IFN response plays an important role during hantavirus infection [16–21] and, not surprisingly, hantaviruses replicate better in IFN-deficient than in IFN-competent cells [19, 22, 23].
NSs ORF is found in many but not in all hantaviruses . Both nonpathogenic hantaviruses (e.g. TULV and Prospect Hill virus) and pathogenic ones (e.g. Sin Nombre virus (SNV) and Andes virus) have NSs ORF, and presumably produce the NSs protein. Thus this protein is probably not the sole determinant of hantavirus pathogenicity. An NSs ORF is present also in the S segments of bunyaviruses of the genera Orthobunyavirus, Tospovirus, and Phlebovirus . The NSs proteins of orthobunya- and phleboviruses counteract the IFN response by inhibiting RNA polymerase II and hence downregulate the general transcription in infected cells [8–11]. By analogy one would assume a similar anti-IFN function for hantaviral NSs protein. According to our data, host protein synthesis is not severely affected by infection with TULV and PUUV. The NSs proteins of these viruses decrease the IFN response by inhibiting the activation of IFN-β promoter via NF-κB and IRF-3 pathways . Thus the suppression of IFN-β induction by TULV, PUUV, and also Prospect Hill virus, New York virus, SNV, and Andes virus reported by several research groups [17–21] could be, at least in part, attributed to the inhibitory activity of the NSs protein. In hantaviruses lacking the NSs ORF, the IFN response could be antagonized by other means, e.g. by glycoproteins [21, 23].
Here we have studied the competitiveness of two TULV isolates, TULV/Lodz and TULV/Moravia, after double infection in IFN-deficient and IFN-competent cells. These two TULV isolates differ in the length of their NSs ORF, which provided an opportunity to gain insights on function(s) of the NSs protein in vivo. TULV/Lodz isolate was expected to be more resistant to the IFN response than TULV/Moravia. This appeared to be the case indeed, supporting our earlier conclusion that the NSs protein is involved in the counteraction of IFN response, and suggesting that the N-terminal aa residues in the molecule are needed for the full activity of the NSs protein of TULV. It would be interesting to examine the anti-IFN activity of the NSs proteins of other hantaviruses, especially of SNV and SNV-like viruses that possess shorter NSs ORFs than PUUV and TULV .
Interestingly, even the more resistant of two TULV isolates, TULV/Lodz, failed to survive in MRC5 cells for more than five consequent passages. This temporary survival is in sharp contrast to the persistent, life-long infection, which TULV causes in its natural rodent host [24, 25]. One possible explanation is that, in the course of natural infection, the virus infects only a few IFN-competent cells and thus can avoid an immediate clearance by the host innate immunity. In Vero E6 cells the full-length NSs protein of TULV/Lodz did not appear beneficial for the competitiveness of this isolate suggesting that the full-length NSs protein is not essential for the virus in IFN-deficient cells.
So far no hantavirus with the entire NSs ORF deleted has been found in nature or engineered using reverse genetics. However, an interesting clone of PUUV strain Sotkamo was recently obtained by focus purification technique from the original Vero E6 cell culture isolate . This clone, Sotkamo-delNSs, carries a stop codon instead of Trp-21 codon in the NSs ORF, and thus could produce a truncated NSs protein (transcription presumably starts from Met-24), which is of the same size as in TULV/Moravia isolate. Most notably, Sotkamo-delNSs clone grows to substantially lower titers (about 10 times) than parental virus in IFN-competent A549 cells while in IFN-deficient Vero cells both viruses replicated with the same efficacy (Andreas Rang, personal communication). This is in agreement with our results on TULV and supports the idea that the production of the full-length NSs protein is beneficial for the viral growth in IFN-competent cells but not vital in IFN-deficient cells.
Reassortant variants could have been formed in the course of double infection with two TULV isolates. One could also assume that the reassortants possessing the S segment of TULV/Lodz isolate would have higher chances to survive in MRC5 cells (provided that the full-length NSs protein is a potent pro-survival factor). Unfortunately, our current isolate-specific RT-PCR assays are not quantitative and thus this hypothesis could not be properly evaluated. We are currently trying to develop real-time PCR assays to clarify this issue.