The success of the innate cellular defense to viral infection is dependent on the capacity of the host to detect the presence of the invading pathogen. Upon infection, many cellular sensors recognise different components of the virus and initiate in turn signal transduction cascades (such as IRF-3) producing in the end cytokines, including IFNs. The RNA helicase RIG-1 has been recently discovered to recognise viral RNA. RNA recognition leads to IRF-3 and NF-kB activation and finally to the induction of type I IFNs and the antiviral state of the cell. To circumvent the detection of their own RNA genome, the mononegalevirales possess the nucleocapsid (NCs) that surrounds completely the RNA viral genomes (and antigenomes). It is likely that their nucleocapsid never disassemble during genome expression. This protection seems to be quite strong considerating that the RNA genomes (within the nucleocapsid) are resistant to nuclease attack at any salt concentration and that the NC is very stable, as it withstands the high salt and gravity forces of cesium chloride density gradient centrifugation (Lamb and Kolakofsky, 1996). Further more, the N proteins are believed to be “sticky”, in the way that they are tightly bound to the viral RNA genomes and can also encapsidate free RNA independently of whether it comes from the virus or the cell.
It has been shown in 2002 that MeV nucleocapsid (NC) protein was the major component of IRF-3 activation and triggered the induction of IFN (Tenoever et al., 2002). The ability to activate IRF-3 during the course of infection has also been observed in other single stranded, enveloped RNA viruses such as RSV, NDV, VSV and SeV (Casola et al., 2001; Servant et al., 2001; Sundstrom et al., 2001). This suggests that the IRF-3 cascade could be involved in the viral NC detection. Since NCs are the first viral elements that enter the cell, it is logical to make the hypothesis that, like MeV, SeV NCs act as PAMPs and consequently induce IFNβ activation upon SeV infection.
1) SeV N protein expressed alone does not activate IFNβ. When N is expressed alone (in the absence of the viral P protein which forms a complex with N and prevent
“illegitimate” non-specific binding to cellular RNA), it is often found in high number and aggregates together with non genome RNAs, which represent a bogus NCs. To analyze the direct effect of the SeV N protein alone, MEF cells were first transfected
with a plasmid containing a luciferase reporter gene under the control of the IFNβ promoter and 24h later with a plasmid expressing SeV N protein (Fig. A). We have examined the ability of the N protein to induce IFNβ activation after transfection and compared the level of IFNβ activation with those of the copyback DI-H4 infection that we used as a positive control. As shown in figure A, the expression of N protein alone does not induce IFNβ activation. By contrast, DI-H4 infection activates stongly the activation of the IFNβ in this experiment. These results show that under these conditions, SeV viral N protein does not induce IFNβ activation.
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ctl SeV-DI-H4 infection pEBS_N
A.
Relative Luciferase
Activity
Figure A: Transfected SeV N proteins do not induce IFNβ activation.
Parallel cultures of MEFs were first transfected with pIFNβ-lucff and pTK-lucr. After 24h, the cells were either infected with SeV-DI-H4, or transfected with plasmids expressing SeV N protein (pEBS_N).
Cytoplasmic extracts were prepared after 20h of incubation, and used to determine firefly and renilla luciferase levels.
2) Transfected encapsidated SeV (NCs) genomes induce IFNβ activation. To further investigate the role of the NCs, 24 hours SeV infected cells were collected and the NCs purified by CsCl centrifugation (cf. M&M). Two doses of purified SeV NCs (or PolyI/C) were transfected into MEF cells and tested for their ability to activate IFNβ by using the reporter plasmid in which the IFNβ promoter expresses a luciferase reporter gene. Surprisingly, we observed that transfected purified NCs induce strongly
IFNβ activation and that the transfection of 10μl of purified NCs (corresponding to 250000 infected cells) is almost as efficient as a DI-H4 infection. Moreover, we can see that NCs transfection is more competent than the synthetic dsRNA (poly I/C) treatment in activating IFNβ. These results suggest that induction of IFNβ requires the presence of the NCs complex (fig. B).
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_ 5ug 5ul 10ul
mock DI-H4 infection poly IC SeV NC
B.
Relative Luciferase
Activity
Figure B: Transfected NCs induce IFNβ activation.
Parallel cultures of MEFs were first transfected with pIFNβ-lucff and pTK-lucr. After 24h, the cells were either infected with SeV-DI-H4, or transfected either with poly I/C (5ug) or purified SeV NCs (5-10ul) isolated by CsCl density gradients centrifugation (cf.M&M). Cell extracts were prepared after further 20h of incubation, and equal amounts were used to determine luciferase activities.
3) SeV C and V proteins expression decreases the IFNβ activation upon NCs transfection. Since SeV C and V proteins are responsible of counteracting the host innate immune response and because we have shown in paper three that IFNβ activation upon DI-H4 infection was in part due to a downregulation of the viral C and V proteins, we decided to over-express the V or C proteins in NCs-transfected MEF cells. We observed that IFNβ activation was strongly reduced by C expression (4 folds), and more modestly by V expression (2 folds) (Fig C). Thus, the SeV V and C proteins inhibit IFNβ activation induced by NCs transfection.
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