Expression of the severe acute respiratory syndrome coronavirus 3a protein and the assembly of coronavirus-like particles in the baculovirus expression system

Abstract

The Bac-to-Bac Baculovirus expression system was used to generate a recombinant baculovirus capable of expressing the severe acute respiratory syndrome (SARS)-coronavirus (CoV) 3a protein. Using the same expression system, two structural proteins, membrane (M) and envelope (E), were co-expressed to form SARS-CoV virus-like particles (VLPs) within an insect cell. Expression of viral proteins was confirmed by Western blot analysis and the formation of VLPs was studied by transmission electron microscopy.

Fig. 1.

Schematic drawing of pFastBac1 (Invitrogen).

Fig. 2.

PCR amplification of the recombinant bacmid DNA. Lanes 1, 1 kb marker (New England, Biolabs); lane 2, pFastBac1-myc-3a plasmid; lane 3, pXJ40myc-3a plasmid with gene specific primers (3a forward and 3a reverse); lanes 4 and 5, recombinant bacmid DNA with primer combinations of M13 Reverse and 3a forward, and M13 Forward (−40) and 3a reverse, respectively. Lane 6 was the water control.

Fig. 3.

Expression of severe acute respiratory syndrome (SARS)-coronavirus (CoV) 3a protein in insect cells. Sf9 cells were infected with a recombinant myc-3a baculovirus at a multiplicity of infection of 1 (lane 1). Cells were harvested at 72 h positinfection, lysed, and the cell lysate subjected to Western blot analysis using (A) anti-3a antibody and (B) anti-myc antibody. Two forms of myc-3a were detected by anti-3a antibody as previously reported (–21). Mock infected Sf9 cells were used as a negative control (lane 2).

Fig. 4.

Expression of severe acute respiratory syndrome (SARS)-coronavirus (CoV) M (A) and E (B) proteins in insect cells. Sf9 cells were infected with M only at a multiplicity of infection (MOI) of 10 (A, lane 1) and E only at an MOI of 2 (B, lane 1). The cells were also co-infected with the two recombinant baculoviruses, M and E, at an MOI of 5∶1 respectively (A, B, lane 2). Cells were harvested at 72 h postinfection, lysed, and the cell lysate subjected to Western blot analysis using (A) anti-M and (B) anti-E antibody. Mock infected Sf9 cells were used as a negative control (lane 3).

Fig. 5.

Analysis of virus-like particles formed by co-infecting Sf9 cells with M and E and a multiplicity of infection of 5∶1, respectively. Bar = 100 nm.