Inhibition of Influenza Virus Infection in Human Airway Cell Cultures by an Antisense Peptide-Conjugated Morpholino Oligomer Targeting the Hemagglutinin-Activating Protease TMPRSS2

• DOI: 10.1128/jvi.01294-10
• Publication Date: 2010-12-02T05:34:01Z
• Authors: Eva Böttcher-Fri...; David A. Stein; Hans-Dieter Klenk; Wolfgang Garten
• Keywords: 0301 basic medicine; Influenza A Virus, H3N2 Subtype; Morpholines; RNA Splicing; Serine Endopeptidases; Metalloendopeptidases; Bronchi; Epithelial Cells; Hemagglutinin Glycoproteins, Influenza Virus; Chick Embryo; Cell Line; Morpholinos; 3. Good health; 03 medical and health sciences; Dogs; Influenza A Virus, H1N1 Subtype; Influenza A virus; RNA Precursors; Animals; Humans; Influenza A Virus, H7N1 Subtype; RNA, Messenger; Cells, Cultured

ABSTRACT

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.