Interferon (IFN)-induced 2'-5' oligoadenylate synthetase (2-5A synthetase)/RNase L, PKR, and Mx proteins are considered to be the principal antiviral protein pathways through which IFN induces an antiviral state. It was previously reported that human parainfluenza virus type 3 (HPIV3) multiplication was inhibited by IFN-alpha in human lung epithelial cells A549 and that MxA was found to contribute to the inhibition process (Zhao et al., Virology 220:330-338, 1996). Viral primary transcription was dramatically inhibited in A549 cells after IFN-alpha treatment, but a step following primary transcription was inhibited in U87-MxA cells constitutively expressing MxA. Here we have investigated the role of MxA, believed to be cell type specific, and other antiviral pathways in the inhibition of viral primary transcription. Our data indicate that a novel IFN-induced pathway(s) is involved in the inhibition of primary transcription. This is based on the following findings: (i) IFN-alpha inhibited viral primary transcription in U87-MxA and other cell types including cells lacking MxA; (ii) cells constitutively expressing 2-5A synthetase had no antiviral effect against HPIV3; and (iii) primary transcription occurred in the absence of protein synthesis, a step of PKR target. The novel antiviral pathway(s) was induced by both IFN-alpha and IFN-gamma to establish an effective antiviral state against HPIV3. By using IFN-alpha-signaling mutant cells, we found that IFN-gamma could elicit antiviral effect against HPIV3 without cross talk with the IFN-alpha-signaling pathway. These data provide the first evidence that a novel antiviral pathway(s) contributes to the antiviral action of IFN against a nonsegmented negative-strand RNA virus by targeting the primary transcription.