The ability of the host cell to fight viral infection requires recognition of the invading viral pathogen as such and subsequent induction of signaling cascades that lead to the production of innate and adaptive antiviral responses. IFNβ plays a crucial role in priming neighbouring cells for possible infection and is also involved (with other IFN-stimulated genes) in recruiting cells of the host immune system to the site of infection, providing a way to eliminate infected cells. In the present study, we investigated the mechanisms used by the host cell to recognize SeV infection, and those used by SeV to counteract the cellular innate immune system.
By using different SeV stocks carrying mutations either in the promoter region or in the C proteins, we demonstrated that the presence of the leader RNA transcript (or the overexpression of the trailer) as well as the expression of the viral C and V proteins have an important role in counteracting the IFNβ activation and the expression of the chemokine IL-8 (c.f. Paper 1). As opposed to the V protein and the leader RNA, a number of investigations have been made on the SeV C protein. As mentioned in the first paper, this particular protein is in charge of many functions during infection: (i) it stimulates viral RNA synthesis early in infection (Latorre et al., 1998a); (ii) it inhibits viral RNA synthesis late in infection by binding to the viral polymerase (Cadd et al., 1996b); (iii) it has a role in the virus particle budding, which is facilitated by its binding to Alix, and in virion assembly, possibly by interaction with the matrix (M) protein (Mottet et al., 1996; Sakaguchi et al., 2005); (iv) The C protein interacts with Stat1 in two ways: counteracting IFN signaling and inducing Stat1 instability (Garcin et al., 2003). (v) Finally, the C protein inhibits the IRF-3-dependent activation of IFNβ as well as the activation of IL-8 expression (Strahle et al., 2003). These multiple functions of SeV C protein during infection fit well with its property to interact with various viral and cellular proteins, such as Stat1, Alix and Rig-I (unpublished). However, these interactions remain to be closely analyzed. Additionally, the reason why IL-8 expression has been only recently discovered could be explained by the fact that standard SeV induces very little IL-8. Indeed IL-8 is only expressed upon infection of SeV bearing mutations in the C and V proteins, and in the leader region. Moreover, it has been already reported that SeV and MeV infections induce an other CC-chemokine RANTES via the activation of IRF-3 (Genin et al., 2000; Tenoever et al., 2002). These data confirm the results suggesting that SeV targets the inflammatory and adaptive immune responses (IL-6 and IL-8) as well as the IFN-induced intracellular antiviral state (IFNβ and STAT1).
SeV is commonly used by many laboratories for its capacity to strongly induce IFN. This ability was long known to be associated with the presence of DI genomes in the SeV stock.
The third paper provides evidence that the strong induction of IFNβ activation upon SeV infection (SeV stock containing DI genomes) is mainly due to the presence of copyback DI genomes. Moreover, the level of IFNβ activation was found to be proportional to that of DI genome replication. Apparently, the ability of DI-H4 to induce IFNβ is not only related to its strong interference with the helper, which leads to lower levels of V and C proteins intracellularly but also to the relative content of copyback DI genomes, which can presumably form dsRNA. (cf. paper 2).
It is important to emphasize the difference between a standard SeV-WT infection from an infection of SeV that contains DI genomes. In the case of a SeV-WT infection, the RNA genomes and antigenomes are tightly assembled into NCs, meaning that they are never free to anneal (like other NNV). Furthermore, Weber et al. have shown recently that no detectable amounts of dsRNA produced by NNV have been found in infected cells (Weber et al., 2006).
This result can be explained by the fact that the amount of dsRNA produced by NNV are below their detection limit, since one molecule of dsRNA per cell can be effective in triggering an antiviral response. If it is the case, we can imagine two different ways for SeV-WT (devoided of DI genomes) to produce dsRNA: 1) During SeV replication, the polymerase ignores occasionally the stop signal at the end of the trailer template, which results in the extension of the trailer beyond the trailer/L gene junction. This event will produce a long trailer whose extended 3’sequences can anneal to those of the L gene (Vidal and Kolakofsky, 1989). 2) dsRNA can also be the result of a readthrough by the transcriptase of the L gene-end site until the end of the genome (an event that can happen at 5% of the time at all gene junctions). In this case the genome 5’end can anneal to the trailer RNAs (Le et al., 2002).
Finally, if we consider that there is a small amount of dsRNA that is generated in SeV infection, it is evident that the presence of dsRNA will be dampen by the SeV C and V proteins expressed during ND genome replication and that the expression of IFNβ will consequently be blocked.
In the case of copyback DI infections, it is a different story. Because DI-H4 contains two antigenomic promoters located at both the genomes and antigenomes ends, it has strong replicative advantage over the standard virus. Consequently, a huge amount of DI genomes is rapidly generated and the level of viral proteins (including the level of the nucleocapsid protein) is also strongly attenuated. This rapid generation of DI genomes and the low level of
NC proteins could explain why the genomes and antigenomes may not assemble into nucleocapsids and would consequently form dsRNA. If DI-H4 genomes and antigenomes are not encapsidated during infection, two forms of dsRNA can be found (in addition to those mentioned for a SeV infection). First, the extremities of the DI genome (complementary on 110 nucleotides) can self anneal in a concentration independent manner and form dsRNA, resulting in the shape of panhandles. Secondly, the genome and the antigenome can anneal together, which remains unlikely since this would require the melting of intramolecular secondary structure.
Recent evidence suggests that NNV infections could activate the innate immune response by producing viral signatures other than dsRNA, namely 5’tri-phosphorylated single-stranded RNA transcripts (5’pppRNAs). This discovery suggests that dsRNA may not always be implicated in the IFNβ induction upon viral infection and that there is probably more than one factor able to induce this primary cellular induction. Two cytoplasmic helicases, namely RIG-I and Mda-5, have been recently found to respond to dsRNA and, at least for RIG-I, to 5’pppRNAs. These RNAs are generated in the cytoplasm during RNA virus replication. Upon detection of specific viral RNAs RIG-I and Mda-5 interact with Cardif which is present in the mitochondrial membrane, and this interaction is thought to lead to the recruitment and activation of the TBK1, IKKε and other IKK kinases that activate NF-kB and IRF3, thereby activating the IFNβ promoter.
In the third paper, we investigated different potential inducers of IFNβ, including the synthetic dsRNA (polyI/C) and 5’pppRNAs and the involvement of RIG-I in these inductions.
We showed evidences that i) transfection of poly-I/C and ii) a SeV coinfection that can produce GFP dsRNA, as well as iii) transfections of small 5’pppRNAs can all induce IFNβ.
In all cases, the IFNβ induction was dependent on RIG-I. Not all 5’pppRNAs generated by SeV can act as PAMPs: 1) SeV mRNAs carry 5’ ppp ends, but because they are capped, these mRNAs cannot be potential targets. 2) SeV genomes and antigenomes also carry triphosphorylated 5’ends, but because the RNA is tightly and entirely encapsidated, it cannot be seen. 3) Finally, leader and trailer RNA transcript are the most susceptible to induce IFNβ, because they are short unencapsidated 5’pppRNAs that are independent on on-going (N) protein synthesis. For this reason, we suspect that, in the case of DI-H4 infection, there is a real overproduction of pppRNAs (trailer) resulting in the induction of the IFNβ activation.
Alternatively, other viral structures may take the role of RNA as a danger signal for the host cell. Indeed, it was previously shown that the NCs from VSV and MeV are capable of
triggering IFN induction. It was interesting to observe in our experiments that NCs purified from SeV infection could strongly induce IFNβ. However, RNase treatment revealed that it was probably not the NCs that were responsible for this activation, but more likely RNA products that have contaminated the CsCl banded NCs. Because the NC is the first viral element that enters the cell, one can imagine that viruses have evolved to avoid its detection.
Thus, it is reasonable to think that NCs do not act as PAMPs. Concerning MeV, it was shown recently that the NCs were not responsible for the IFNβ activation after all and that it was the leader transcript of MeV that was responsible for the activation of IFNβ via the RIG-I pathway (Helin et al., 2001; Plumet et al., 2007). This again comforts the idea that SeV leader and trailer RNAs are more likely to be the main PAMPs responsible for triggering the antiviral responses upon SeV infection.
Regarding the C and V proteins, we presented evidence that for SeV infection of MEFs, it is the C protein (and not V) that is primarily responsible for this effect, and that C acts by countering RIG-I dependent signaling to IFNβ. For example, independent expression of either the C or the V proteins inhibited IFNβ activation due to RIG-I over-expression. In addition to this, both proteins inhibited IFNβ activation due to DI-H4 infection, although C was always more effective than V (in our experiments). However, only C expression effectively inhibited IFNβ activation due to GFP(+/-) infection, or transfected poly-I/C or pppRNAs (cf paper 3).
These new findings, suggesting that the 5’pppRNA is a PAMP specific to the NNV infections, lead us to reconsider the way the cell could detect the presence of viruses.
However, no proof has been found concerning the role of 5’pppRNA products in the activation of IFNβ in SeV infection. Further analyses of the precise RNAs involved in the IFNβ activation need to be done. Additionally, more investigations are necessary to determine how these PAMPs are detected and the mechanisms that drive cellular responses. Finally, learning more about the protein targets of SeV proteins also appear important in order to understand the details of the IFN suppressive activities of SeV and virus infection in general.
VIII.
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