RESUMEN
Antisense oligonucleotides (ODNs) are therapeutic molecules that hybridize to complementary target mRNA sequences. To further overcome the poor cellular uptake of ODNs, we proposed a novel strategy to deliver ODNs by conjugating the anti-influenza A virus (IAV) ODN with a peptide showing high affinity to the hemagglutinin (HA) on the surface of IAV particles or the IAV-infected host cells. The HA-specific binding peptides were selected by phage display, and the individual binding clones are characterized by DNA sequencing, and the selected phage was further assayed by enzyme-linked immunosorbent assay. The final selected HA-binding peptide, SHGRITFAYFAN, was conjugated to an anti-IAV ODN. The delivery efficiency and the anti-IAV effects of the conjugated molecule were evaluated in a cell-culture and a mouse-infection model. The conjugated molecule was successfully delivered into IAV-infected host cells more efficiently than the anti-IAV ODN in vitro and in vivo. Furthermore, the conjugated molecule protected 80% of the mice from lethal challenge and inhibited the plaque count by 75% compared to the unconjugated molecule (60% and 40%). These findings demonstrate that the delivery of antisense oligodeoxynucleotides to infected tissues by a virus-binding peptide-mediated system is a potential therapeutic strategy against IAV.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Oligonucleótidos Antisentido/uso terapéutico , Animales , Hemaglutininas/metabolismo , Humanos , Inmunoconjugados , Subtipo H1N1 del Virus de la Influenza A/química , Alphainfluenzavirus/química , Ratones , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/inmunología , Biblioteca de Péptidos , Péptidos/metabolismoRESUMEN
The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.
Asunto(s)
Células Epiteliales/virología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Pulmón/virología , MicroARNs/metabolismo , Proteínas de la Matriz Viral/metabolismo , Regiones no Traducidas 3' , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Biología Computacional , Regulación hacia Abajo , Células Epiteliales/citología , Interacciones Huésped-Patógeno , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/genética , Pulmón/citología , Pulmón/metabolismo , MicroARNs/genética , Análisis por Micromatrices , ARN Mensajero , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Regulación hacia Arriba , Replicación ViralRESUMEN
Natural compounds provide a large reservoir of potentially active anti-hepatitis B virus (HBV) agents. We examined the direct effects of protocatechuic aldehyde (PA; derived from the Chinese herb, Salvia miltiorrhiza) on HBV replication in HepG2 2.2.15 cell line and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA, respectively. DHBV in duck serum was analyzed by dot blot. PA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner at concentrations between 24 and 48 microg/mL. PA (25, 50, or 100 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. We provide the first evidence that PA, a novel anti-HBV substance derived from traditional Chinese herb S. miltiorrhiza, can efficiently inhibits HBV replication in HepG2 2.2.15 cell line in vitro and inhibit DHBV replication in ducks in vivo. PA therefore warrants further investigation as a potential therapeutic agent for HBV infections.
