Host–rabies virus protein–protein interactions as druggable antiviral targets

Abstract

We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a hostcatalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.assembly intermediate | viral-host interaction | whole pathway screen | drug discovery paradigm | protein heterogeneity

Small-Molecule Probes Targeting the Viral PPxY-Host Nedd4 Interface Block Egress of a Broad Range of RNA Viruses

“…To our knowledge, small molecules targeting the viral PPxYhost Nedd4 interaction have not been reported; however, a number of previous studies have reported on antivirals targeting virushost interactions and/or budding of filoviruses, arenaviruses, rhabdoviruses, and others (7,9,12,31,(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81). Host Tsg101 and its recruitment by viral PTAP type L domains of RNA viruses have been targeted for the development of antivirals in a number of recent studies (7,9,31,39,71,(82)(83)(84)(85)(86)(87)(88).…”

How HIV-1 Gag assembles in cells: Putting together pieces of the puzzle

“…Thus, the cell-free system provided a “road map” for detection of assembly intermediates in cells, which are transient and present in very small quantities. Additionally, the cell-free system is currently being used as a screen for inhibitors of capsid assembly of a variety of viruses (Lingappa et al, 2013), and therefore holds promise for identifying novel antiviral small molecules in the future (see Section 6.4).…”

“…A small molecule screen based on a cell-free system for assembly of rabies virus capsids identified a compound that inhibits replication of infectious rabies virus. Interestingly, this relatively non-toxic inhibitor targets a subfraction of ABCE1 (Lingappa et al, 2013). Thus, ABCE1 may play a broad role in viral replication, and could be a target for antiviral compounds that selectively inhibit its action in viral replication.…”

Rabies vaccine preserved by vaporization is thermostable and immunogenic

“…Vials were removed at different time points and reconstituted with 0.4 ml PBS (0.01M, pH 7.4). Virus titers were measured in an 8-well chamber slide as described [15]. The mean focus forming units (ffu)/ml and standard deviation were calculated from at least three statistical replicates.…”