Potentially Killing Two Viruses with One Drug

Based on this paper: Outlaw, V. K., Bottom-Tanzer, S., Krietler, D. F., … Moscona, A. (2019) Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus with a Single Agent

            Human Parainfluenza Virus Type 3 (HPIV3) and Respiratory Syncytial Virus (RSV) are two common viruses that often infect the respiratory systems of young children and, in extreme cases, can lead to death. There are currently no vaccines for either infection and, while there are some treatments being used for each, they are limited by the ability to distinguish between the similar immunologic responses caused by both viruses, making targeted treatment difficult. Recent research, such as conducted by this group, has been to find methods to inhibit fusion of viral membranes to cell membranes in order to prevent infections from ever taking hold. This study found that the HPIV3 C terminal heptad repeats (HRCs), the end section of the fusion protein, and their mutated derivatives were able to block the fusion of the viral protein with cell membranes in both HPIV3 virions and RSV virions.

            Previous research has demonstrated that during normal viral fusion, the F proteins act by extending the C terminal domain of their protein into the membrane of the host cell. Then, the protein folds back onto itself so the C terminus is adjacent to the N-terminus, the other side of the protein that is still stuck in the viral envelope. This interaction between the HRN and HRC is extremely stable because three proteins come together to form a 6-helix bundle (6HB).¹ The research group was interested to determine if derivatives of the HRC peptide, a smaller portions of the protein, of the HPIV3 virus would inhibit RSV viral fusion. They were specifically using the HRC domain of HPIV3 because previous studies had shown it to be a potent inhibitor of HPIV3 fusion and fusion of other paramyxoviruses.² Although RSV is not a paramyxovirus, researchers were hopeful that its clinical relatedness to HPIV3 may be indicative of similar fusion mechanisms.

Figure 1: Demonstration of viral fusion mechanism. Orange structures are HRN domains, red structures are insertion portions of the viral protein, and green structures are HRC domains. (D) shows the 6HB being formed. The goal of the research group was to make an inhibitor that would replace the green interactions in part (D) such that the HRC of the virus protein could not come together with the HRN of the virus protein.

            The first thing that the researchers needed to do was to find an inhibitor that would have the potential to obstruct viral fusion. Knowing that the HRC of HPIV3 needed to become bound the the HRN region for fusion, the research group made two mutants of the HRC, one with 2 amino acid residue changes (VI) and one with 5 amino acid residue changes (VIQKI). An amino acid residue is simply the building block unit of a protein and the letters are used to denote the changes of specific amino acids from the natural peptide sequence to the mutant sequence. These particular mutations were essential to maintain the ability of the inhibitor to form the aforementioned 6-helix bundle with the HRN. The researchers found that the VI mutant was ineffective at binding with the HRN of RSV, however, the VIQKI mutant had a strong binding affinity to create 6HB with the HRN of both HPIV3 and RSV.

            Next, the group conjugated (joined) the VIQKI mutant with lipid particles, which can insert into the cell membranes and virion envelopes, because lipid conjugation has previously been shown to increase inhibition³. VIQKI was conjugated with two different lengths of lipids and it was found that VIQKI-PEG₂₄-Chol, the longer lipid conjugate, was very effective at inhibiting both HPIV3 and RSV fusion in cell cultures of human airway epithelium, the native target cell of both viruses.

Figure 2: Demonstrates the different amount of inhibition of lipid conjugated VIQKI derivatives. VIQKI-PEG₂₄-Chol is the best inhibitor of both HPIV3 (A) and RSV (B) in cells. Viral infection is undetectable in human epithelial cells until Day 9 when treated with the artificially produced inhibitor.

            Later characterization of the structures that the VIQKI inhibitor formed with both the HPIV3 and RSV HRN peptides confirmed the 6HB that is reminiscent of the native HRC binding for the virion fusion to cell membranes. Interestingly, the opposite end of the mutant VIQKI inhibitor creates different structures in order to stabilize independently with HPIV3 and RSV. The research group then experimented with different derivatives of the mutant VIQKI in order to determine what amino acid sequence would form the most favorable interactions with both virion HRN domains. They found only one derivative that was able to minimally increase the stability of interactions between the VIQKI inhibitor derivative with both of the virus HRNs being examined.

            Overall, this study is an extremely important step in creating a therapeutic drug for both HPIV3 and RSV in young children. This study demonstrated that a peptide derivative of HPIV3, namely the VIQKI-PEG₂₄-Chol derivative, is highly effective at decreasing the cytopathic effects of both viruses by inhibiting viral fusion and entry into potentially infectable cells. Essential to the importance of this study is that a single agent can be used to inhibit both because it would allow for faster treatment because exact diagnosis would not be required prior to the start of treatment. This can hopefully lead to decreases in infant mortality as a result of infection by HPIV3 and RSV.

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1. Outlaw, V. K., Bottom-Tanzer, S., Krietler, D. F., … Moscona, A. (2019). Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus with a Single Agent. Journal of the American Chemical Society, 141, 12648-12656.
2. Chang, A & Dutch, R. E. (2012). Paramyxovirus Fusion and Entry: Multiple Paths to a Common End. Viruses, 4(4), 613-636.
3. Mathieu, C., Augusto, M. T., Niewiesk, S., Horvat, B., … Moscana, A. (2017). Broad spectrum antiviral activity for paramyxoviruses is modulated by biophysical properties of fusion inhibitory peptides. Scientific Reports, 7(43610), 1-15.
4. Porotto, M., Yokoyama, C. C., Palermo, L. M., Mungall, B., … Moscona, A. (2010). Viral entry inhibitors targeted to the membrane site of action. Journal of Virology, 84(13), 6760-6768.