siRNA against the G gene of human metapneumovirus
© Preston et al.; licensee BioMed Central Ltd. 2012
Received: 3 November 2011
Accepted: 7 June 2012
Published: 10 July 2012
Human metapneumovirus (hMPV) is a significant viral respiratory pathogen of infants and children, the elderly and immunocompromised individuals. Disease associated with hMPV infection resembles that of human respiratory syncytial virus (RSV) and includes bronchiolitis and pneumonia. The glycosylated G attachment protein of hMPV is required for viral entry in vivo and has also been identified as an inhibitor of innate immune responses.
We designed and validated two siRNA molecules against the G gene using A549 cells and demonstrated consistent 88-92% knock-down for one siRNA molecule, which was used in subsequent experiments. Significant reduction of G mRNA in A549 cells infected with hMPV did not result in a reduction in viral growth, nor did it significantly increase the production of type I interferon (α/β) in response to infection. However, there was a moderate increase in IFN-β mRNA expression in response to infection in siG-transfected cells compared to untransfected and si-mismatch-transfected cells. Expression of G by recombinant adenovirus did not affect type I IFN expression.
G has been previously described as a type I interferon antagonist, although our findings suggest it may not be a significant antagonist.
Human metapneumovirus (hMPV) is a member of the Pneumoviridae subfamily of the Paramyxoviridae. hMPV infects half of all infants under the age of 1 year and almost all children have been infected by the age of 10 years [2, 3]. hMPV causes acute respiratory disease in children worldwide [4–6]. Currently, there are no vaccines or targeted therapies for hMPV infection. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing found in almost all eukaryotes and is triggered by endogenous small non-coding microRNAs (miRNAs) or small interfering RNAs (siRNAs) . The recently development of synthetic sequence-specific siRNAs has allowed a variety of host and pathogen genes to be targeted for mRNA cleavage, effectively silencing or significantly reducing gene expression . Reports for RSV and hMPV suggest that RNAi can inhibit viral mRNA expression, and is a potentially effective therapy for respiratory infections [8–13].
siRNA molecules targeting the nucleoprotein (N) and phosphoprotein (P) mRNA of hMPV have been found effective at inhibiting the hMPV genome . Here we have designed and validated a siRNA molecule against the G gene, which encodes a principal attachment protein required for replication in vivo and also identified as a type I interferon (IFN) antagonist .
Design and validation of siRNA against the G gene
The hMPV isolate CAN97-83 G gene was used as target sequence to design siRNA molecules by Dharmacon, using their algorithm and ON-TARGETplus ® sense strand modification to avoid off-target effects. Two siRNA molecules were chosen for analysis and validation:
G1: sense - GCUCAAAGCAAGAGUGAAA
G2: sense - AGGUGAAAGUAGAGAACAUUU
The molecules do not target all isolates of hMPV as identified by BLAST, due to the high level of divergence in the G sequence [6, 15]. A mis-matched siRNA molecule (siMM; sense - CUAAAGUGGUAGUUGAUAUUU) was also generated as a control for the effect of transfection and off-target effects. hMPV (CAN97-83) was propagated in LLC-MK2 cells, concentrated through a 30%/60% w/v sucrose gradient, pelleted by centrifugation at 12,000 x g for 2 hours at 4 °C, and the viral titre quantified using a TCID50 assay and anti-hMPV M monoclonal antibody (Chemicon) to identify positive cells. Preliminary transfection experiments (not shown) using a fluorescein labeled RNAi delivery control (Mirus) demonstrated that transfection of 200 mM siRNA resulted in 100% transfection efficiency at 24 h. Preliminary infection experiments established that 100% of cells were infected by 24 h post-infection (pi) when exposed to hMPV at a multiplicity of infection (MOI) of 2 in the absence of trypsin.
A549 cells were transfected with siG1, siG2 or siMM 6 h prior to infection with hMPV. At 12 h and 24 h pi, total RNA was extracted from cells using TRIzol/chloroform separation and RNeasy spin columns (QIAGEN). cDNA was generated using superscript III reverse transcriptase (Invitrogen) and oligo d(T)20 primers to select for mRNA. Quantitative (q) RT-PCR was performed to quantify G mRNA reduction using the following oligonucleotides: forward primer 3′- GCAGCAATAGACATGCTCAAA-5′, reverse primer 3′-GAGCTGGTGTGGTGTTCTGA-5′, hybridization probe 3′-HEX-AAATCGTGTGGCACGTAGCAAATGC-BHQ-5′. N mRNA was also quantified using the following oligonucleotides: forward primer 3′- TTACGGTGCTGGTCAAACAA-5′, reverse primer 3′-TTTGGGCTTTGCCTTAAATG-5′, hybridization probe 3′-HEX-CTATGACCTGGTGCGAGAAATGGGC-BHQ-5′. qPCR was performed using QuantiTECT mastermix (QIAGEN) and a Rotorgene 3000 (Roche). G and N mRNA in hMPV-infected cells was quantified in relation to β-actin mRNA for each sample and in relation to untransfected cells using the ΔΔCt formula. The ΔΔCt value was then converted to percent reduction of G or N mRNA (Applied Biosystems Technotes vol 15 p. 10).
We had planned to investigate the effect of siG2 transfection on G protein expression. An antibody to detect G protein in lysed cell preparations has not been described in the literature, nor is commercially available. Affinity purified rabbit antisera were generated to a G peptide considered suitable for this purpose. However, the sera detected only a ~32 kDa band, which may have been a truncated form of G, and did not detect the non-truncated 90 kDa G protein. Due to the ambiguity of G protein detection we were not able to quantify the effect of G mRNA reduction on G protein expression. However, other studies that involve the validation of siRNA molecules against viral mRNAs demonstrate that a reduction in target mRNA similar to that demonstrated here correlate to a significant reduction in protein [9, 10, 16].
Knockdown of G does not modulate viral growth in vitro or the induction of type I interferon
In summary, we have designed and validate a siRNA molecule that is effective against the G gene of hMPV in vitro. A significant reduction in G mRNA did not reduce viral growth in vitro or induce a significant type I IFN response, suggesting that G may not be a significant type I IFN antagonist, and other hMPV proteins may play a role in modulating type I IFN induction. However hMPV G may still be a valid target for RNAi as G is required for viral replication in vivo.
This work was funded by the Australian National Health and Medical Research Council, grant number 508601.
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