La Crosse virus nonstructural protein NSs counteracts the effects of short interfering RNA - PubMed (original) (raw)

La Crosse virus nonstructural protein NSs counteracts the effects of short interfering RNA

Samantha S Soldan et al. J Virol. 2005 Jan.

Abstract

Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Specific inhibition of LACV replication. 293T and C6/36 cells were pretreated with LACV L, S, and M segment siRNAs. LACV (A and B) and TAHV (C and D) replication was measured in cell culture supernatants obtained from 293T (A and C) and C6/36 (B and D) cells 12, 24, 48, and 72 h after infection with LACV or TAHV at an MOI of 0.0001 PFU/cell. This representative experiment demonstrates virus replication in mock-transfected cells, cells that were pretreated with LAC528c (a control for the LACV S segment), and cells that were treated with LACS103, LACM1566, and LACL1949 siRNAs, designed for the S, M, and L segments of LACV, respectively. Error bars represent the variabilities (standard errors of the means [SEM]) between triplicate samples for each time point. TAHV replication was not inhibited by pretreatment with LACV siRNAs, except for LACM2860, which inhibited TAHV replication 138-fold at 48 h postinfection of 293T cells (data not shown).

FIG. 2.

FIG. 2.

siRNA-mediated inhibition of LACV replication. The percent decrease in LACV titer in LACV siRNA-treated cells was compared to that in mock-transfected cells at 48 h for infections with various MOIs (0.01, 0.001, and 0.0001 PFU/cell). The pretreatment of 293T cells with LACV siRNAs resulted in an up to 99% inhibition of virus replication when the cells were challenged with LACV at a low MOI (0.0001). The efficiency of LACV siRNAs decreased as cells were challenged with increasing MOIs (0.001 and 0.01). Error bars represent variabilities (SEM) between triplicate samples for each time point.

FIG. 3.

FIG. 3.

Inhibition of LACV replication is reduced when siRNAs are delivered after virus infection. 293T (A) and C6/36 cells (B) were infected with LACV at an MOI of 0.0001 PFU/cell and were transfected 24 h later (arrow) with LACV L1949, S103, and M1566 segment siRNAs or control siRNAs (LAC528c and LACM1566c). Error bars represent variabilities (SEM) between triplicate samples for each time point.

FIG. 4.

FIG. 4.

LACV glycoprotein expression is decreased in 293T cells that are pretreated with LACV siRNAs. LACV antigen accumulation in 293T (A to C) and C6/36 (D to F) cells was measured by FACS with a panel of LACV G1-specific antibodies 48 h after siRNA transfection. As shown in these representative plots, LACV-specific siRNAs, including M1627 (A and D) and S356 (B and E), inhibited LACV G1 expression. The LACV control siRNA 528c (C and F) did not affect LACV glycoprotein expression in 293T cells. LACV siRNAs did not decrease TAHV glycoprotein expression in either 293T or C6/36 cells, with the exception of M2860 (data not shown).

FIG. 5.

FIG. 5.

RNAi resistance of LACV. Supernatants harvested 72 h after LACV infection of 293T cells that had been pretreated with LACV siRNAs were used to infect a second round of 293T cells that were pretreated with the same LACV siRNAs (LACM1566 [A], LACL1949 [B], and LACS103 [C]). Plaque assays were performed 12, 24, 48, and 72 h after infection with either LACV or LACV cultured for 72 h in 293T cells that were pretreated with LACM1566. In all instances, green lines represent mock siRNA-treated wild-type LACV while red lines represent LACV siRNA-treated wild-type LACV (A), LACL1949 (B), or LACS103 (C). (A) Green, mock siRNA-treated wild-type LACV; purple, mock siRNA-treated, LACM1566-pretreated LACV; red, LACM1566 siRNA-treated wild-type LACV; blue, LACM1566 siRNA-treated, LACM1566-pretreated LACV. (B) Green, mock siRNA-treated LACV; purple, mock siRNA-treated, LACL1949-pretreated LACV; red, LACL1949 siRNA-treated LACV; blue, LACL1949 siRNA-treated, LACL1949-pretreated LACV. (C) Green, mock siRNA-treated LACV; purple, mock siRNA-treated, LACS103-pretreated LACV; red, LACS103 siRNA-treated LACV; blue, LACS103 siRNA-treated, LACS103-pretreated LACV. A significant (P < 0.01 by Student's t test) increase in titer was observed at 12 to 72 h for viruses that were previously exposed to LACS103 compared to naïve, wild-type LACV.

FIG. 6.

FIG. 6.

NSs inhibition of GAPDH RNAi. (A) Expression of NSs FLAG in 293T cells and inhibition of RNA silencing. LACV NSs-FLAG (1.0 μg of DNA/well) was transfected into 293T cells by use of the ProFection mammalian calcium phosphate transfection system (Promega) 24 h prior to GAPDH and control siRNA treatment. GAPDH expression in 293T cells was assessed 72 h after treatment with GAPDH siRNA (+) and controls, as measured by Western blotting (see Materials and Methods). Effective silencing of GAPDH was observed for 293T cells that were transfected with a GAPDH siRNA (+) (Ambion) (lane 2) compared to mock-transfected cells (lane 1) or cells that were transfected with a negative control for GAPDH siRNA (−) (lane 3) (Ambion). A similar inhibition of GAPDH expression was observed for 293T cells that were transfected with a vector containing NSs in the wrong orientation (NSs-WO; lane 6) or with the BAP-FLAG vector (lane 10). GAPDH expression was not silenced in 293T cells that were transfected with NSs in the correct orientation (NSs-Co; lane 4) or with the NSs-FLAG fusion protein (lane 8). (B) Quantitative real-time TaqMan PCR was used to examine the effects of GAPDH siRNA on GAPDH expression. RNAs were extracted from NSs-FLAG-, BAP-FLAG-, and mock-transfected 293T cells 72 h after GAPDH siRNA treatment. Quantitative real-time PCRs for GAPDH were performed on an ABI Prism 770 sequence detection system (Perkin-Elmer). A normalization 18S RNA experiment was performed simultaneously for each sample by the use of 18S RNA primers and probe. CT values were calculated for the target (GAPDH CT) and the standard (18S RNA CT), and the results are expressed as ΔCTs (mean GAPDH CT − mean 18S RNA CT). High CT values are equated with low copy numbers. Therefore, low ΔCTs correlate with high GAPDH expression levels and high ΔCTs correlate with low GAPDH expression levels. GAPDH mRNA expression was significantly reduced in 293T cells that were transfected with the GAPDH siRNA (GAPDH+) compared to mock-transfected cells and cells that were transfected with a GAPDH siRNA negative control (GAPDH−). In addition, GAPDH mRNA was significantly increased in GAPDH+ siRNA-transfected 293T cells that were cotransfected with NSs-FLAG compared to mock- and BAP-FLAG-transfected 293T cells (asterisk; P < 0.01 by ANOVA). (C) BAP-FLAG and NSs-FLAG expression. As controls, 293T cells were mock transfected (lane 2), transfected with the 3X-FLAG vector alone (lane 3), and transfected with a control FLAG fusion protein, BAP-FLAG (lane 1). With an anti-FLAG monoclonal antibody, NSs FLAG was detected (lane 4) at approximately 16 kDa, the predicted size for the NSs-FLAG fusion protein.

FIG. 7.

FIG. 7.

Inhibition of LACV M segment RNAi by NSs. 293T cells were transfected with either NSs-FLAG or BAP-FLAG, were treated 24 h later with the LACM1566 siRNA, and were cotransfected with the LACV M segment (G1/G2). LACV glycoprotein expression was measured by FACS with a cocktail of LACV G1-specific antibodies (807.31, 807.33, 813.13, and 807.35). Representative FACS plots from one of three experiments are shown (A to C). (A) Transfection with NSs-FLAG and BAP-FLAG did not affect LACV glycoprotein expression in LACM (G1/G2)-cotransfected cells. (B) LACV glycoprotein expression was not inhibited significantly in 293T cells that were pretreated with an M segment control siRNA. (C) LACV glycoprotein expression was downregulated in mock-transfected or BAP-FLAG-cotransfected 293T cells that were pretreated with the LACM1566 siRNA. Importantly, LACM expression was partially recovered in 293T cells that were cotransfected with NSs. (D) This experiment was performed on three separate occasions, and the data were analyzed as percentages of maximum expression of the mean fluorescence intensities. LACV glycoprotein expression was significantly increased in NSs-FLAG-transfected 293T cells that were treated with the LACM1566 siRNA compared to mock- and BAP-FLAG-transfected 293T cells (asterisk; P < 0.05 by ANOVA).

References

    1. Al-Kaff, N. S., S. N. Covey, M. M. Kreike, A. M. Page, R. Pinder, and P. J. Dale. 1998. Transcriptional and posttranscriptional plant gene silencing in response to a pathogen. Science 279:2113-2115. - PubMed
    1. Arunagiri, C. K., L. P. Perera, S. B. Abeykoon, and J. S. Peiris. 1991. A serologic study of California serogroup bunyaviruses in Sri Lanka. Am. J. Trop. Med. Hyg. 45:377-382. - PubMed
    1. Bridgen, A., F. Weber, J. K. Fazakerley, and R. M. Elliott. 2001. Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis. Proc. Natl. Acad. Sci. USA 98:664-669. - PMC - PubMed
    1. Brigneti, G., O. Voinnet, W. X. Li, L. H. Ji, S. W. Ding, and D. C. Baulcombe. 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17:6739-6746. - PMC - PubMed
    1. Bucher, E., T. Sijen, P. De Haan, R. Goldbach, and M. Prins. 2003. Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions. J. Virol. 77:1329-1336. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources