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

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.

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

“…HA is a trimeric glycoprotein that is present in the membrane envelope of all Influenza viruses and requires cleavage into the subunits HA1 and HA2 by a specific host cell protease. To further complicate the issue, these cleavage sites can often vary between viral strains 13 . As the expression of proteases capable of cleaving HA is restricted to specific tissues, proteases are often added to cell culture media in order to facilitate viral fusion and entry into the selected host cell 13 .…”

“…To further complicate the issue, these cleavage sites can often vary between viral strains 13 . As the expression of proteases capable of cleaving HA is restricted to specific tissues, proteases are often added to cell culture media in order to facilitate viral fusion and entry into the selected host cell 13 . TPCK-trypsin is one example of a commonly used protease that is used in cell culture with both MDCK and Vero cells: facilitating multi-cycle replication (in the absence of any trypsin inactivating serum) through the proteolytic activation of viral HA 14,15 .…”

Matriptase, HAT, and TMPRSS2 Activate the Hemagglutinin of H9N2 Influenza A Viruses

“…TMPRSS2 is expressed in epithelial cells of the respiratory, gastrointestinal, and urogenital tracts, with high expression levels in prostate and colon (47,48). Recently, TMPRSS2 was shown to support proteolytic activation of HA with a monobasic cleavage site in human Calu-3 airway epithelial cells and Caco-2 intestinal epithelial cells (49,50). Matriptase (also known as epithin, ST14, and MT-SP1) is one of the best-studied members of the TTSP family and plays essential roles in the formation and integrity of the oral and intestinal epithelium and the epidermis (30,47,51).…”

Pathogenesis and Transmission Assessments of Two H7N8 Influenza A Viruses Recently Isolated from Turkey Farms in Indiana Using Mouse and Ferret Models

“…Calu-3 cells express serine proteases, such as TMPRSS2, to cleave HA0 and, therefore, are able to support the replication of most influenza viruses without the addition of exogenous trypsin (26). The ability of H7N8 LPAI and HPAI viruses to replicate in Calu-3 cells was compared with that of several human isolates from the Eurasian lineage (H7N7 HPAI NL/219 virus and H7N9 LPAI Anhui/1 virus), human and avian isolates from the North American lineage (H7N3 HPAI Can/504 virus and H7N9 LPAI Gs/NE/11 virus), and one human seasonal strain (H3N2 Pan/99 virus).…”