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. 2011;6(10):e25816.
doi: 10.1371/journal.pone.0025816. Epub 2011 Oct 3.

Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation

Stefan W Metz  1 , Femke Feenstra, Stephane Villoing, Marielle C van Hulten, Jan W van Lent, Joseph Koumans, Just M Vlak, Gorben P Pijlman
Affiliations

Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation

Stefan W Metz et al. PLoS One. 2011.

Abstract

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.

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Conflict of interest statement

Competing Interests: SV, MH, and JK are employees of Intervet. JV is an employee of Wageningen University and a consultant for Intervet. All experimental work was carried out at Wageningen University, the Netherlands. This did not alter the authors' adherence to all PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. SAV3 structural cassette expression, using recombinant baculoviruses.
A) Schematic representation of the SAV3 structural cassette as it is expressed by recombinant baculoviruses. The molecular mass of the proteins are indicated and shaded areas represent transmembrane domains or signal sequences (ss). Autocatalytic (A), furin (F) and signalase (S) cleavage sites are indicated, asterisks represent N-linked glycosylation sites. B) Protein expression in Sf9 cells was analyzed by CBB and WB using SAV α-E1 and SAV α-E2 mabs. C) Whole cell lysates were treated with/without PNGase F and analyzed with SAV α-E2 mabs. D) Cells infected with Ac-GFP and Ac-SAV3 and stained with Hoechst. CPE was evaluated by brightfield and fluorescence microscopy. Arrows indicate dense nuclear bodies in Ac-SAV3-infected insect cells.
Figure 2
Figure 2. Nuclear localization and assembly of SAV3-nucleocapsids.
Sf9 infection with Ac-SAV3 and Ac-GFP was analyzed by A) light-microscopy at 2 d.p.i. B) Ac-SAV3-infected cells were fixed and embedded in gelatin and ultrathin coupes were analyzed by TEM, with C, N, VS and NC indicating cytoplasm, nucleus, virogenic stroma and SAV nucleocapsids, respectively.
Figure 3
Figure 3. Temperature-dependent processing and secretion of SAV-E2.
A) Sf9 cells were infected with Ac-SAV3 with an MOI of 10 at 27°C, 18°C, 15°C and 12°C. SAV-E2 expression in the cell-fraction was analyzed by WB using SAV α-E2 mab (17H23). B) Relative percentages of SAV-E3E2 and E2, indicating more efficient processing of E3E2, with decreasing temperatures. C) Secretion of E2 as a function of the temperature. The medium fraction of infected Sf9-cell cultures was PEG-precipitated and analyzed by WB using SAV α-E2 mab.
Figure 4
Figure 4. SAV3-E2 detection on the surface of Sf9 cells after recombinant baculovirus expression.
Cells were infected with Ac-SAV3 at 12°C, 15°C, 18°C and 27°C. Cells were fixed with 4% paraformaldehyde and subjected to immunostaining with α-E2 mabs. Cells were analyzed by confocal microscopy and positive staining indicates the presence of E2 at the surface of infected cells.
Figure 5
Figure 5. SAV-E2 antigenic mass determination and VLP production.
A) SAV-E2 antigenic mass was determined using the SAV-neutralizing mab 17H23 on cell and medium fractions of infected Sf9-cells, incubated at 27°C, 18°C, 15°C and 12°C. B) The medium fraction of cells infected with Ac-SAV3 at 15°C was analyzed by TEM to analyze SAV VLP production. Medium fractions of Ac-GFP infected Sf9 cells and SAV3 infected Chinook salmon embryo cells were used as control samples.
Figure 6
Figure 6. SAV3 structural cassette expression and VLP formation by temperature-shift in Sf9 insect cells.
Sf9 cells were infected with Ac-SAV3, incubated for 2 days at 27°C and subsequently transferred to 12°C for 3 days. A) Cell cultures were treated with/without PNGase F and analyzed by WB using α-E2 mabs. B) Infected cells were treated with 4% paraformaldehyde and subjected to surface immunostaining with α-E2 mabs. C) The medium fraction the infected cell culture was analyzed by TEM for the presence of VLPs.
Figure 7
Figure 7. Comparative temperature-shift assay on Sf9-cells expressing the SAV3 structural polyprotein by recombinant baculoviruses.
Cells were infected with Ac-SAV3 at 27°C for 1, 2 or 3 days. Next, cells were transferred for 3 days to 12°C, 15°C or 18°C. Whole cell lysates and PEG-precipitated medium fractions were analyzed by WB using α-E2 mabs. Protein fraction quantities, relative to 1 day 27°C followed by 3 days 15°C, are indicated.
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