Assembly of human severe acute respiratory syndrome coronavirus-like particles

Abstract

Viral particles of human severe acute respiratory syndrome coronavirus (SARS CoV) consist of three virion structural proteins, including spike protein, membrane protein, and envelope protein. In this report, virus-like particles were assembled in insect cells by the co-infection with recombinant baculoviruses, which separately express one of these three virion proteins. We found that the membrane and envelope proteins are sufficient for the efficient formation of virus-like particles and could be visualized by electron microscopy. Sucrose gradient purification followed by Western blot analysis and immunogold labeling showed that the spike protein could be incorporated into the virus like particle also. The construction of engineered virus-like particles bearing resemblance to the authentic one is an important step towards the development of an effective vaccine against infection of SARS CoV.

Fig. 1

Electron microscopic analysis of VLPs in insect cells. (A) The formation of VLPs in the cytoplasm of an insect cell upon co-infection with E- and M-expressing recombinant baculoviruses. Bundles of baculoviruses were found in the nucleus of the cell (indicated by Bac). C, cytoplasm; N, nucleus; and NE, nuclear envelope. Bar=2 μm. (B) The boxed region of (A) was further magnified to show the formation of VLPs in a vesicle (solid arrow). Smaller dense vesicles filled with VLPs were also found in this region (open arrow). Bar=0.6 μm. (C) Extensive cytoplasmic spreading of VLPs in a cell. Bar=2 μm. (D) Higher magnification of the boxed region in (C). Some of VLPs were arbitrarily indicated by arrowheads. Bar=0.4 μm.

Fig. 2

Isolation and identification of VLPs. The VLPs were isolated from cells co-expressing S, E, and M proteins. After sucrose gradient centrifugation, 28 fractions were collected and their chromatograph was shown in (A). Fractionation was monitored with a wavelength of 280 nm; the dotted line represents weight percentage of sucrose. (B) Equal amounts of gradient fractions were separately resolved in 8% (anti-S) and 14% (anti-E) of polyacrylamide gels, followed by Western analyses. Numbers on the left sides of the sub-panels are the molecular weights of protein markers (M). Western analysis confirmed the presence of S and E proteins mainly in fractions 16–18.

Fig. 3

Ultrastructural characterization of VLPs. (A) VLPs purified by sucrose gradient centrifugation were analyzed by negative staining. (B) Immunogold labeling of purified VLP. A baculovirus particle (Bac) was shown to serve as a negative control. VLP, virus-like particle.