Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Elsevier Science full text link Elsevier Science Free PMC article
Full text links

Actions

Share

.2007 Mar;73(3):219-27.
doi: 10.1016/j.antiviral.2006.10.008. Epub 2006 Nov 7.

Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific SARS proteins, 7a/7b, 3a/3b and S

Affiliations

Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific SARS proteins, 7a/7b, 3a/3b and S

Sara Akerström et al. Antiviral Res.2007 Mar.

Abstract

The severe acute respiratory syndrome coronavirus (SARS CoV) genome has 14 potential open reading frames (ORFs). The first ORF is translated from the full-length genomic mRNA while the remaining ORFs are translated from eight subgeomic RNAs (sgRNAs). In this study, we designed small interference RNAs (siRNAs) targeting sgRNA 2, 3 and 7 and tested their efficiency and specificity in silencing the protein translated from the targeted sgRNA. Our results demonstrated that siRNA 7 could inhibit sgRNA 7, which showed 19/19 nucleotides (nt) matching, and sgRNA 8, which showed 18/19 nt matching; but, it did not inhibit the full-length genomic mRNA which showed 17/19 nt matching. Overall, each of the siRNAs can inhibit the targeted sgRNA without affecting the full-length genomic mRNA or the other sgRNAs that showed mismatch of two or more nt. Thus, siRNA could be designed so as to knockdown the expression of viral protein(s) from a targeted sgRNA during viral infection, thereby allowing the contribution of individual viral proteins to viral infection to be delineated. When Vero E6 cells expressing siRNA 2, 3 or 7 were infected with SARS-CoV, a significant reduction in the yield of progeny virus was observed. Indirect immunofluorescence assays showed that in the infected cells expressing each of the siRNAs, there was aspecific silencing of S, 3a and 7a, respectively, but the expression of nucleocapsid protein was not affected. Thus, our data suggests that the accessory proteins, i.e. 3a and 7a, could play an important role during the replication cycle of the SARS-CoV.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Schematic diagram showing the expression of viral proteins from the severe acute respiratory syndrome coronavirus (SARS-CoV) genome and subgenomic RNAs. Replicase genes (ORFs 1a and 1b) encode for two large polyproteins, pp1a and pp1ab (white solid boxes). These are expressed from the full-length genomic mRNA (RNA 1). Expression of the ORF1b gene involves ribosomal frameshifting into the −1 frame just upstream of the ORF1a translation termination codon. Open reading frames (ORFs) in the last 1/3 of the SARS-CoV genome are translated from eight subgenomic mRNAs (RNA 2 to RNA 9). Four of these encode the structural proteins (checked boxes), spike (S), membrane (M) and envelope (E) and nucleocapsid (N). Another eight SARS-CoV-unique ORFs (grey solid boxes) encode for accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b) with no significance sequence homology to viral proteins of other coronaviruses. All the mRNAs have a common leader sequence (represented by ♦), which is derived from the 5′ end of the genome.
Fig. 2
Fig. 2
The specificity of designed siRNAs was demonstrated using cDNA constructs that mimic the subgenomic RNAs of SARS-CoV. (A) Stable cell lines harboring empty vector or siRNA 2, 7 or 3 were transiently transfected with pXJ-CS-7a and harvested after 16 h. Western blot analysis was performed using anti-7a antibody to determine if the expression of 7a is silenced by the siRNA. Anti-GFP antibody was used to determine the amount of siRNA in each cell line and anti-actin antibody was used in the Western Blot to check for equal loading of samples in each well. (B) The same cell lines were transfected with pXJ-CS-8a-HA (lanes 1–4) or pXJ-CS-nsp1-HA (lanes 5–8). Western blot analysis was performed using anti-HA, anti-GFP and anti-actin monoclonal antibodies.
Fig. 3
Fig. 3
SARS-CoV subgenomic RNA-specific siRNAs can inhibit the production of progeny virus. (A) Stable cell lines harboring siRNA 2, 3 and 7, which are targeted to subgenomic RNA 2, 3 and 7 of SARS-CoV, were infected with SARS CoV at an MOI of 0.01. Viruses were harvested at 24 hpi and titers were determined by TCID50, calculations were made from the CPE induced in cell culture by different dilutions of the harvested virus. Cells transfected with empty vector were used as a control. The values given are average of three independent experiments. Error bars indicate standard deviations. (B) Vero E6 cells transiently transfected with empty vector or plasmid carry siRNA 2, 3 or 7 were infected with SARS CoV at an MOI of 0.01. Viruses were harvested and titers were determined as described above. Mock-infected cells were used as a control. The values given are average of three independent experiments. Error bars indicate standard deviations.
Fig. 4
Fig. 4
siRNA 2, 3 and 7, which targeted against subgenomic RNA 2, 3 and 7, can specifically silence the expression of S, 3a and 7a, respectively. Vero E6 cells were transfected with siRNA against S, 3a and 7a as indicated in the figure. Transfected cells were mock-infected or infected with SARS CoV at an MOI of 0.01. At 24 hpi cells were fixed with 4% formaldehyde and then incubated with specific antibodies. The cells were incubated with anti-rabbit or mouse TRITC-conjugated antibodies and analyzed by immunoflourescence assay. GFP expression used for tracking the transfected cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Åkerström S., Mousavi-Jazi M., Klingström J., Leijon M., Lundkvist A., Mirazimi A. Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus. J. Virol. 2005;79:1966–1969. - PMC - PubMed
    1. Chan W.S., Wu C., Chow S.C., Cheung T., To K.F., Leung W.K., Chan P.K., Lee K.C., Ng H.K., Au D.M., Lo A.W. Coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (SARS) Mod. Pathol. 2005;18:1432–1439. - PMC - PubMed
    1. Chen Y.Y., Shuang B., Tan Y.X., Meng M.J., Han P., Mo X.N., Song Q.S., Qiu X.Y., Luo X., Gan Q.N., Zhang X., Zheng Y., Liu S.A., Wang X.N., Zhong N.S., Ma D.L. The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line. Chin. Med. J. 2005;118:267–274. - PubMed
    1. Elbashir S.M., Martinez J., Patkaniowska A., Lendeckel W., Tuschl T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 2001;20:6877–6888. - PMC - PubMed
    1. Fielding B.C., Tan Y.J., Shuo S., Tan T.H., Ooi E.E., Lim S.G., Hong W., Goh P.Y. Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus. J. Virol. 2004;78:7311–7318. - PMC - PubMed

MeSH terms

Substances

Related information

LinkOut - more resources

Full text links
Elsevier Science full text link Elsevier Science Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp