Nuclear localization of the Nipah virus W protein allows for inhibition of both virus- and toll-like receptor 3-triggered signaling pathways.

The Nipah virus V and W proteins, which are encoded by the P gene via RNA editing, have a common N-terminal domain but unique C-terminal domains. They localize to the cytoplasm and nucleus, respectively, and have both been shown to function as inhibitors of JAK/STAT signaling. Here we report that V and W proteins also block virus activation of the beta interferon (IFN-) promoter and the IFN regulatory factor 3 (IRF3)-responsive IFN-stimulated gene 54 promoter. Surprisingly, only W protein shows strong inhibition of promoter activation in response to stimulation of Toll-like receptor 3 (TLR3) by extracellular double-stranded RNA. This activity is dependent on the nuclear localization of W protein. Within the unique C-terminal domain of W protein, we have identified a nuclear localization signal (NLS) that requires basic residues at positions 439, 440, and 442. This NLS is responsible for mediating the preferential interaction of W protein with karyopherin- 3 and karyopherin- 4. Nuclear localization of W protein therefore enables it to target both virus and TLR3 pathways, whereas the cytoplasmic V protein is restricted to inhibiting the virus pathway. We propose that this discrepancy is in part due to the V protein being less able to block signaling in response to the kinase, TBK-1, whereas both V and W can prevent promoter activation in response to IKK. We demonstrate that, when the TLR3 pathway is stimulated, the levels of phosphorylated IRF3 are reduced in the presence of W protein but not V protein, confirming the differential effects of these proteins and illustrating that W protein-mediated inhibition is due to a loss of active IRF3. A major role of the host’s innate immune response upon viral infection is to sense the presence of the invading virus and to respond by establishing an antiviral state within the infected cell. This response is mediated primarily by alpha/beta interferon (IFN-/) and can be divided into three basic stages (reviewed in references 22 and 56). First, virus infection stimulates the production of IFN-, which is released from the infected cell. In the second stage, the released IFN binds to the IFN-/ receptor and initiates a signaling cascade (termed the JAK/STAT pathway) that results in the transcriptional upregulation of IFN-stimulated genes (ISGs). The products of these genes are antiviral proteins (e.g., PKR and Mx) that function in a variety of ways to halt virus replication in the infected cells and prevent infection of neighboring uninfected cells. Induction of IFN- synthesis is a hallmark of virus infection, but it can also be induced by treating cells with doublestranded RNA (dsRNA) alone. dsRNA is produced as a replication product during most virus infections, and therefore it is thought to be the virus-associated molecule that triggers IFN synthesis. This implicates Toll-like receptor 3 (TLR3) as a sensor of virus infection, since it has been shown to specifically recognize dsRNA, an event that leads to IFN- induction (2). TLR3 signaling is mediated by association of its Toll/interleukin-1 receptor (TIR) domain with a TIR domain-containing adaptor protein called TRIF (46, 73, 74). Signaling down