces apoptosis in tissue culture, in contrast to wt PIV5 that does not induce cytopathic effect (CPE). This apoptosis induction by the mutant virus can be prevented by expression of wt V, confirming the ability of V protein to prevent PIV5-induced apoptosis [105]. Another study reported that PIV5 containing mutations in^{the shared N termini of V and P protein (known as CPI–virus) accelerated viral gene expression and caused increasedcell death with characteristics of apoptosis, suggesting that multiple regions in V are required for blocking apoptosis [109,110].}

In the case of Respiroviruses, both hPIV1 and SeV C^{proteins have been shown to act as inhibitors of apoptosis through the characterization of C-deficient viruses. Early duringinfection in vitro, rhPIV1-P(C–), which is deficient in C protein expression, replicated as efficientlyas wt hPIV1, but its titer subsequently decreasedcoincident with the onset of extensive CPEnot observed with wt hPIV1. The rhPIV1-P(C–) infection, butnot wt hPIV1 infection, induced caspase 3 activation and nuclearfragmentation in LLC-MK₂ cells, identifying the hPIV1 C proteinsas inhibitors of apoptosis [21]. Similarly, SeV Cdeletion mutants have been shown to induce apoptosis in vitro, and longer C proteins (C’ and C) are more effective for the anti-apoptotic activity [93,107].}

Infection with recombinant MeV lacking either V or C also causes more cell death than infection with the parental vaccine-equivalent virus [89,111]. Infection with C-deficient MeV causes significantly less apoptosis in PKR-knockdown cells than in PKR-sufficient cells, suggesting a principal function of the MeV C protein is to antagonize the proapoptotic and antiviral activities of PKR [108].

It is clear that anti-IFN and anti-apoptosis activities play a major role in viral pathogenicity to their specific hosts. These activities have also been suggested to contribute to host range restriction because of their specificity. It has been demonstrated that while PIV5 can enter murine cells and initiate viral protein synthesis, the infection is rapidly cleared by the endogenous innate system. PIV5 does not cause STAT1 degradation in murine cell culture [112,113], however, expression of human STAT2 in mouse fibroblasts enabled PIV5 to effectively disrupt murine IFN signaling and to support specific degradation of the endogenous murine STAT1 [114]. These findings demonstrated a unique role for STAT2 as a species-specific host range determinant and corroborated its importance as a cofactor for STAT1 targeting by PIV5. Like PIV5, NDV V protein antagonizes the avian IFN system via STAT1 destabilization [10]. Recombinant NDV deficient in V expression grows poorly in embryonated chicken eggs and chicken embryo fibroblasts (CEFs). Insertion of influenza virus NS1 gene restores impaired growth to wt levels in eggs and CEFs, indicating that NDV V and influenza NS1 are functionally interchangeable. However, in human cells, wt NDV grows poorly compared to NDV V(-)/NS1 virus, suggesting that the anti-IFN activity of NDV V protein is species specific [106]. While the molecular basis has not yet been revealed, it is possible that, like PIV5, avian STAT2 might be involved in this specificity.

Specificity of anti-innate activity was also suggested from studies using closely related Respiroviruses, SeV (a murine parainfluenza virus type 1) and hPIV1. A major difference in the anti-IFN activity of these viruses is that SeV expresses both C proteins and a V protein, while hPIV1 expresses only C proteins. A recombinant SeV, rSeVhP, whose P gene was replaced with that of hPIV1, has been rescued. This virus allows the determination of whether hPIV1 C proteins can substitute the function of SeV accessory C and V proteins. rSeVhP infection prevents nuclear translocation of STAT1 upon IFN stimulation, indicating that hPIV1 C protein can inhibit the Jak/STAT pathway in murine cells [102]. Similarly, SeV C protein blocks the IFN signaling pathway in human cells [101], suggesting no specificity in the inhibitory activity of the Jak/STAT pathway between these two viral C proteins. Infection of rSeVhP in murine cells, however, strongly activates IRF-3 and NFκ-B, resulting in an increased level of IFN-β production compared with wt SeV. Insertion of SeV V, but not SeV C gene into rSeVhP partially restores the ability to suppress IFN induction, suggesting that V contributes to the suppression of IFN induction in SeV-infected murine cells [102].

This minireview has illustrated a variety of functions and strategies of paramyxovirus accessory proteins in antagonizing host cell innate activities. Although this group of viruses has similar genome structure and replication patterns, their methods to circumvent the host cell innate activities vary between the viruses of this group. Most of the paramyxoviruses have been shown to antagonize both IFN induction and Jak/STAT signaling pathways by their various accessory proteins. In addition, a study on SeV suggests the accessory proteins can block IFN-independent antiviral activity directly induced in infected cells, the mechanism of which is not known. Much remains to be clarified about the complex interplay between host and viruses, especially, host or tissue specificity and its role in viral pathogenicity and host range. Elucidation of a detailed mechanism is essential for understanding viral pathogenicity and disease development, and will also provide key information that will lead to the development of a safe and effective attenuated live vaccine to clinically important paramyxoviruses.