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Molecular and cellular studies of host-mediated proteolytic maturation of dengue virus serotypes 1–4 McArthur, Steven J.


The four serotypes of dengue virus (DENV-1–4) are viruses of global concern. Although it is a key step in the lifecycle of these viruses, the host-mediated proteolytic maturation of the structural membrane precursor (prM) glycoprotein is an enigmatic molecular event. Maturation of prM is required for DENV infectivity. This proteolysis is thought to be mediated by human furin, a member of the proprotein convertase family of endoproteases that cleaves a wide variety of host cell molecules and is often hijacked by infectious agents to facilitate their lifecycle. DENV prM maturation is enigmatic for three reasons. First, a cleavage sequence that would be poorly processed by furin has been selected in all four serotypes, resulting in a large proportion of uncleaved immature prM on nascent virus particles. Second, it is unknown whether furin is the sole host enzyme responsible for cleaving prM. Third, while this event has been studied in the context of DENV-2, it is unknown whether the other three serotypes behave similarly with regard to prM maturation rate and its dependence on host furin. Research into these biological questions has been hindered by a lack of molecular tools to accurately quantify DENV-1–4 prM maturation. Here, we developed a novel adaptation of multiple reaction monitoring mass spectrometry (MRM-MS) that uses N-terminal acetyl (NTAc) labelling to differentially quantify cleaved M and uncleaved prM. We applied our NTAc-MRM methodology to determine the relative maturation rate of DENV-1–4 derived from cultured human cells and found significant differences among the serotypes. We also found that prM maturation of DENV-1 does not require active furin. Finally, we applied NTAc-MRM to quantify DENV-1–4 prM maturation in the presence of an adenovirus-expressed serine protease inhibitor (serpin), Spn4A, which stoichiometrically inhibits furin-like proteases. We found that the ER-retained form of Spn4A inhibited DENV-1–4 prM maturation, but a constitutively secreted form of Spn4A produced a robust inhibition of the DENV lifecycle, including intracellular vRNA synthesis, which cannot be explained solely in terms of prM maturation. We therefore hypothesize that host cellular targets of furin-like proteases play an important part in the DENV lifecycle.

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