C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response

Abstract

Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.

Figure 1. Size and abundance of small RNA reads Mmapping to the viral genomes.

The abundance of 19–30-mer sRNA reads mapping to the WNV (A), SINV (B) and LACV (C) genomes based on size. Abundance is represented as a percentage of the total viRNAs from each sample. The black bars correspond with samples collected from S2 cells and white bars from C6/36 cells.

Figure 2. viRNA coverage of the WNV genome in C6/36 and S2 cells.

Complete genome of WNV (11,029 nt.) showing intensity at each nucleotide of the genome in C6/36 (A) and S2 (B) cells. Plotted are the 19–30-mer viRNA reads. Reads originating from the genomic, positive strand are represented in blue above the x-axis and those originating from the negative strand are represented in red below the x-axis.

Figure 3. viRNA coverage of the TE3′2J SINV genome in C6/36 and S2 cells.

Complete genome of TE3′2J SINV (11,385 nt.) showing intensity at each nucleotide of the genome in C6/36 (A) and S2 (B) cells. Plotted are the 19–30-mer viRNA reads. Reads originating from the genomic, positive strand are represented in blue above the x-axis and those originating from the negative strand are represented in red below the x-axis. The green vertical line represents the location of the subgenomic promoter.

Figure 4. viRNA coverage of the LACV/Human/1960 strain genome in C6/36 and S2 cells.

Complete genome of LACV/Human/1960 strain showing intensity at each nucleotide of the genome in C6/36 (A,C,E) and S2 (B,D,F) cells. A and B correspond with the L gene segment (6,980 nt), C and D the M gene segment (4,526 nt), and E and F to the S gene segment (984 nt). Plotted are the 19–30-mer viRNA reads across the length of each segment represented by the x-axis. Reads originating from the genomic, negative strand are represented in red below the x-axis and those originating from the positive strand are represented in blue above the x-axis.