Rice stripe virus protein NS3 exploits synergistically insect vector importin and ubiquitin systems to promote viral replication

Plant viruses pose significant threats to agriculture, with many vectored by insect pests. The entry of viruses and their encoded proteins into the host nucleus is a critical step for promoting some viral replication and enabling systemic infection. Laodelphax striatellus, also known as the small brown planthopper (SBPH), is an efficient vector for rice stripe virus (RSV), one of the most damaging viruses of rice. In this study, we demonstrate that RSV infection induces the expression of genes in both the classical and non-classical nuclear import pathways of SBPH. A gene belonging to the importin β family, importin 5 (LsIPO5), was upregulated by 84% in SBPH midguts infected with RSV. The nuclear localization signal (NLS, ¹⁶⁸YRSPSKKRHKYV¹⁷⁹) is located within the nonstructural protein NS3 directly bound to LsIPO5, thereby facilitating NS3 nuclear entry. Moreover, a RING-type E3 ligase (LsRING) in SBPH, which mediated the ubiquitination of NS3 in the insect vector, enhanced NS3 binding to LsIPO5 and facilitated NS3 perinuclear localization. Combined treatment of SBPH with both dsIPO5 and dsRING significantly reduced RSV loads, highlighting the importance of LsIPO5 and NS3 ubiquitination cooperation in facilitating viral replication. Our findings provide new insights into synergistic molecular mechanisms that govern RSV infection and suggest potential therapeutic targets to control viral transmission through their insect vectors.