A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon
infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which
protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during
infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV
polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A
proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for
virus infection required coexpression of an NS4A-NS4B cassette with the viral
protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B
proteins. Individual expression of NS4A
protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A
protein to the Golgi apparatus. The results show that cleavage of the KUNV
polyprotein NS4A-4B by the viral
protease is the key initiation event in the induction of membrane rearrangement and that the NS4A
protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.