Small interfering RNA inhibition of Andes virus replication

Abstract

Andes virus (ANDV) is the most common causative agent of hantavirus pulmonary syndrome (HPS) in the Americas, and is the only hantavirus associated with human-to-human transmission. Case fatality rates of ANDV-induced HPS are approximately 40%. There are currently no effective vaccines or antivirals against ANDV. Since HPS severity correlates with viral load, we tested small interfering RNA (siRNA) directed against ANDV genes as a potential antiviral strategy. We designed pools of 4 siRNAs targeting each of the ANDV genome segments (S, M, and L), and tested their efficacy in reducing viral replication in vitro. The siRNA pool targeting the S segment reduced viral transcription and replication in Vero-E6 cells more efficiently than those targeting the M and L segments. In contrast, siRNAs targeting the S, M, or L segment were similar in their ability to reduce viral replication in human lung microvascular endothelial cells. Importantly, these siRNAs inhibit ANDV replication even if given after infection. Taken together, our findings indicate that siRNAs targeting the ANDV genome efficiently inhibit ANDV replication, and show promise as a strategy for developing therapeutics against ANDV infection.

Figure 1. siRNA inhibits ANDV protein synthesis.

Vero-E6 cells were transfected with 100 nM of either scrambled siRNA (Ctrl) or pools of siRNAs targeting the small (S), large (L), or medium (M) segment of ANDV using DharmaFECT 1. After 6 h, siRNAs were removed, and cells were infected with ANDV at multiplicity of infection (MOI)  = 0.5 for 48 h. (A) Cells were lysed, and expression of the N protein was quantitated by an immunolabeling assay. (B) The levels of Gc glycoprotein, N protein, and β-actin were analyzed by Western blotting, and quantified by densitometry. (C) Data are presented as % of scrambled siRNA control, and are normalized to β-actin internal controls. (D) N protein was detected by immunofluorescence staining 48 h post-infection. Green: N protein; blue: DAPI.

Figure 2. siRNA inhibits production of infectious ANDV.

(A) Viral protein levels determined by Western blot analysis. Vero-E6 cells were transfected with 33 nM of each siRNA pool (S, L, or M) alone or in combination (S+L, S+M, L+M, or S+L+M) for 6 h. The total concentration of each transfection was brought up to 100 nM with scrambled siRNA. Cells treated with only scrambled siRNA were used as control. The cells were then infected with ANDV at MOI  = 0.5. ANDV Gc and N protein levels were determined by densitometry. (B) ANDV production and release were determined by immunofocus assays. All experiments were performed in quadruplicate.

Figure 3. siRNA inhibits ANDV replication and release when administered post-infection in Vero-E6 cells.

Vero-E6 cells were infected with ANDV at MOI  = 0.5. After virus adsorption for 2 h, the virus inoculum was removed and replaced with fresh media. Cells were then transfected with 100 nM siRNA 2, 6, 12, or 24 h post-infection. Time shown in parentheses indicates the total h post-infection. (A) N protein levels as determined by immunolabeling assays. (B) Infectious virus release as measured by immunofocus assays. After virus adsorption for 2 h, virus was removed and fresh media replaced. The cells were transfected with 100 nM siS for 6, 12, 24, or 48 h post-infection. Cell supernatants were harvested after 2 days, and infectious virus production was determined by immunofocus assays. All experiments were performed in triplicate. Data indicated above each bar represent percentage reduction in comparison to scrambled siRNA control.

Figure 4. siRNA inhibits ANDV replication in human primary lung endothelial cells (HMVEC-L).

HMVEC-L were transfected with siRNAs for 6 h, and then infected with ANDV at MOI  = 0.5. After 2 h of virus adsorption, the virus inoculum was removed and replaced with fresh media. (A) Viral protein levels as determined by Western blotting. Percent downregulation (%↓) of N or Gc protein represents percent decrease compared to non-targeting siRNA control. (B) Viral production as measured by immunofocus assays 48 h post-infection.

Figure 5. siRNA administered to HMVEC-L post-infection inhibits ANDV replication and infectious virus release.

Cells were infected with ANDV at MOI  = 0.5. After virus adsorption for 2 h, the virus inoculum was removed and replaced with fresh media. The cells were then transfected 6, 12, or 24 h post-infection with 100 nM of siS, siL, or siM using DharmaFECT 1. Time shown in parentheses indicates total h post-infection. (A) Viral protein levels as determined by Western blotting. (B) Viral release as determined by immunofocus assays. All experiments were performed in triplicate. Data shown above each bar indicates percent decrease in comparison to the scrambled siRNA control.