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2B). That LASV Z, but not PICV Z, interacts specifically with RIG-i and MDA5 was also confirmed by the inverse co-IP with an anti-HA antibody followed by Western blotting with an anti-FLAG antibody (Fig. 2C). Both RIG-i and MDA5 harbor the N-terminal tandem CARD domains and a central ATPase-containing DExD/H box helicase domain (3). That LASV Z can strongly inhibit RIG-iN- and MDA5N-induced IFN production suggests that the Z-interactive region is located within the N-terminal CARD domains. Indeed, we demonstrated that LASV Z associated specifically with RIG-iNbut not RIG-iC by co-IP (Fig. 2D). To determine whether LASV Z directly binds to RLR, we expressed and purified GST-tagged RIG-iN protein from bacteria. We then incubated the purified GST-RIG-iN or GST control protein with the biotinylated LASV or PICV Z protein that was generated by an in vitro transcriptiontranslation system. After GST pulldown with glutathione-Sepharose 4B beads, the associated Z protein was detected by the use of streptavidin-horseradish peroxidase followed by chemiluminescent detection. As shown in Fig. 2E, LASV Z but not PICV Z specifically binds to GST-RIG-iN, demonstrating the direct protein-protein interaction between LASV Z and RIG-iN. Taking the results together, LASV Z protein inhibits RLR signaling by interacting directly with the RLR CARD domains. (iii) Pathogenic but not nonpathogenic Z proteins can interact with RLRs and disrupt the RLR-MAVS binding. We then examined whether the other pathogenic Z proteins could also interact with RLRs in a manner similar to that seen with LASV Z. Using the same co-IP experiment as described in Fig. 2B, we showed that the Z proteins of all other pathogenic arenaviruses (LUJV, MACV, LCMV, CHPV, DANV, GTOV, JUNV, and SABV), but not of nonpathogenic ones (PICV, TCRV, AMAV, IPPYV, MOBV, MOPV, and PIRV), could interact with RIG-i and MDA5 (Fig. 3A), correlating with their differential abilities to inhibit RLR-induced IFN- production. We also showed that the interaction of pathogenic Z with RIG-i/MDA5 was not due to the potential aggregation-prone nature of the pathogenic Z protein, as LASV Z and LCMV Z did not associate with IKK under the same co-IP assaying conditions (Fig. 3B). RLRs are known to activate the downstream target MAVS/ IPS-1 via CARD-CARD interactions (45). We hypothesized that pathogenic Z proteins bind to the RLR CARD to disrupt the RLRMAVS association and thus block MAVS activation. To test this, we transfected cells with HA-tagged Z and FLAG-tagged RIG-iN or MDA5N, together with myc-tagged MAVS. We pulled down the RIG-i complex and the MDA5 complex using anti-FLAG antibody and detected the presence of Z and MAVS by Western blotting using anti-HA and anti-myc antibodies, respectively (Fig. 3C). As predicted, in cell lysates that contained the pathogenic Z proteins (LASV, LUJV, MACV, LCMV, CHPV, and DANV), the RIG-i or MDA5 complex included Z but not MAVS. In contrast, in cell lysates of viruses that contained nonpathogenic Z proteins (PICV, TCRV, AMAV, IPPYV, MOBV, MOPV, and PIRV), the RIG-i or MDA5 complex included MAVS but not Z (Fig. 3C). Taking the data together, we showed that pathogenic Z proteinsFIG 1 The Z proteins of pathogenic arenaviruses but not nonpathogenic ones can block RLR-induced IFN- activation.