1 Special Pathogens Laboratory, Department of Virology I, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan
2 Laboratory of Respiratory Viral Diseases and SARS, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan
3 Laboratory of Influenza Virus, Department of Virology III, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan
Correspondence
Shuetsu Fukushi
fukushi{at}nih.go.jp
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ABSTRACT |
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Detection of expression of SARS-CoV-S protein and soluble mutated ACE2 are available as supplementary material in JGV Online.
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MAIN TEXT |
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Entry of SARS-CoV into susceptible cells is mediated by binding of the viral spike (S) protein to receptor molecules. The SARS-CoV-S protein has a 13 aa signal peptide at its N terminus, a single ectodomain of 1182 aa and a transmembrane region followed by a cytoplasmic domain of 28 aa (Marra et al., 2003; Rota et al., 2003
). Recently, pseudotyped retroviruses bearing SARS-CoV-S protein have been generated by several laboratories (Hofmann et al., 2004
; Nie et al., 2004
; Simmons et al., 2004
). It has been shown that these pseudotyped viruses have a cell tropism identical to SARS-CoV and that their infection is dependent on a receptor molecule, angiotensin-converting enzyme 2 (ACE2), indicating that infection is mediated solely by SARS-CoV-S protein. Pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus. A quantitative assay of pseudotyped virus infection could facilitate research on SARS-CoV entry, cell tropism and neutralizing antibodies. Interestingly, it was reported that a pseudotyped retrovirus bearing a SARS-CoV-S protein variant with a truncation in the cytoplasmic domain was incorporated more efficiently into retrovirus particles than the full-length S protein (Giroglou et al., 2004
; Moore et al., 2004
).
Another pseudotyping system with a vesicular stomatitis virus (VSV) particle [the VSVG* system, in which the VSV G gene is replaced by the green fluorescent protein (GFP) gene] was reported previously to produce pseudotype VSV particles incorporating the envelope glycoproteins of several RNA viruses (i.e. measles virus, hantavirus, Ebola virus or hepatitis C virus; Matsuura et al., 2001
; Ogino et al., 2003
; Takada et al., 1997
; Tatsuo et al., 2000
). This system may be useful for research on viral envelope glycoproteins due to the ability of the pseudotype to grow at high titres in a variety of cell lines. The pseudotype virus titre obtained with the VSV
G* system is generally higher than that of the pseudotype retrovirus system (Ogino et al., 2003
). Furthermore, infection of target cells with pseudotype VSV can be readily detected as GFP-positive cells by 16 h post-infection (p.i.) because of the high level of GFP expression in the VSV
G* system (Ogino et al., 2003
). In contrast, the time required for infection in the pseudotype retrovirus system is 48 h (Moore et al., 2004
; Nie et al., 2004
), which is similar to the time required for SARS-CoV to replicate to a level that results in plaque-forming or cytopathic effects in infected cells. To date, there have been no reports of VSV pseudotyped with the S protein of a coronavirus. Pseudotyping of SARS-CoV-S protein using the VSV
G* system may have advantages for studying the functions of SARS-CoV-S protein, as well as for developing a rapid detection system to examine neutralizing antibodies specific for SARS-CoV infection.
To generate VSV pseudotyped with SARS-CoV-S protein, we first constructed an expression plasmid encoding full-length SARS-CoV-S protein. The cDNA of SARS-CoV-S protein was amplified using forward primer S-Bam-f (5'-GGATCCAAGTGATATTCTTGTTAACAAC-3') and reverse primer S-Bam-r (5'-GGATCCAAGAGTAAAAAATCTCATAAAC-3') and cloned into the expression vector pKS336 (Saijo et al., 2002). The resulting plasmid, pKS-SARS-S, was transfected into 293T cells (see Supplementary Fig. S1, available in JGV Online), followed by infection with VSV
G* (Matsuura et al., 2001
). When the culture supernatants of the infected 293T cells were inoculated on to Vero E6 cells, commonly used for SARS-CoV propagation, only small numbers of GFP-expressing cells were observed (data not shown). This result indicated that the VSV pseudotype bearing the full-length SARS-CoV-S protein was not very infectious. Next, we generated an expression plasmid encoding a C-terminal-truncated version of the SARS-CoV-S protein. The cDNA of C-terminal-truncated SARS-CoV-S protein was amplified from pKS-SARS-S using forward primer S-Bam-f and reverse primer S-Bam19r (5'-GGGATCCTTAGCAGCAAGAACCACAAGAGCATG-3'), followed by cloning into pKS336. The resulting plasmid, pKS-SARS-St19, encoded the full-length SARS-CoV-S protein except for the C-terminal 19 aa. When 293T cells were transfected with the expression plasmid pKS-SARS-St19, expression of the S protein was detected on the cell membrane (Supplementary Fig. S1). In contrast, transfection of the plasmid pKS-SARS-St19rev, in which the cDNA of C-terminal truncated SARS-CoV-S protein was inserted in the reverse orientation, did not show expression of SARS-CoV-S protein and was therefore used as a negative control for generating the VSV pseudotype. To generate a VSV pseudotype with the C-terminal truncated SARS-CoV-St19 protein, we inoculated VSV
G* on to 293T cells transfected with either pKS-SARS-St19 or pKS-SARS-St19rev. VSV
G*-G was used as a positive control, in which the deleted VSV-G protein was provided in trans encoded by pCAG-VSV-G (a kind gift from Dr Y. Matsuura, Osaka University, Japan) (Fig. 1a
). The SARS-CoV-S protein-bearing VSV pseudotype, referred to as VSV-SARS-St19, obtained from 293T cells transfected with pKS-SARS-St19, efficiently infected Vero E6 cells (Fig. 1a
). The titre of VSV-SARS-St19 was 5·0x105 infectious units (IU) ml1. Since partial deletion of the SARS-CoV-S protein cytoplasmic domain allowed efficient incorporation into VSV particles and led to the generation of pseudotype at high titre, it was suggested that the intact cytoplasmic domain of SARS-CoV-S protein may have interrupted correct assembly of the pseudotype particles.
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To determine the optimal incubation period for detection of VSV-SARS-St19 infection, we performed a time-course analysis of GFP expression. Vero E6 monolayers on 24-well glass slides were infected with VSV-SARS-St19. At various time points p.i., cells were photographed under a fluorescent microscope. The number of GFP-expressing cells in the photographs was counted using ImageJ software (http://rsb.info.nih.gov/ij/). Interestingly, GFP expression was detected clearly at 7 h p.i., although the fluorescence intensity of GFP expression was increased at 9 h p.i. (Fig. 1c). Infected cells detached from culture slides, presumably due to a cytopathic effect of VSV proteins, after incubation for 16 h (data not shown). As pseudoviruses are unable to produce progeny viruses, this system would be useful for the evaluation of cell entry mechanisms mediated by SARS-CoV-S protein. Therefore, the infection efficiency of VSV-SARS-St19 can be evaluated without incubation for up to 16 h p.i. Time-course analysis of the number of GFP-positive cells showed that quantification of VSV-SARS-St19 infection was possible at 7 h p.i. (Fig. 1c
). Therefore, we counted the number of GFP-positive cells infected with VSV-SARS-St19 at 7 h p.i. for further study.
To confirm the specificity of infection, a rabbit anti-SARS-CoV antibody was tested for its ability to neutralize VSV-SARS-St19. The antibody was raised against UV-inactivated, purified SARS-CoV and reacted with SARS-CoV-S protein in an immunofluorescence assay (see Supplementary Fig. S1); the antibody showed neutralizing activity against SARS-CoV infection on Vero E6 cells at a dilution of 1 : 2560 (data not shown). As a negative control, we used a rabbit antibody raised against SARS-CoV-N protein-specific oligopeptides (Mizutani et al., 2004). Culture medium containing 500 IU VSV-SARS-St19 was pre-incubated with serially diluted antibodies followed by inoculation on to Vero E6 cells. Pre-incubation with anti-SARS-CoV antibody showed a significant reduction in the titre of VSV-SARS-St19 in a dose-dependent manner (Fig. 2a
). Fifty per cent neutralizing activity was calculated at a dilution of 1 : 2000. In contrast, infection of Vero E6 cells with VSV
G*-G was not affected by the anti-SARS-CoV antibody, even at a lower dilution (1 : 80 dilution) (Fig. 2a
). When VSV-SARS-St19 was pre-incubated with control anti-SARS-CoV-N antibody, no neutralizing activity was observed (Fig. 2a
). The possibility of carry-over of VSV-G during the production of VSV-SARS-St19, leading to infection of Vero E6 cells, was excluded, since an anti-VSV-G monoclonal antibody (P2F3, a kind gift from Dr. S. Nagata), which showed specific neutralizing activity against VSV infection (Nagata et al., 1992
), had no effect on infection of Vero E6 cells by VSV-SARS-St19 (Fig. 2b
), but prohibited infection by VSV
G*-G. These results indicated that infection of Vero E6 cells with VSV-SARS-St19 was mediated solely by SARS-CoV-S protein.
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We further analysed whether soluble ACE2 protein could inhibit infection by VSV-SARS-St19. We introduced two amino acid substitutions at aa 374 and 378 within the putative catalytic domain of ACE2 (soACE2-NN; Supplementary Fig. S2). Purified soACE2-NN protein expressed in a baculovirus expression system was pre-incubated with VSV-SARS-St19 or VSVG*-G and the mixtures inoculated on to Vero E6 cells. As shown in Fig. 3
(a), soluble ACE2 protein strongly inhibited VSV-SARS-St19 infection in a dose-dependent manner, but did not affect VSV
G*-G infection. These observations indicated that purified soluble ACE2 specifically inhibited VSV-SARS-St19 infection. The highest concentration of soACE2 showed partial inhibition (7080 %) of VSV-SARS-St19 infectivity. However, the possibility that ACE2-independent entry of the pseudotype virus also existed could be excluded, since anti-ACE2 antibody completely inhibited VSV-SARS-St19 infectivity and almost all of the infection was dependent on ACE2 (Fig. 2c
). The amount of soluble ACE2 that neutralized 50 % of the VSV-SARS-St19 infection (50 % neutralizing dose; ND50) was estimated to be 12·5 nM. The neutralization activity of soACE2 in the present study was somewhat lower than that reported by Moore et al. (2004)
. This may have been due to the different strategies used for soACE2 preparation. The neutralization activity of soACE2 against SARS-CoV infection was lower than that of soluble mouse hepatitis virus receptor (soMHVR) against MHV infection (ND50=1 nM; Miura et al., 2004
; Zelus et al., 1998
). These observations suggested that SARS-CoV infection is regulated less strictly by the receptor function than MHV infection. Alternatively, a subdomain of ACE2 protein might represent increasing neutralization activity if bound to the SARS-CoV-S protein more strongly than the full-length protein. Indeed, neutralization of MHV-A59 infection was enhanced when the N-terminal subdomain of MHVR was used for neutralization experiments (Zelus et al., 1998
). It is noteworthy that an alternative splice variant of mouse ACE2 mRNA, consisting of the 5' half of mouse ACE2 mRNA and not containing the metalloproteinase catalytic domain, has been reported (Komatsu et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 9 February 2005;
accepted 21 April 2005.