Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures

Abstract

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immunogold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.

A Guide to Viral Inclusions, Membrane Rearrangements, Factories, and Viroplasm Produced During Virus Replication

“…The detection of dsRNA and viral NSPs (NS1, NS2A, NS3, and NS4A) within the vesicle packets points strongly to this being the site of RNA replication (Mackenzie et al, 1998;Westaway et al, 1997b). The vesicle packets associate closely with the convoluted membranes and paracrystalline arrays, which are thought to be the sites of proteolytic processing of NS3 and NS2B (Westaway et al, 1997b). These modified membranes are linked with the ER, and ultrastructural studies have shown virions present in the ER, cytoplasmic vesicles, Golgi cisternae, and vacuoles.…”

“…For historical reasons, these are called vesicular packets [reviewed in Mackenzie (2005)]. They are larger (80-to 100-nm diameter) than the 50-nm alphavirus spherules, and form from the limiting membrane of the trans-Golgi network (TGN) (Uchil and Satchidanandam, 2003;Westaway et al, 1997b). Infection by Kunjin virus leads to unique membrane structures thought to be derived from both the early and late secretory pathways.…”

“…Infection by Kunjin virus leads to unique membrane structures thought to be derived from both the early and late secretory pathways. These include convoluted membranes and paracrystalline arrays derived from the rough ER and ERGIC, and vesicle packets derived from the TGN (Mackenzie et al, 1999;Ng, 1987;Roosendaal et al, 2006;Westaway et al, 1997b). The detection of dsRNA and viral NSPs (NS1, NS2A, NS3, and NS4A) within the vesicle packets points strongly to this being the site of RNA replication (Mackenzie et al, 1998;Westaway et al, 1997b).…”

“…These include convoluted membranes and paracrystalline arrays derived from the rough ER and ERGIC, and vesicle packets derived from the TGN (Mackenzie et al, 1999;Ng, 1987;Roosendaal et al, 2006;Westaway et al, 1997b). The detection of dsRNA and viral NSPs (NS1, NS2A, NS3, and NS4A) within the vesicle packets points strongly to this being the site of RNA replication (Mackenzie et al, 1998;Westaway et al, 1997b). The vesicle packets associate closely with the convoluted membranes and paracrystalline arrays, which are thought to be the sites of proteolytic processing of NS3 and NS2B (Westaway et al, 1997b).…”

Kunjin replicon-based simian immunodeficiency virus gag vaccines

“…The titre of infectious VLPs in infectious units (IUs) was determined by infection of Vero cells with 10-fold serial dilutions of the culture fluid. At 30-40 h post-infection the cells were fixed with methanol/acetone (1:1, v/v) and analysed by immunofluorescence antibody staining using the 4G4 monoclonal anti-NS1 antibody [33,34] and the 55-2F12 anti-SIV Gag monoclonal antibody (catalogue # 1610, NIH AIDS Research & Reference Reagent Program) and the number of positive cells counted using Zeiss Axiophot 2 fluorescent microscope (Carl Zeiss Microimaging GmbH, Berlin, Germany).…”