Host Factors Required by Human Parainfluenza Virus 3: Determinants of entry and viral spread

Project Summary Human parainfluenza viruses (HPIV) cause significant lower respiratory diseases, including croup, bronchiolitis, and pneumonia, that lead to illness or death in millions of infants and young children worldwide. Viral infection is initiated when the viral fusion complex, comprised of the surface glycoproteins HN (receptor-binding protein; hemagglutinin-neuraminidase) and F (fusion protein), mediates fusion upon HN engaging sialic acid receptor. Bound-HN triggers F to undergo conformational rearrangements that promote viral entry when F is sufficiently close to insert into the host membrane. To mediate fusion, the fusion protein precursor (F0) must first be cleaved by host proteases into its active form. F0 cleavage has been thought to be executed intracellularly by the ubiquitously expressed furin, because F0 contains a dibasic cleavage site. However, the sponsor’s laboratory found that the dibasic cleavage site underlying the assumption is an artifact of laboratory adaptation, whereas circulating strains of HPIV3 have a monobasic cleavage site that is cut by an unidentified extracellular protease in the lung. F0 cleavage by extracellular proteases opens the possibility that catalytic activation of F may be delayed until a target cell is reached, thereby enabling the fusion protein to remain in a more stable uncleaved state until reaching specific protease expressing target cells. The goal of this proposal is to identify the proteases required for HPIV3 spread in the human lung and novel mechanisms by which the virus exploits these proteases to selectively target cells, proliferate in the respiratory system, and survive host-to-host transmission. Aim 1 will identify host proteases that are necessary for circulating HPIV3 F0 cleavage and assess the impact of their expression on viral tropism in authentic lung models. Aim 2 will determine how the timing of F cleavage affects fusion efficiency, and how a delay in F cleavage until the time of infection may enable virions to evade inactivation of the viral fusion machinery by host factors during traversal of the human respiratory tract. The proposed project employs an array of cell biological, biochemical, and imaging techniques to apply complementary, yet independent approaches for understanding critical host protease determinants of HPIV3 infectivity and novel viral mechanisms for maintaining fusion protein functionality. The breadth of approaches will contribute to the project’s scientific rigor, since each question will be answered in several different ways, and provide technical and theoretical learning opportunities. By the time of completion, this project will have identified F cleaving proteases, characterized the role of cleavage in regulating F activity, and explored the broader implications of F0 for viral evasion of virucidal host factors, like mucins. Data gleaned from these studies will clarify an essential step of HPIV life cycles and our overall understanding of the HN-F viral fusion machinery. For these reasons, the project serves as an ideal training vehicle, providing an interdisciplinary path to greater independence.