Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2.
Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products; however, the emergence of drug-resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and therefore presents a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking cRNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently the expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by the T-ex5 PPMO prevented HA cleavage in different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2 to 3 log(10) units, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections.
Institute of Virology, Philipps University Marburg, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany. garten@staff.uni-marburg.de
Eva
E
Böttcher-Friebertshäuser
David A
DA
Stein
Hans-Dieter
HD
Klenk
Wolfgang
W
Garten
D000818:Animals; D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D004285:Dogs; D004847:Epithelial Cells / Q000821:virology*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000201:enzymology; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000201:enzymology; D057916:Influenza A Virus, H7N1 Subtype / Q000187:drug effects / Q000201:enzymology; D009980:Influenza A virus / Q000187:drug effects* / Q000201:enzymology / Q000472:pathogenicity*; D008666:Metalloendopeptidases / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology*; D060172:Morpholinos; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009025:Morpholines; D060172:Morpholinos; D012322:RNA Precursors; D012333:RNA, Messenger; D012697:Serine Endopeptidases; C421305:TMPRSS2 protein, human; D008666:Metalloendopeptidases; C039814:hemagglutinin-protease