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The PIV5-w3, PIV5-CPI+, PIV5-CPI- and rPIV5-VΔC strains of PIV5, which express V proteins with different biological properties, differed in their ability to induce IFN. rPIV5-VΔC was the best inducer while PIV5-w3 was the poorest, in accordance with a previous study (Poole et al., 2002). PIV5-CPI+ and PIV5-CPI- viruses induced significant levels of IFN in infected cells, despite the ability of their V proteins to inhibit mda-5 activity (Childs et al., 2007, Andrejeva et al., 2004, Poole et al., 2002). This may be explained by reports that paramyxoviruses activate RIG-I, but not mda-5 signalling (Kato et al., 2006, Loo et al., 2008); since PIV5 is unable to block RIG-I signalling (Andrejeva et al., 2004, Childs et al., 2007), these viruses will still induce IFN. One obvious question is raised by these observations: why do paramyxoviruses actively inhibit mda-5 activity if paramyxovirus infection predominantly signals through RIG-I? It may be that PIV5 activates both mda-5 and RIG-I, but only RIG-I activity can be detected in infected cells due to mda-5 inhibition by V. In support of this, rPIV5-VΔC, whose V protein is unable to inhibit mda-5 signalling due to a C- terminal truncation (Poole et al., 2002, He et al., 2002a), induces large amounts of IFN: rPIV5-VΔC may be activatingboth RIG-I and mda-5 signalling. Comparisons of IFN induction by different virus strains may additionally be complicated by the presence of defective interfering (DI) particles in virus stocks. DIs lack one or more functions required for genome replication or synthesis/assembly of virus particles and can therefore replicate only in the presence of a replication-competent (“helper”) virus. DIs are potent inducers of IFN and apoptosis, presumably because they are able to efficiently produce the inducer of IFN/apoptosis but are unable to antagonise these responses (Johnston, 1981, Sekellick and Marcus, 1982, Aoki et al., 2001, Strahle et al., 2006, Fuller and Marcus, 1980b, Fuller and Marcus, 1980a, Strahle et al., 2007). DIs are difficult to correct for, as virus titres obtained by plaque assay are representative of the number of non-defective (plaque-forming) particles, but not DIs. Estimates of DI numbers in a particular virus stock can be obtained from the ratio of

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haemagglutinin titre to plaque-forming units; however, in this experiment the levels of DIs in virus stocks were unknown. The large amounts of IFN induced by rPIV5-VΔC could therefore be due to the presence of large amounts of DIs.

PIV5-CPI-, but not PIV5-CPI+, induced significant amounts of apoptosis in A549 cells. While PIV5-CPI- and PIV5-CPI+ induced similar levels of IFN, PIV5-CPI- is unable to block STAT1 signalling, so IFN produced during PIV5-CPI- infection can upregulate pro-apoptotic genes and promote apoptosis. Accordingly, in A549/BVDV/NPro and A549/PIV5/V cells, which are unable to produce or respond to IFN respectively, PIV5-CPI- and PIV5-CPI+ viruses cause a similar amount of cell death. Somewhat surprisingly, rPIV5-VΔC, which is reported to be apoptogenic (Sun et al., 2004), did not cause cell death in A549 naïve cells, despite inducing large amounts of IFN. In IFN-defective A549/BVDV/NPro and A549/PIV5/V cells however, rPIV5-VΔC induced significant apoptosis. These results suggest that the large amount of IFN produced during rPIV5-VΔC infection induced an antiviral state in naïve cells: rPIV5-VΔC is unable to antagonise the IFN response, so the virus is unable to replicate efficiently and is unable to cause cell death. In contrast, the antiviral state is not established in A549/BVDV/NPro and A549/PIV5/V cells so rPIV5-VΔC can replicate efficiently, resulting in apoptosis induction. The ability of PIV5 to induce cell death therefore seems to depend on the balance between limitation of virus replication by the host cell, and the ability of the virus to circumvent host cell responses to enhance its replication. Since A549/BVDV/NPro cells are IRF3-deficient, rPIV5-VΔC-induced cell death did not require IRF3; this is a surprising result since IRF3 is a requirement for apoptosis induced by dsRNA, reovirus, Bunyamwera virus, NDV and SeV (Heylbroeck et al., 2000, Peters et al., 2008, Weaver et al., 2001, McAllister and Samuel, 2008, Kohl et al., 2003, Holm et al., 2007). This indicates the existence of IRF3-independent pathways of apoptosis induction by PIV5. IRF3-independent modes of apoptosis induction may also be activated by infection with other viruses: while Bunyamwera virus is still able to induce significant cell death in cells with low levels of IRF3 expression (Kohl et al., 2003).

PIV5-w3 was unable to induce apoptosis in IFN-competent or IFN-compromised cells; this indicates that this virus may not produce significant amounts of the viral inducer of apoptosis. Work by Parks and colleagues revealed that the 6 amino acid differences between the shared P/V N-terminus of PIV5-w3 and PIV5-CPI- are responsible for differences in viral RNA production and subsequent PKR activation

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(Gainey et al., 2008a). During PIV5-w3 infection, Gainey et al. suggest a model by which P/V gene products decrease activity and increase fidelity of the viral polymerase, thereby limiting production of aberrant viral RNA to prevent activation of antiviral responses. In contrast, the P/V gene products from PIV5-CPI- are unable to do this, so PIV5-CPI- induces IFN and apoptosis (Gainey et al., 2008a). In fact, the three amino acid differences between the PIV5-w3 and PIV5-CPI+ P/V proteins are sufficient to affect RNA production by these viruses (Timani et al., 2008). Both PIV5- CPI+ and PIV5-CPI- are therefore unable to limit viral RNA synthesis. This would go some way to explaining why PIV5-CPI+ and PIV5-CPI- induce similar levels of IFN and apoptosis and why both viruses induce more IFN/apoptosis than PIV5-w3 (Figures 3.12 and 3.13). Interestingly though, ectopic expression of V(w3) could only partially prevent PIV5-CPI- or PIV5-CPI+ induced cell death in this study. This may indicate that while V(w3) expression levels in A549/PIV5/V are sufficient to deplete STAT1, they are insufficient to limit viral RNA production by PIV5-CPI- and PIV5- CPI+. In conclusion, there may be at least four factors dictating induction of IFN and apoptosis by PIV5 infection: the ability of the V protein to target STAT1 for degradation, the ability of V to bind and inhibit mda-5, the numbers of DIs in virus stocks and the ability of V to limit viral RNA production.

Despite observations that PIV5/V(w3) can inhibit apoptosis when stably expressed in cell-lines, apoptosis induction by FasL/CHX, TNF-α/CHX and STS was not significantly affected in PIV5-infected cells. PIV5-CPI- and PIV5-VΔC-infected cells did not go into apoptosis faster with these inducers than cells infected PIV5-w3 and PIV5-CPI+ (Figure 3.14), despite differences in STAT1 expression between these cells. IFN induced during virus infection may be affecting apoptosis induction, so it would be interesting to repeat this experiment in IFN-deficient cells.