25The flavivirus Zika virus (ZIKV) activates ribonuclease L (RNase L) catalytic antiviral 26 function during infection, yet deletion of RNase L decreases ZIKV production, suggesting 27 a proviral role of RNase L. In this study, we reveal that latent RNase L supports ZIKV 28 replication factory (RF) assembly. Deletion of RNase L induced broader cellular 29 distribution of ZIKV dsRNA and NS3 compared with densely concentrated RFs detected 30 in WT cells. An inactive form of RNase L was sufficient to contain ZIKV genome and 31 dsRNA within a smaller area, which increased levels of viral RNA within RFs as well as 32 infectious ZIKV released from the cell. We used a microtubule stabilization drug to 33 demonstrate that RNase L deletion impaired the cytoskeleton rearrangements that are 34 required for proper generation of RFs. During infection with dengue or West Nile Kunjin 35 viruses, RNase L decreased virus production, suggesting that RNase L proviral function 36 is specific to ZIKV. 37 38 39 Keywords 40 Zika virus, flavivirus, RNase L, replication factories, dengue virus, West Nile Kunjin virus 41 42 43 44 45 46 47 All rights reserved. No reuse allowed without permission.