Antiviral compounds displaying remarkable features have been identified by an unconventional drug screen and advanced through animal validation. Efficacy is observed against the six viral families causing most human respiratory viral disease, irrespective of strain, including both influenza (FLUV) and SARS-CoV-2, with cell culture EC50 at or below 100 nM. Survival benefit is demonstrated in pigs against another member of family Coronaviridae, porcine epidemic diarrhea virus (PEDV), and shown equally effective in mild and severe disease. Respiratory syncytial virus (RSV) titer is reduced by drug treatment in cotton rats. A substantial barrier to viral resistance is demonstrated for FLUV. Drug resin affinity chromatography (DRAC) reveals a novel drug target: a multi-protein complex (MPC) formed transiently, in an energy-dependent fashion, and composed of host proteins implicated in both viral lifecycles and manipulation of innate immunity. The protein composition of this host MPC is modified upon viral infection, with increase or decrease of some proteins and appearance or complete loss of others. Valosin-containing protein, also known as Transitional endoplasmatic reticulum ATPase (VCP/p97), is present in the target MPC of uninfected cells and significantly increased in both FLUV and CoV infection. SQSTM1/p62, a key regulator of the autophagy pathway of innate immunity whose dysfunction is implicated in cytokine storm, is i) found in the target MPC from uninfected cells, ii) diminished in DRAC eluates by infection, and iii) restored by drug treatment of infected cells. 14-3-3 is one of likely several proteins that comprise the drug-binding site. Advanced compounds with improved pharmacokinetic (PK) properties and lung exposure are approaching criteria for a Target Product Profile. We propose these novel drug targets to comprise a previously unappreciated molecular basis for homeostasis that is modified by viruses to allow exploitation for viral propagation and is restored by treatment with the therapeutic compounds presented. This discovery has transformative implications for treatment of respiratory viral-related disease, applicable to everything from seasonal FLUV to COVID-19 and future novel respiratory viruses, due to the pan-family nature of drug activity and barrier to resistance development.

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In an attempt to produce a protein that will allow determination of the native human immunodeficiency virus type 1 (HIV-1) gp120 (Env) structure in its trimeric state, we fused the globular head of gp120 to the stalk region of influenza virus A (X31) hemagglutinin (HA). The chimeric protein (EnvHA) has been expressed by using a recombinant vaccinia virus system, and its functional characteristics were determined. EnvHA is expressed as a 120-to 150-kDa protein that can oligomerize to form dimers and trimers. It retains the low-pH (5.2 to 5.4) requirement of X31-HA to trigger membrane fusion but, unlike X31-HA, it is not absolutely dependent on exogenously added trypsin for protein processing to release the HA2 fusion peptide. In terms of receptor binding the chimeric protein retains specificity for human CD4 but, in relation to the membrane fusion event, it appears to lose the Env coreceptor specificity of the parental HIV-1 strains: NL43 for CXCR4 and JRFL for CCR5. These properties suggest that stable, functional EnvHAs are being produced and that they may be exploited in terms of structural studies. Further, the potential of introducing the envHA genes into influenza viruses, by use of reverse genetics, and their use as a therapeutic vaccine for HIV are discussed.Attachment and entry of human immunodeficiency virus type 1 (HIV-1) to a host cell is mediated by the envelope (Env) glycoproteins. Env monomers are synthesized as 160-kDa glycoproteins (gp160) in the endoplasmic reticulum, where oligomerization takes place, with trimers being the predominant form (24). The gp160 precursor is transported to the Golgi, where it is proteolytically processed by furin into gp120 and gp41 subunits, which are held together noncovalently (24,34,69). The processed Env glycoproteins are then presented on the surface of the infected cell and subsequently, through budding, form the envelopes of progeny virions (69).HIV-1 attachment to target cells is mediated by the binding of gp120 to CD4, the primary HIV-1 receptor (20). This binding exposes a site on gp120 that enables interactions with secondary coreceptors and further conformational changes (39). Thiol/disulfide exchange mediated by protein disulfide isomerase is involved in these conformational changes (2, 42). The two major HIV-1 coreceptors are the chemokine receptors CCR5 and CXCR4; CCR5 is generally considered to be the coreceptor utilized by HIV-1 virions in early, asymptomatic stages of infection, whereas viruses utilizing CXCR4 usually emerge during late stages of disease (5, 54). Binding of gp120 to the appropriate coreceptor triggers a conformational change in gp41 (15, 66). gp41 contains a short N-terminal hydrophobic sequence of amino acids, the fusion peptide, which mediates fusion of viral and host cell membranes, enabling entry of the virus into the host cell (26, 27). The conformational change in gp41, induced by the gp120-coreceptor interaction is necessary in order for this fusion to take place (15,66).It is generally accepted that knowledge of the structu...

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