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.
KeywordssiRNA hMPV Type I interferon
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.
- van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, Osterhaus AD: A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001, 7: 719-724. 10.1038/89098PubMedView ArticleGoogle Scholar
- Principi N, Bosis S, Esposito S: Human metapneumovirus in paediatric patients. Clin Microbiol Infect 2006, 12: 301-308. 10.1111/j.1469-0691.2005.01325.xPubMedView ArticleGoogle Scholar
- Williams JV, Harris PA, Tollefson SJ, Halburnt-Rush LL, Pingsterhaus JM, Edwards KM, Wright PF, Crowe JE: Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children. N Engl J Med 2004, 350: 443-450. 10.1056/NEJMoa025472PubMedPubMed CentralView ArticleGoogle Scholar
- Biovin G, Abed Y, Pelletier G, Ruel L, Moisan D, Cote S, Peret TC, Erdman DD, Anderson LJ: Virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups. J Infect Dis 2002, 186: 1330-1334. 10.1086/344319View ArticleGoogle Scholar
- Greensill J, McNamara PS, Dove W, Flanagan B, Smyth RL, Hart CA: Human metapneumovirus in severe respiratory syncytial virus bronchiolitis. Emerg Infect Dis 2003, 9: 372-375. 10.3201/eid0903.020289PubMedPubMed CentralView ArticleGoogle Scholar
- van den Hoogen BG, Herfst S, Sprong L, Cane PA, Forleo-Neto E, de Swart RL, Osterhaus AD, Fouchier RA: Antigenic and genetic variability of human pneumoviruses. Emerg Infect Dis 2004, 10: 658-666. 10.3201/eid1004.030393PubMedPubMed CentralView ArticleGoogle Scholar
- Barik S: Control of nonsegmented negative-strand RNA virus replication by siRNA. Virus Research 2004, 102: 27-35. 10.1016/j.virusres.2004.01.012PubMedView ArticleGoogle Scholar
- Barik S, Bitko V: Prospects of RNA interference therapy in respiratory viral diseases: update 2006. Expert Opin Biol Ther 2006, 6: 1151-1160. 10.1517/14712518.104.22.1681PubMedView ArticleGoogle Scholar
- Bitko V, Barik S: Phenotypic silencing of cytoplasmid genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiology 2001, 1: 34. 10.1186/1471-2180-1-34PubMedPubMed CentralView ArticleGoogle Scholar
- Bitko V, Musiyenko A, Shulyayeva O, Barik S: Inhibition of respiratory viruses by nasally administered siRNA. Nature Medicine 2005,11(1):50-55. 10.1038/nm1164PubMedView ArticleGoogle Scholar
- Zhang W, Yang H, Knog X, Mahapatra S, San Juan-Vergara H, Hellermann G, Behera S, Singam R, Lockey RF, Mahapatra SS: Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene. Nature Medicine 2005,11(1):56-62. 10.1038/nm1174PubMedView ArticleGoogle Scholar
- Kong X, Zhang W, Lockey RF, Auais A, Piedimonte G, Mohapatra : Respiratory syncytial virus infection in Fisher 344 rats is attenuated by short interfering RNA against the RSV-NS1 gene. Genetic Vaccines and Therapy 2007, 5: 4. 10.1186/1479-0556-5-4PubMedPubMed CentralView ArticleGoogle Scholar
- Deffrasnes C, Cavahagh MH, Goyette N, Cui K, Ge Q, Seth S, Templin MV, Quay SC, Johnson PH, Boivin G: Inhibition of human metapneumovirus replication by small interfering RNA. Antiviral therapy 2008, 13: 821-832.PubMedGoogle Scholar
- Bao X, Liu T, Shan Y, Li K, Garofalo RP, Casola A: Human metapneumovirus glycoprotein G inhibits innate immune responses. PLoS Pathog 2008,4(5):.View ArticleGoogle Scholar
- Peret TC, Abed Y, Anderson LJ, Erdman DD, Boivin G: Sequence polymorphism of the predicted human metapneumovirus G glycoprotein. J Gen Virol 2004, 85: 679-686. 10.1099/vir.0.19504-0PubMedView ArticleGoogle Scholar
- Munir S, Kaur K, Kapur V: Avian metapneumovirus phosphoprotein targeted RNA interference silences the expression of viral proteins and inhibits virus replication. Antivir Res 2006, 69: 46-51. 10.1016/j.antiviral.2005.09.004PubMedView ArticleGoogle Scholar
- Biacchesi S, Skiadopoulos MH, Yang L, Lamirande EW, Tran KC, Murphy BR, Collins PL, Buchholz UJ: Recombinant human metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate. J Virol 2004, 78: 12877-12887. 10.1128/JVI.78.23.12877-12887.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Spann KM, Tran KC, Chi B, Rabin RL, Collins PL: Suppression of the induction of alpha, beta and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages. J Virol 2004, 78: 4363-4369. 10.1128/JVI.78.8.4363-4369.2004PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.