Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants

M. Otilia Delgadillo, Pilar Sáenz{dagger}, Beatriz Salvador, Juan Antonio García and Carmen Simón-Mateo

Centro Nacional de Biotecnología (CSIC), Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain

Correspondence
Juan Antonio García
jagarcia{at}cnb.uam.es


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RNA silencing has a well-established function as an antiviral defence mechanism in plants and insects. Using an Agrobacterium-mediated transient assay, we report here that NS1 protein from human influenza A virus suppresses RNA silencing in plants in a manner similar to P1/HC-Pro protein of Tobacco etch potyvirus, a well-characterized plant virus silencing suppressor. Moreover, we have shown that NS1 protein expression strongly enhances the symptoms of Potato virus X in three different plant hosts, suggesting that NS1 protein could be inhibiting defence mechanisms activated in the plant on infection. These data provide further evidence that an RNA silencing pathway could also be activated as a defence response in mammals.

{dagger}Present address: Medplant Genetics, 48901 Baracaldo, Vizcaya, Spain.


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Higher eukaryotes are involved in a continuing battle against viruses. To minimize the effects of viral infection, a number of defence mechanisms have been developed based on the recognition of specific molecular patterns produced only in infected cells (Plasterk, 2002). This is the case with double-stranded RNA (dsRNA) molecules, which are not normally found in eukaryotic cells but are generated as an intermediate molecule during virus replication (Hutvagner & Zamore, 2002). In mammalian cells, dsRNA appears to play a major role in the induction of the interferon (IFN) response following viral infection (Stark et al., 1998). In recent years, another dsRNA-mediated defence mechanism known as RNA silencing has been described (Dougherty & Parks, 1995; Montgomery & Fire, 1998).

RNA silencing is a sequence-specific RNA degradation process that leads to elimination of the targeted RNA mediated by cytoplasmic nucleases and plays a natural antiviral role in plants (for reviews, see Baulcombe, 2002; Carrington et al., 2001; Vance & Vaucheret, 2001; Vazquez Rovere et al., 2002; Waterhouse et al., 2001). Recently, this mechanism of antiviral defence has also been reported in insect cells (Li et al., 2002).

In response to these types of host antiviral defences, it is not unexpected that viruses have devised counteracting mechanisms that interfere with them at different levels. Thus, many animal viruses are known to prevent or inhibit IFN-mediated defence (García-Sastre, 2001). The same is true for RNA silencing: many plant viruses (Carrington & Whitham, 1998; Li & Ding, 2001; Voinnet et al., 1999) and one insect virus (Li et al., 2002) have been shown to encode silencing suppressor proteins. Nearly 20 silencing suppressors described so far show no sequence similarity and hence appear to have evolved independently to overcome silencing-mediated defence. Thus, it is not easy to predict which protein is going to behave as an RNA silencing suppressor based on sequence analysis.

Since this RNA silencing has been described in species from different kingdoms (fungi, animals and plants), it has been proposed that it can also play an antiviral role in mammalian cells (Cullen, 2002; Gitlin et al., 2002). Here, we have investigated whether the NS1 protein, a multifunctional protein of human influenza A virus, could act as an RNA silencing suppressor. This protein has at least two features that make it a good candidate for such activity; first, it is able to bind dsRNA in vitro (Hatada & Fukuda, 1992), and secondly, it prevents the type I IFN-mediated antiviral response of the host induced by dsRNA during viral infection (García-Sastre et al., 1998).

To test whether NS1 protein is a suppressor of locally induced RNA silencing, we used the Agrobacterium tumefaciens silencing suppression assay that has been widely used to identify both plant and animal suppressors (Li et al., 2002; Voinnet et al., 1999). Infiltration of Agrobacterium containing a plasmid with the green fluorescence protein (GFP) gene controlled by the 35S promoter (provided by Dr D. Baulcombe) produces transient expression of the mRNA at high levels followed by induction of RNA silencing. The co-infiltration of this strain together with another harbouring a plasmid encoding a silencing suppressor blocks the onset of RNA silencing, and GFP is easily detected under UV light for a long time. The constructs 35S–NS1 and 35S–noNS1 were obtained by PCR from the plasmid pGem3NS1 provided by J. Ortín (Madrid) by using the forward primers 5'-ACCATGGATTCCAACACTGTG-3' (for 35S–NS1) and 5'-ACCACGGATTCCAACACTGTG-3' (for 35S–noNS1) and the reverse primer 5'-GCGTCGACTCAATCAGCCATCTTATC-3') and inserted between the 35S promoter and the Nos terminator of the pBIN19 binary vector T-DNA (Frisch et al., 1995). An empty vector (pBIN19) and a plasmid encoding the previously characterized Tobacco etch virus (TEV) RNA silencing suppressor, P1/HC-Pro (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau & Carrington, 1998), were used as negative and positive controls, respectively. The P1/HC-Pro cistron of TEV was obtained by PCR from plasmid pTEV7DA, described by Dolja et al. (1992), and inserted in the pBIN19 vector. We will refer to each Agrobacterium strain by the name of the plasmid it carries.

We first analysed the RNA silencing suppression in Nicotiana benthamiana plants. Leaves co-infiltrated with the mixture of p35S-GFP and pBIN19 showed bright green fluorescence (Fig. 1A) with a maximum peak at 2–3 days and then declined as a consequence of RNA silencing activation. This response was clearly inhibited when p35S-GFP was co-infiltrated with p35S-P1/HC-Pro or p35S-NS1 and an intense green fluorescence was observed, which persisted for at least 12 days (Fig. 1A). The intensity and the length of time of the fluorescence observed in the NS1-infiltrated leaves were similar to those obtained with P1/HC-Pro. Northern blot analysis of RNA extracted from the infiltrated patches verified that persistence of the fluorescence correlated with high steady-state levels of GFP mRNA (Fig. 1B). In addition, the GFP-specific siRNAs, a hallmark of RNA silencing (Hamilton & Baulcombe, 1999), remained at an extremely low level in the leaves where there was expression of either P1/HC-Pro or NS1, in contrast to their high level of accumulation in the plants co-agroinfiltrated with p35S-GFP and pBIN19 (Fig. 1C). RNA silencing produces two classes of siRNA, short (21–22 nt) and long (24–26 nt) siRNA (Hamilton et al., 1999); both were affected by P1/HC-Pro and NS1. These results showed that NS1 acts as a silencing suppressor in this transient assay.



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Fig. 1. Effect of potential suppressor activity on 35S–GFP expression after agroinfiltration of N. benthamiana leaves. (A) The level of silencing suppression was monitored visually by assessing GFP fluorescence with a hand-held long-wavelength UV lamp at different times (2, 6 and 9 days) after agroinfiltration. Photographs of infiltrated leaves co-infiltrated with the strains indicated to the left of each panel are shown. (B, C) RNA blot analysis showing the GFP mRNA levels (B) and GFP-specific siRNAs (C) extracted from the patches co-infiltrated with the different strains. Five µg total RNA extracted as described (Simón-Mateo et al., 2003) was loaded per lane and a GFP 32P-labelled full-length antisense RNA transcribed by T7 polymerase was used in the hybridization. Low molecular mass RNA was analysed as described (Hamilton & Baulcombe, 1999; Llave et al., 2000). The positions of the 21 and 24 nt DNA markers are shown on the left. Ethidium bromide-stained rRNA and tRNA and 5S rRNA are shown in (B) and (C), respectively, as loading controls.

 
A remarkable feature of RNA silencing is that it is not cell-autonomous, since signals are generated and move through the plant to induce silencing at a distance (Palauqui et al., 1997; Voinnet & Baulcombe, 1997). We assessed whether NS1 was able to suppress systemic RNA silencing by using the co-agroinfiltration method described above in a GFP-expressing transgenic N. benthamiana (line 16c) obtained by D. Baulcombe (Sainsbury Laboratory, Norwich, UK) (Voinnet & Baulcombe, 1997). At 9 days post-infection (p.i.), the patches infiltrated with p35S-GFP plus pBIN19 (see Fig. 3A) or plus p35S-noNS1, which express a non-translatable NS1 gene (data not shown), a red fluorescence was observed, indicating that the newly infiltrated transgene and the resident GFP transgene had both become locally silenced. As a result of silencing suppression, this local silencing did not take place when p35S-P1/HC-Pro or p35S-NS1 were co-infiltrated together with p35S-GFP and a bright green fluorescence was observed for at least 15 days (Fig. 2A). Northern blot analysis of RNA from the 16c-infiltrated tissue showed that the GFP mRNA levels, as in non-transformed N. benthamiana plants, sharply declined in leaves that did not express a silencing suppressor, but remained high in leaves expressing P1/HC-Pro or NS1, even at 17 days p.i., although some decay appeared to be taking place at that time (Fig. 2B).



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Fig. 3. Phenotypic effects associated with the expression of NS1 protein from PVX vector in plants. N. benthamiana (A), N. clevelandii (B) and N. tabacum (C) were agroinfiltrated with PVX vector containing no insert (PVX) or containing the NS1 gene (NS1). After 7 days (20 days shown here), tissues inoculated with PVX-NS1 showed severe necrosis.

 


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Fig. 2. Effect of potential suppressor activity on 35S–GFP expression after agroinfiltration of transgenic GFP N. benthamiana 16c leaves. (A) The level of silencing suppression was monitored visually by assessing GFP fluorescence with a hand-held long-wavelength UV lamp at different times (2, 6, 9 and 15 days) after agroinfiltration. Photographs of half-leaves co-infiltrated with 35S–GFP and the strains indicated above the images are shown. (B, C) RNA blot analysis showing the GFP mRNA levels (B) and GFP-specific siRNAs (C) extracted from the patches co-infiltrated with the different strains. Five µg total RNA extracted as described (Simón-Mateo et al., 2003) was loaded per lane and a GFP 32P-labelled full-length antisense RNA transcribed by T7 polymerase was used in the hybridization. Low molecular mass RNA analysis was as described (Hamilton & Baulcombe, 1999; Llave et al., 2000). The positions of the 21 and 24 nt DNA markers are shown on the left. Ethidium bromide-stained rRNA and tRNA and 5S rRNA are shown in (B) and (C), respectively, as loading controls.

 
We also analysed the presence of siRNA in the infiltrated patches (Fig. 2C). In samples without a viral suppressor, the two classes of short siRNAs (Hamilton et al., 2002) were detected at late times, when GFP mRNAs from the stably integrated transgene and from the transiently expressed DNA were at very low levels. We found that NS1, as well as P1/HC-Pro, mostly affected long siRNAs (24–26 nt). These data are in agreement with results obtained by Bucher et al. (2004) (accompanying paper) showing that NS1 protein binds siRNAs with preference for the larger (25 nt) species. A similar preferential decay has been previously shown for tissue expressing a potyviral HC-Pro (Hamilton et al., 2002), although in this case accumulation of small siRNAs was associated with silencing overcoming the suppressor activity of P1/HC-Pro at late times post-infiltration. However, detection of siRNAs at significant levels, despite strong silencing suppression by HC-Pro, has been also reported (Qu et al., 2003). Thus, in some circumstances, P1/HC-Pro, and in our case NS1, appear not to be able to completely make small siRNAs disappear. The efficiency of silencing suppression most likely depends on the amount of siRNA that is still left.

The disappearance of long siRNAs has been found to be associated with loss of systemic silencing activity (Hamilton et al., 2002). In agreement with this correlation, all (8/8) of the GFP-expressing N. benthamiana 16c transgenic plants infiltrated with p35S-GFP plus pBIN19, which accumulates high levels of long siRNA at the infiltrated leaves, became completely silenced at 20 days, whereas the systemic silencing of plants infiltrated with p35S-GFP plus either p35S-P1/HC-Pro or p35S-NS1, with much lower levels of long siRNA, was delayed. The number of completely silenced plants infiltrated with p35S-P1/HC-Pro was 5/8, and 2/8 in the case of p35S-NS1, and no silencing was detected in some plants, even at 30 days p.i., for both suppressors. This result indicated that NS1 protein, like P1/HC-Pro, prevents systemic silencing to a certain extent. We also tested the ability of NS1 protein to interfere with the RNA silencing maintenance by infiltration of leaves from systemically silenced GFP 16c plants. Neither P1/HC-Pro nor NS1 could reverse the established silencing in this system (data not shown).

Numerous observations have associated silencing suppressors with symptom modulation, systemic virus movement and pathogenesis functions (Kasschau & Carrington, 2001; Pruss et al., 1997). Since PVX produces a mild infection in different hosts, we decided to use a PVX–NS1 chimera to check whether NS1 affects viral infection as a result of its silencing suppression activity in planta. pPVX-NS1 was constructed making use of the pP2C2S clone (Chapman et al., 1992; Hammond-Kosack et al., 1995) (a gift of D. Baulcombe, Sainsbury Laboratory, Norwich, UK; http://www.jic.bbsrc.ac.uk/Sainsbury-Lab/david-baulcombe/Services/vigsprotocol.htm). Three different host plants, Nicotiana clevelandii, N. benthamiana and Nicotiana tabacum, were inoculated with RNA obtained by in vitro transcription of pP2C2S (PVX) and pPVX-NS1 as described (Sáenz et al., 2001). PVX–NS1-infected plants displayed much more severe symptoms than those infected with wild-type PVX (Fig. 3) or PVX expressing a non-translatable NS1 (data not shown). In the case of PVX–NS1-infected N. benthamiana plants (Fig. 3A), the enhancement of PVX infection was so strong that plants died in 9 days, while the control plants showed only a very mild infection. It is unlikely that the effect of NS1 in the infected plants could be the result of a toxic non-specific effect, because its transient expression in the agroinfiltrated leaves did not cause any apparent phenotype different from that of the controls. Similar enhancements of pathogenicity, including death in N. benthamiana, have been described for PVX-based chimeras expressing different plant silencing suppressors (for example, Brigneti et al., 1998; Chu et al., 2000; Pfeffer et al., 2002; Sáenz et al., 2001; Thomas et al., 2003). However, as in other cases, a direct demonstration that the pathogenicity enhancement associated with NS1 expression is the consequence of a more efficient virus replication due to RNA silencing suppression was precluded by the strong damage of the tissue infected by PVX–NS1.

In summary, we have shown that NS1 protein from influenza A virus behaves as a bona fide silencing suppressor in plants. In all the assays tested, NS1 protein acted in the same manner as a well-characterized plant silencing suppressor, the potyviral P1/HC-Pro protein. Both inhibited the onset of local RNA silencing, affected systemic RNA silencing and did not reverse RNA silencing in a silenced tissue. Furthermore, NS1 protein had a dramatic effect on the infection phenotype of a mild virus, leading to rapid tissue necrosis and even plant death. Similar enhanced disease phenotypes are typical of synergistic reactions following co-infections with two viruses and have been associated with an increased suppression of gene silencing (Pruss et al., 1997).

It is well established that dsRNA is a potent activator of the host's response to virus infection activating both the IFN system and RNA silencing. Therefore, it is plausible that some of the dsRNA-binding proteins encoded by viruses could impede host defences prevalent in the cell. During the progress of this work, Lichner et al. (2003) have published evidence that two dsRNA-binding proteins from E. coli (RNase III protein) and reovirus ({sigma}3 protein) are able to overcome GFP silencing in the A. tumefaciens infiltration assay. Interestingly, the two proteins encoded by mammalian viruses described as suppressing gene silencing ({sigma}3 and NS1 proteins) have also been described as counteracting the type I IFN response that takes place in the cell following virus infection (Beattie et al., 1995; García-Sastre et al., 1998; Kaufman, 1999). This is unlikely to be a coincidence and opens up the possibility that both mechanisms of viral defence activated by dsRNA might be counteracted by the same viral protein. Since the dsRNA-binding domain of this type of protein has been reported not only to bind dsRNA but also to mediate protein–protein interactions (Saunders & Barber, 2003), {sigma}3 and NS1 proteins could interfere either by sequestering dsRNA or by affecting a protein that mediates both pathways. A putative candidate is the dsRNA-dependent protein kinase (PKR), which functions in dsRNA signalling and host defence against virus infection in mammals (Williams, 1999) and has also been described as having a role in pathogenesis in plants (Hiddinga et al., 1988; Hu & Roth, 1991; Langland et al., 1995). There are many questions that have to be addressed but the results presented in this work further support the idea that RNA silencing reported as an antiviral defence mechanism in plants most likely also plays an antiviral role in mammalian cells.


   ACKNOWLEDGEMENTS
 
We thank Juan Ortín and Amelia Nieto for the pGem3NS1 clone and valuable discussions, David Baulcombe for the pP2C2S clone and the N. benthamiana 16c seeds, César Llave and James Carrington for plasmid pTEV7DA, Elvira Domínguez for technical assistance and Inés Poveda for helping with the photographic work. We acknowledge Rob Goldbach and Marcel Prins for sharing results before publication. M. O. D, P. S., B. S. and C. S.-M. were recipients of fellowships from CONCYTEA (M. O. D.), Comunidad de Madrid (P. S. and C. S.-M.) and Ministerio de Ciencia y Tecnología (B. S.). This work was supported by grants BIO2001-1434 from CICYT and QLG2-2002-01673 and QLK2-2002-01050 from the European Union.


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Received 20 October 2003; accepted 8 December 2003.