Subcellular Localization and Some Biochemical Properties of the Flavivirus Kunjin Nonstructural Proteins NS2A and NS4A

Abstract

In a previous study on the replication of Kunjin virus using immunoelectron microscopy (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, 1997, J. Virol. 71, 6650-6661), NS1 and NS3 were found associated with double-stranded RNA (dsRNA) within vesicle packets (VP) in infected Vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of RNA replication. NS2B and NS3 (comprising the virus-encoded protease) were colocalized within distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the sites of proteolytic cleavage. In this study we found by immunofluorescence (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at both 16 and 24 h postinfection, partially coincident with dsRNA foci. In cryosections of infected cells at 24 h, NS2A was located by immunogold labeling primarily within VP, associated with labeled dsRNA. NS2A fused to glutathione S-transferase (GST) bound strongly to the 3' untranslated region of Kunjin RNA and also to the proposed replicase components NS3 and NS5 in cell lysates. NS4A was localized by immunogold labeling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3' untranslated region of Kunjin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the results indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3.

Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells

“…Translation and replication of flaviviruses occurs in close association with cellular membranes (Mackenzie et al, 1998;Welsch et al, 2009;Westaway et al, 1997Westaway et al, , 1999Westaway et al, , 2003. The viral proteins associated with the replication complex are the two hydrophobic viral proteins, NS2A and NS4A, the abundant luminal protein NS1, and the NS5 and NS3 proteins.…”

West Nile virus NS2A protein facilitates virus-induced apoptosis independently of interferon response

“…The flavivirus NS2A protein is~25 kDa, hydrophobic, membrane-associated protein shown to be involved in RNA replication (Mackenzie et al, 1998;Rossi et al, 2007), membrane permeabilization (Chang et al, 1999), virion formation (Kümmerer & Rice, 2002;Leung et al, 2008;Liu et al, 2003), viral dissemination from mosquito midgut (McElroy et al, 2006) and regulation of host antiviral responses (Liu et al, 2005(Liu et al, , 2006. Our previous work demonstrated that a single amino acid substitution from alanine to proline at position 30 (A30P) of the NS2A gene attenuated virus virulence in mice and inhibited the virus' ability to downregulate type I interferon (IFN-a/b) production (Liu et al, 2006).…”

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

“…It was reported that NS2A binds with high specificity to the 3' untranslated region (UTR) of viral genomic RNA and to other components of the replication complex (Mackenzie et al, 1998). In addition, NS2A is considered to play roles in modulating the host-antiviral interferon response (Liu et al, 2004;Liu et al, 2006;Liu et al, 2005;Munoz-Jordan et al, 2003) and assembly/ secretion processes of virus particles (Kummerer and Rice, 2002;Leung et al, 2008;Liu et al, 2003).…”