Seed sequence-matched controls reveal limitations of small interfering RNA knockdown in functional and structural studies of hepatitis C virus NS5A-MOBKL1B interaction

Abstract

Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to the case for other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with small interfering RNA (siRNA) knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a cocrystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 Å, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18-amino-acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, which indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches, we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data.

Importance: Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA, treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNA interference (RNAi)-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.

FIG 1

MOBKL1B interacts with NS5A. Yeast two-hybrid analysis of the interaction between MOBKL1B and 10 HCV proteins was performed. MOBKL1B-AD (GAL4 activation domain) fusion or a control AD construct (empty) was coexpressed with core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, or NS5B-DBD (GAL4 DNA binding domain) fusions or control DBD construct (empty) and tested for positive yeast two-hybrid interactions under selective nutritional conditions (lacking leucine, tryptophan, histidine, and adenosine). Each viral protein was derived from the Jc1 sequence.

FIG 2

Treatment with MOBKL1B siRNA inhibits HCV RNA replication. (A) HCV growth in culture is inhibited by MOBKL1B depletion. Huh-7.5 cells were transfected twice with MOBKL1B-specific or irrelevant (IRR) siRNA at 24-h intervals and were infected with HCV Jc1 at 48 h after the second transfection. Depletion of endogenous MOBKL1B protein was analyzed by Western blotting (top panel); β actin levels are shown as a control. The results shown are representative of three independent experiments. (B) MOBKL1B depletion blocks viral RNA replication. Depletion of MOBKL1B in CD81 knockdown Huh-7.5 cells was performed, as described above. Two individual MOBKL1B-specific siRNAs (1 and 2) were used. Jc1FLAG(p7-nsGluc2A) RNA was transfected at 48 h after the second siRNA transfection. At each indicated time point, released luciferase was measured to analyze viral RNA replication (mean of n = 3; error bars, SD). RLU, relative light units. (C) MOBKL1B knockdown does not affect the entry of pseudoparticles. After siRNA 1 treatment, Huh-7.5 cells were infected with luciferase reporter pseudoparticles at 48 h after the second siRNA transfection. Luciferase was measured at 48 h after infection. Values are normalized to RLU measured in irrelevant-siRNA-treated cells (mean of n = 3; error bars, SD). VSV-G, vesicular stomatitis virus glycoprotein; H77-HCV, HCV strain H77 glycoproteins. (D) MOBKL1B is also required for the replication of J6/JFH1-based HCV NS5A recombinants. MOBKL1B depletion and viral genome transfection were performed as described above. Recombinant HCV variants were as follows: 1a, J6/JFH1(H77-NS5A); 1b, J6/JFH1(J4-NS5A)_{R867H, C1185S}; 2a, J6/JFH1(J6-NS5A)_F772S; 3a, J6/JFH1(S52-NS5A)_D1975G; 4a, J6/JFH1(ED43-NS5A)_{F772S, Y1644H, E2267G}; 5a, J6/JFH1(SA13-NS5A)_{R1978G, S2416G}; 6a, J6/JFH1(HK6a-NS5A)_I2268N; 7a, J6/JFH1(QC69-NS5A). The results shown are representative of two independent experiments. The dashed line indicates the quantitation limit of the assay.

FIG 3

Mapping the MOBKL1B binding site in NS5A. (A) Yeast two-hybrid mapping of the MOBKL1B binding motif in NS5A. An MOBKL1B-AD fusion or a control AD construct (empty) was coexpressed with serially deleted NS5A-DBD fusion or control DBD construct (empty) and tested for positive yeast two-hybrid interactions under selective nutritional conditions (lacking leucine, tryptophan, histidine, and adenosine). NS5A deletion mutants were derived from genotype 1b, Con1. Numbers indicate the Con1 NS5A amino acid positions. (B) A schematic of NS5A is shown in the top panel. JFH1 NS5A amino acid positions are indicated. Yeast two-hybrid interactions are shown in the bottom panel. Numbers indicate the JFH1 NS5A amino acid positions. Note that JFH1 NS5A residues 310 to 335 are equivalent to Con1 NS5A residues 314 to 339. (C) Biosensor binding isotherms for full-length MOBKL1B binding to GST-JFH1 NS5A peptides. Triplicate data points are shown for each condition. Apparent binding affinities (K_ds) derived after fitting to a simple one-to-one binding model were as follows: NS5A_310-335, 24 ± 1 μM; NS5A_310-327, 18 ± 1 μM; NS5A_310-324, 182 ± 39 μM; NS5A_310-320, 830 ± 100 μM; NS5A_314-329, no binding detected (means ± SD). Note that adaptive mutations in 1b, 2a, 3a, 4a, 5a, and 6a recombinant HCV variants (Fig. 2D) are not located in the NS5A MOBKL1B binding motif. (D) Sequence alignment of the MOBKL1B minimal binding motif of NS5A for the seven HCV genotypes. Asterisks indicate residues absolutely conserved in all genotypes. NS5A amino acid positions (JFH1 numbering) are indicated.

FIG 4

Structure of core MOBKL1B-NS5A_308-327 complex. (A) Core MOBKL1B (residues 33 to 216) with the NS5A_308-327 peptide depicted in ribbon representation as two views related by a 180° rotation. Nine α-helices and two β-strands are numbered as previously reported (27). There are two MOBKL1B-NS5A peptide interactions, interaction 1 and interaction 2, in which NS5A_308-327 peptide was determined from L309 to E323 in interaction 1 and from A308 to D316 in interaction 2. H, α-helix; S, β-strand; N, N terminus; C, C terminus. (B) Biosensor binding isotherms for full-length MOBKL1B binding to GST-NS5A peptides. Triplicate data points are shown for each condition. No binding was detected for NS5A_308-327 W312A and Y317A or for NS5A_305-316 Wt in interaction 2. (C) Close-up views of interaction 1, showing the NS5A peptide (stick) binding to the shallow hydrophobic patch of MOBKL1B in space-filling models or ribbon representation (left and right panels, respectively). Amino acid residues participating in the MOBKL1B-NS5A interaction are highlighted by showing their side chains and labeled with the corresponding amino acid sequence number. Left panel, NS5A residues labeled in black. Right panel, MOBKL1B residues in blue. Black arrows in the left panel point out trans conformational amide bonds at P319 and P320 NS5A residues that are putative CypA substrates.

FIG 5

MOBKL1B depletion reduces CypA-independent HCV replication despite lack of MOBKL1B-NS5A interaction. (A) NS5A bearing the DEYN mutation does not bind to MOBKL1B. One-STrEP-tag (OST)-fused NS5A was purified using Strep-Tactin Sepharose from Jc1 NS5A-OST wild-type- or DEYN mutant-transfected Huh-7.5 cell lysates. Left panels, Western blot signals of MOBKL1B and NS5A in the input lysates using anti-MOBKL1B and anti-NS5A antibodies. Right panels, Western blot signals following affinity purification of NS5A. (B) Depletion of endogenous CypA or MOBKL1B protein in CD81 knockdown Huh-7.5 cells was analyzed by Western blotting; β-actin is included as a control. (C) Viral genome replication following CypA or MOBKL1B knockdown. At each indicated time point, released luciferase was measured to analyze viral RNA replication (mean of n = 3; error bars, SD). RLU, relative light units.

FIG 6

Seed sequence-matched siRNA shows that MOBKL1B is not required for HCV RNA replication in Huh-7.5 cells. (A) Three MOBKL1B-specific siRNAs (1, 2, and 3), their seed sequence-matched control siRNAs (1 C911, 2 C1012, and 3 C911), or irrelevant (IRR) siRNA was transfected into Huh-7.5 cells as described for Fig. 2. Depletion of endogenous MOBKL1B protein was analyzed by Western blotting. β-Actin levels are shown as a control. (B) MOBKL1B siRNA treatment is not cytotoxic. Huh-7.5 cells were transfected twice with three individual MOBKL1B-specific siRNAs (1, 2, and 3), their seed sequence-matched siRNAs (1 C911, 2 C1012, and 3 C911), or irrelevant (IRR) siRNA at 24-h intervals. Toxicity of the MOBKL1B knockdown was evaluated with a luminescent cell viability assay (mean of n = 3; error bars, SD). RLU, relative light units. (C) MOBKL1B depletion does not inhibit HCV genome replication. Depletion of MOBKL1B in CD81 knockdown Huh-7.5 cells was performed, as described above. Jc1FLAG(p7-nsGluc2A) was transfected at 48 h after the second siRNA transfection. Medium was collected 48 h later for luciferase quantitation. (D) MOBKL1B depletion and infectious virus production. Naive Huh-7.5 cells were infected from the media from cells treated with IRR, siRNA 3, and siRNA 3 C911 (shown in panel C), and luciferase was measured to analyze infectious virus production. Values are normalized to RLU measured in irrelevant siRNA-treated cells (mean of n = 3; error bars, SD).