Targeting Flu Through a Host Protein
By: Carol A. Rouzer, VICB Communications
Published: August 20, 2014
Inhibition of host phospholipase D suppresses influenza replication in cell culture and in vivo.
Despite widespread vaccination programs, influenza and pneumonia together are the eighth leading cause of death in the United States, costing $40 billion dollars to the economy in 2005. While pneumonia can be treated with antibiotics, therapies for flu remain limited. Drugs currently available for treatment of flu target proteins coded by the influenza virus. Thus, selection pressure may lead to rapid acquisition of resistance through mutation of the genes for these proteins. This led Vanderbilt Institute of Chemical Biology members Alex Brown and Craig Lindsley to take a novel approach to antiviral therapeutics, namely targeting a host protein required for flu infection. They now report that a selective inhibitor of human phospholipase D (PLD) suppresses influenza virus replication in cells and in vivo (T. H. Oguin, et al., (2014) J. Biol. Chem., published online July 27, DOI: 10.1074/jbc.M114.558817).
The influenza virus enters a host cell following interaction between the viral hemagglutinin protein (HA) and a sialic acid-containing protein on the cell surface, which triggers endocytosis of the viral particle (Figure 1). Acidification of the endosome as it matures leads to a conformational change in the HA protein that results in fusion of the viral membrane with the endosomal membrane. This allows the viral RNA and associated proteins to escape into the cell cytoplasm, which is followed by movement of the viral components to the nucleus. There, translation and replication of the viral RNA produces new viral macromolecules, which are ultimately assembled into complete viral particles for release and infection of new cells. The involvement of PLD in endocytosis and membrane trafficking led the investigators to postulate that the enzyme may be required for successful virus infection. Their discovery of potent and selective PLD inhibitors provided the necessary tools to test this hypothesis.
Figure 1. Process of influenza virus infection. The virus binds to sialic acid-containing proteins on the cell surface through association with the viral hemagglutinin proteins (HA1, HA2). The virus is then taken up through endocytosis. Acidification of the endosome causes a conformational change in the HA proteins that leads to fusion between the viral membrane and the endosomal membrane. This allows escape of the viral RNA and proteins into the cytoplasm. These viral components then travel to the nucleus for translation and replication. Also shown is the entry mechanism for the human immunodeficiency virus (HIV-1), which is not discussed in this article. Image reproduced by permission from Macmillan Publishers Ltd, from G. G. Karlsson Hedestam et al., (2008) Nat. Rev. Microbiol., 6, 143. Copyright 2008.
The two PLD isoforms, PLD1 and PLD2, catalyze the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) (Figure 2A). However, if available, the enzyme will preferentially use a small primary alcohol in the place of water, yielding a phosphatidylalcohol and choline as products. Frequently, investigators use this reaction to measure PLD activity or to assess the effect of preventing PA formation. The most commonly used alcohol is n-butanol (Figure 2B).
Figure 2. Reaction catalyzed by phospholipase D (PLD). (A) Under normal circumstances, the substrate, phosphatidylcholine (PC) is hydrolyzed to yield phosphatidic acid (PA) and choline. (B) In the presence of a primary alcohol, such as n-butanol, the alcohol can replace the water yielding a phosphatidylalcohol product (in this case, phosphatidylbutanol) and choline.
The researchers began their investigation by looking at the effects of H1N1 influenza virus infection on PLD activity in A549 human adenocarcinomic alveolar basal epithelial cells. Using the phosphatidylbutanol assay, they discovered that influenza infection elicited an increase in PLD activity, and they found that PLD and the influenza nucleoprotein (NP) accumulate at the periphery of the cell and then move together to a perinuclear region. Treating the cells with the PLD2-selective inhibitor VU0364739 (Figure 3) eliminated the increase in enzyme activity observed upon influenza virus infection. However, complete ablation of PLD activity required siRNA-mediated knockdown of both PLD isoforms.
Figure 3. Chemical structure of VU036739.
Inoculation of A549 cell cultures with influenza virus in the presence of n-butanol resulted in a reduced number of infected cells after 24 h, as compared to cultures inoculated in the presence of t-butanol, which cannot be utilized by PLD. These observations, which applied to infection with several different strains of flu virus, indicate that efficient uptake and/or replication of the virus requires generation of PA. siRNA-mediated knockdown of either PLD1 or PLD2 suppressed influenza replication, but the effect was the most striking in the case of PLD2. Combination of PLD2 knockdown with exposure to VU036739 was not significantly more effective than either treatment alone, confirming that the mechanism of action in both cases is suppression of PLD2 activity. VU036739 suppressed replication of multiple influenza strains, including several that are highly virulent.
To see if VU036739 could suppress influenza virus infection in vivo, the investigators treated mice every 8 h, starting 1 day before and continuing through 8 h after infection with an H1N1 strain of flu. They found reduced viral titres in the lungs of the inhibitor-treated mice compared to those of the vehicle-treated control mice. Similarly, mice treated with VU036739 every 12 h starting one day before and continuing for 3 days after a lethal infection with H1N1 virus showed significantly improved survival and delayed mortality. Under these conditions, VU036739 produced relatively few cures, but its effects were remarkable considering the fact that the compound has not been optimized for in vivo administration.
Treatment of A549 cells with VU036739 resulted in an inhibition of endocytosis kinetics, as observed by a transferrin uptake assay. The inhibitor also suppressed the recruitment of the key membrane trafficking proteins, clathrin, Rab5, and CD63. These results suggest that the suppression of influenza virus replication observed in the presence of the PLD2 inhibitor is due, at least in part, to a suppression of viral endocytosis.
To evaluate the interaction between PLD2 inhibition and the immune response, the investigators used an siRNA screen of important viral immune mediators. They found that siRNA knockdown of IRF3, Rig-I, or MxA led to a failure of influenza virus suppression by VU036739. Thus, it is clear that inhibition of PLD2 works in concert with innate viral defense mechanisms to suppress influenza virus replication.
Together, the results demonstrate that inhibition of PLD2 is a promising host-based target for therapy against influenza virus. It appears that PLD2 suppresses viral uptake and endosomal processing, giving the cell more time to mount an anti-viral defense. VU036739 is remarkably nontoxic in cell culture and in vivo, further supporting the hypothesis that PLD2 is a viable therapeutic target. Further work will be required to reach the full potential of this exciting novel approach to viral infection.