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. 2004 Jun 4;318(3):719-25.
doi: 10.1016/j.bbrc.2004.04.083.

HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus

Norio Yamamoto  1 , Rongge Yang, Yoshiyuki Yoshinaka, Shinji Amari, Tatsuya Nakano, Jindrich Cinatl, Holger Rabenau, Hans Wilhelm Doerr, Gerhard Hunsmann, Akira Otaka, Hirokazu Tamamura, Nobutaka Fujii, Naoki Yamamoto
Affiliations

HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus

Norio Yamamoto et al. Biochem Biophys Res Commun. .

Abstract

A novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quantitative RT-PCR analysis showed that nelfinavir could decrease the production of virions from Vero cells. Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examined clinically for the treatment of SARS and has potential as a good lead compound for designing anti-SARS drugs.

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Figures

Fig. 1
Fig. 1
Effect of nelfinavir on replication of SARS-CoV in Vero E6 cells. (A) Cytopathic effect (CPE) in Vero E6 cells. The cells were treated with phosphate-buffered saline (PBS) as a control, 10 μM nelfinavir, or 10 μM ritonavir for 3 h before infection. CPE was observed 36 h after infection. (B) Immunofluorescence assay (IFA) of infected cells treated with PBS, 10 μM nelfinavir, or 10 μM of ritonavir. Cells were fixed with methanol 24 h after infection and stained with serum samples from SARS patients.
Fig. 2
Fig. 2
Real-time RT-PCR for SARS-CoV RNA. Vero E6 cells had been treated with nelfinavir or ritonavir for 3 h before infection at the concentration of 10 μM. Instead of these drugs, PBS was added as a negative control. Viral RNA in the culture supernatant was collected 24 h after infection and quantified by the use of a fluorogenic probe. All samples were analyzed in triplicate.
Fig. 3
Fig. 3
Effect of various timings of drug addition on SARS-CoV replication in Vero E6 cells. (A) CPE in Vero E6 cells when drugs were added at the time of infection or 3 h after infection. The cells were treated with phosphate-buffered saline (PBS) as a control, 10 μM nelfinavir, or 10 μM ritonavir and CPE was observed 36 h after infection. (B) IFA of infected cells when drugs were added at the time of infection or 3 h after infection. The cells were treated with PBS as a control, 10 μM nelfinavir, or 10 μM ritonavir. The cells were fixed with methanol 24 h after infection and stained with serum samples from SARS patients.
Fig. 4
Fig. 4
Real-time RT-PCR for SARS-CoV RNA with various timings of drug addition. Nelfinavir or ritonavir was added at the time of infection or 3 h after infection at the concentration of 10 μM. Instead of these drugs PBS was added as a negative control. Viral RNA in the culture supernatant was collected 24 h after infection and quantified by the use of a fluorogenic probe. All samples were analyzed in triplicate.
Fig. 5
Fig. 5
Entry inhibition assay. To quantify SARS-CoV RNA which entered the cells, Vero E6 cells were pretreated with the drugs and infected with SARS-CoV. Cells were washed with PBS 3 times 3 h after infection. Subsequently viral RNA and 18S ribosomal RNA in the cells were quantified. All samples were analyzed in triplicate.
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