The G92 NS2B mutant of Tembusu virus is involved in severe defects in progeny virus assembly

Abstract

Although the role of the flavivirus non-structural 2B (NS2B) as a viral protease cofactor has been investigated, its role in viral proliferation has rarely been studied. Moreover, important details on the involvement of the flavivirus NS2B in the virus replication cycle, including attachment, entry, RNA replication, assembly and release, remain unknown. Here, an alignment of flavivirus NS2B was performed to select conserved amino acids for the site-directed mutagenesis study. G92, which is not totally conserved to mutation, was also used. Certain mutations (P32A, G36A and G37A) completely blocked viral RNA synthesis, four mutations (D25A, A43Y, V44A, G92A and P112A) showed a slight defect in virus replication, and only the G92A mutant affected virus proliferation by blocking virus release but not RNA replication. By taking advantage of the tembusu virus (TMUV) replicon that represents viral genome RNA replication, we showed that the G92A mutation did not obviously impair viral replication. Naturally, the NS2B3 G92A mutation protein did not affect the self-cleavage ability or NS2B3 protein-mediated cleavage of the NS2AB G92A mutant. These results indicated that the G92A mutation did not affect viral protease activity. By using the TMUV infection clone and the replicon package system, the effect of G92 on the virus lifecycle was narrowed to viral resemblance or release. Subcellular fractionation further confirmed that G92A attenuated TMUV by reducing virus assembly. Collectively, these studies extend the list of NS2B functions beyond protease activity as a cofactor, show that transmembrane region amino acids play an important role in viral replication and indicate that G92 is involved in the assembly of infectious TMUV particles.