Inhibition of Hazara nairovirus replication by small interfering RNAs and their combination with ribavirin

Abstract

Background: The genus Nairovirus in the family Bunyaviridae contains 34 tick-borne viruses classified into seven serogroups. Hazara virus (HAZV) belongs to the Crimean-Congo hemorrhagic fever (CCHF) serogroup that also includes CCHF virus (CCHFV) a major pathogen for humans. HAZV is an interesting model to study CCHFV due to a close serological and phylogenetical relationship and a classification which allows handling in a BSL2 laboratory. Nairoviruses are characterized by a tripartite negative-sense single stranded RNA genome (named L, M and S segments) that encode the RNA polymerase, the Gn-Gc glycoproteins and the nucleoprotein (NP), respectively. Currently, there are neither vaccines nor effective therapies for the treatment of any bunyavirus infection in humans. In this study we report, for the first time, the use of RNA interference (RNAi) as an approach to inhibit nairovirus replication.

Results: Chemically synthesized siRNAs were designed to target the mRNA produced by the three genomic segments. We first demonstrated that the siRNAs targeting the NP mRNA displayed a stronger antiviral effect than those complementary to the L and M transcripts in A549 cells. We further characterized the two most efficient siRNAs showing, that the induced inhibition is specific and associated with a decrease in NP synthesis during HAZV infection. Furthermore, both siRNAs depicted an antiviral activity when used before and after HAZV infection. We next showed that HAZV was sensitive to ribavirin which is also known to inhibit CCHFV. Finally, we demonstrated the additive or synergistic antiviral effect of siRNAs used in combination with ribavirin.

Conclusions: Our study highlights the interest of using RNAi (alone or in combination with ribavirin) to treat nairovirus infection. This approach has to be considered for the development of future antiviral compounds targeting CCHFV, the most pathogenic nairovirus.

Figure 1

Inhibition of HAZV replication by segment-specific siRNAs in A549 cells. A) Cells were transfected with 100 nM of the different siS (siS1 to siS4), B) siM (siM1 to siM4) or C) siL (siL1 to siL4). Twenty four hours post-transfection, cells were infected with HAZV at a MOI of 0.1. Viral titers were determined 48 hrs post-infection as described in the materials and methods section. Results are expressed as a percentage of average foci counts in siRNAs treated cells to that in siNT (siRNA negative control) transfected cells. Errors bars represent the standard deviation (SD) of the means for at least two independent experiments performed in quadruplicates. * Significant differences compared to the siNT control: Student's t-test; P < 0.05.

Figure 2

Inhibition of HAZV replication with siS1 or siS2 in A549 cells. A) Cells were transfected with 100 nM of siS1 or siS2 and then infected with HAZV at different MOI (i.e. 0.01, 0.1 and 1). B) and C) 24 hrs before HAZV infection (at a MOI of 0.1) cells were transfected with different concentrations of siS1 or siS2 (from 0.01 to 100 nM). Viral titers were determined 48 hrs post-infection. Results are presented as a ratio between the average foci counts from siRNAs treated wells and counts in siNT (siRNA negative control) transfected cells. Errors bars represent the standard deviation (SD) of the means for at least two independent experiments carried out in quadruplicates. * Significant differences compared to the siNT control: Student's t-test; P < 0.05. D) Lysates from A549 cells infected with HAZV and treated with different concentrations of siS2 (or 100 nM of siNT or siM4) were loaded on a 10% SDS-PAGE and electrotransferred on a PVDF membrane. Expression of the ~ 50 kDa NP was detected with an anti-HAZV ascite (GAPDH expression was used as loading control).

Figure 3

Antiviral activities of siS1 and siS2 transfected before and after HAZV infection. A) A549 cells were transfected with 100 nM of siNT (siRNA negative control), siS1 or siS2, 24 hrs, 48 hrs or 72 hrs before HAZV infection (MOI 0.1). B) Cells were infected with HAZV at a MOI of 0.01 and then transfected with 100 nM of siNT, siS1 or siS2, 1 hr, 8 hrs or 24 hrs post-infection. Viral titers were determined 48 hrs post-infection. Results are shown as a ratio between virus titer obtained in siRNAs treated wells and titer determined in siNT transfected cells. Errors bars represent the standard deviation (SD) of the means for at least two independent experiments done in quadruplicates. * Significant differences compared to the siNT control: Student's t-test; P < 0.05.

Figure 4

Inhibitory effect of ribavirin and combination with siS1 and siS2. A) A549 cells were infected with HAZV at a MOI of 0.1 and treated with different concentrations of ribavirin (ranging from 0 to 200 μM) for 48 hrs. Results are presented as a ratio between virus titer obtained in ribavirin treated wells and titer determined in non-treated cells. B) A549 cells were transfected with 1 nM or 10 nM of siS1 or C) with 1 nM or 10 nM siS2. Cells were infected with HAZV at a MOI of 0.1 and then incubated for two days in medium containing 25 μM of ribavirin. Viral titers were determined 48 hrs post-infection. The reduction of virus titer is determined after treatment with either ribavirin or siS1 (or siS2) and with the combination of ribavirin and siS1 or siS2. The white bar represents the theoretical sum of antiviral activity obtained with each compound. The synergistic effect of the combination of ribavirin and each siRNAs is indicated by the grey bar. Nb: no synergy was observed when siS2 was used at 10 nM with ribavirin. * Significant differences between additive and synergistic effect: Student's t-test; P < 0.05. Errors bars represent the standard deviation (SD) of the means for at least two independent experiments performed in quadruplicates.