Ebola (EBOV) and Marburg (MARV) viruses are enveloped, negative-strand RNA viruses within the Filoviridae family and are high priority Category A BSL-4 pathogens for which there are no commercially available vaccines or therapeutic agents. Thus, exploratory and innovative approaches are clearly warranted to obtain a more comprehensive understanding of virus-host interactions that regulate their replication and pathogenesis. As obligate intracellular pathogens, viruses critically depend upon host cell machinery for completion of their replication cycle including late steps of budding. Host mechanisms required for budding of enveloped RNA viruses are highly conserved across virus families, and thereby represent potential broad spectrum antiviral targets. Importantly, we and others have established that the filovirus matrix protein VP40 is both necessary and sufficient to initiate virus-like particle (VLP) assembly and budding from host cells facilitating the study of these host mechanisms under BSL-2 conditions. This approach has been used to identify proteins and pathways (e.g. the ESCRT pathway) that are critical for virus assembly, budding and spread. Moreover, we have identified and validated strategies for disrupting viral-host ESCRT interactions to block the transmission of these deadly pathogens. Our most recent published efforts establish that Ca2+ signals are triggered in host cells by viral matrix proteins and that Ca2+ acts as a master regulator of budding. Moreover, our exciting new preliminary studies reveal a completely novel aspect of this budding mechanism that involves Transient Receptor Potential Mucolipin I (TRPML1). TRPML1 is a Ca2+ activated ion channel that has essential cellular roles in endosomal sorting and exocytosis, and in plasma membrane remodeling and repair. Intriguingly, TRPML1 orchestrates the formation, both in endosomes and on the plasma membrane, of membrane protrusions that bear a provocative resemblance to those induced by eVP40 and infectious EBOV. Here, we propose that TRPML1 represents the ?missing link? in Ca2+ dependent regulation of budding. Our hypothesis is that Ca2+-dependent TRPML1-mediated plasma membrane repair machinery involving lysosomal exocytosis is hijacked by EBOV to direct the growth of plasma membrane protrusions that form VLPs/virions. Consequently, this proposal has broad impact in a critical area of human health as the mechanisms we identify will lead directly to the development of new classes of broad-spectrum host- oriented antiviral therapeutics for filoviruses. Importantly, these mechanisms are also likely to be relevant for a range of enveloped RNA viruses, including arenaviruses (LAFV and JUNV), rhabdoviruses (rabies), retroviruses (HIV-1), and paramyxoviruses (RSV and Sendai virus) that bud via similar mechanisms.
Filoviruses (Ebola and Marburg viruses) are highly lethal pathogens for which there are no commercially available vaccines or therapeutics. We recently established that the Ebola virus matrix protein VP40 triggers a Ca2+ signal in host cells that operates as a master regulator of virus budding. Exciting new data in this proposal provide evidence that an ion channel called TRPML1 is the missing link between VP40/virus induced Ca2+ signals and the generation of viral particles from the host plasma membrane. We will use innovative genetic, biochemical, and live imaging approaches to define how TRPML1 is activated by virus and its role in budding. These studies will directly impact public health, as they will provide crucial insight for the development of broad spectrum host-oriented therapeutics to control the transmission of these and other enveloped RNA viruses.
