Small molecule protein assembly modulators with pan-cancer therapeutic efficacy.

Two structurally unrelated small molecule chemotypes, represented by compounds PAV-617 and PAV-951, with antiviral activity in cell culture against Mpox virus (formerly known as monkeypox virus) and human immunodeficiency virus (HIV) respectively, were studied for anti-cancer efficacy. Each exhibited apparent pan-cancer cytotoxicity with reasonable pharmacokinetics. Non-toxicity is demonstrated in a non-cancer cell line and in mice at doses achieving drug exposure at active concentrations.

A Pan-Respiratory Antiviral Chemotype Targeting a Host Multi-Protein Complex.

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious virus in multiple cell culture models for all six families of viruses causing most respiratory disease in humans. In animals this chemotype has been demonstrated efficacious for Porcine Epidemic Diarrhea Virus (a coronavirus) and Respiratory Syncytial Virus (a paramyxovirus).

Addressing Antiretroviral Drug Resistance with Host-Targeting Drugs-First Steps towards Developing a Host-Targeting HIV-1 Assembly Inhibitor.

The concerning increase in HIV-1 resistance argues for prioritizing the development of host-targeting antiviral drugs because such drugs can offer high genetic barriers to the selection of drug-resistant viral variants. Targeting host proteins could also yield drugs that act on viral life cycle events that have proven elusive to inhibition, such as intracellular events of HIV-1 immature capsid assembly. Here, we review small molecule inhibitors identified primarily through HIV-1 self-assembly screens and describe how all act either narrowly post-entry or broadly on early and late events of the HIV-1 life cycle.

Identification of an Antiretroviral Small Molecule That Appears To Be a Host-Targeting Inhibitor of HIV-1 Assembly.

Given the projected increase in multidrug-resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life cycle stages not targeted by drugs currently in use. Host-targeting compounds are of particular interest because they can offer a high barrier to resistance. Here, we report identification of two related small molecules that inhibit HIV-1 late events, a part of the HIV-1 life cycle for which potent and specific inhibitors are lacking.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S.

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates.

Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

Unlabelled: The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly.

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

During the late stage of the viral life cycle, HIV-1 Gag assembles into a spherical immature capsid, and undergoes budding, release, and maturation. Here we review events involved in immature capsid assembly from the perspective of five different approaches used to study this process: mutational analysis, structural studies, assembly of purified recombinant Gag, assembly of newly translated Gag in a cell-free system, and studies in cells using biochemical and imaging techniques. We summarize key findings obtained using each approach, point out where there is consensus, and highlight unanswered questions.

A temporospatial map that defines specific steps at which critical surfaces in the Gag MA and CA domains act during immature HIV-1 capsid assembly in cells.

Unlabelled: During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known.

Limited recovery of pineal function after regeneration of preganglionic sympathetic axons: evidence for loss of ganglionic synaptic specificity.

The cervical sympathetic trunks (CSTs) contain axons of preganglionic neurons that innervate the superior cervical ganglia (SCGs). Because regeneration of CST fibers can be extensive and can reestablish certain specific patterns of SCG connections, restoration of end organ function would be expected. This expectation was examined with respect to the pineal gland, an organ innervated by the two SCGs.

Host-rabies virus protein-protein interactions as druggable antiviral targets.

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 host-catalyzed 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.

HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly.

To produce progeny virus, human immunodeficiency virus type I (HIV-1) Gag assembles into capsids that package the viral genome and bud from the infected cell. During assembly of immature capsids, Gag traffics through a pathway of assembly intermediates (AIs) that contain the cellular adenosine triphosphatase ABCE1 (ATP-binding cassette protein E1). In this paper, we showed by coimmunoprecipitation and immunoelectron microscopy (IEM) that these Gag-containing AIs also contain endogenous processing body (PB)-related proteins, including AGO2 and the ribonucleic acid (RNA) helicase DDX6.

HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag.

During HIV-1 assembly, Gag polypeptides multimerize to form an immature capsid and also package HIV-1 genomic RNA. Assembling Gag forms immature capsids by progressing through a stepwise pathway of assembly intermediates containing the cellular ATPase ABCE1, which facilitates capsid formation. The NC domain of Gag is required for ABCE1 binding, acting either directly or indirectly.

Innate immune signaling induces high levels of TC-specific deaminase activity in primary monocyte-derived cells through expression of APOBEC3A isoforms.

In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date.

Assembly of immature HIV-1 capsids using a cell-free system.

For many years it has been known that viral capsid proteins are capable of self-assembly, but increasing evidence over the past decade indicates that in cells HIV-1 capsid assembly occurs via a complex but transient series of steps requiring multiple viral-host interactions. To better understand the biochemistry of HIV assembly, our group established a cell-free system that faithfully reconstitutes HIV-1 Gag synthesis and post-translational events of capsid assembly using cellular extracts, albeit more slowly and less efficiently. This system allowed initial identification of interactions that occur very transiently in cells but can be tracked in the cell-free system.

Intracellular destinies: degradation, targeting, assembly, and endocytosis of HIV Gag.

The HIV-1 Gag protein assembles into immature capsids when expressed in human cells. Although self-assembly of Gag was once thought to be sufficient to explain capsid formation, in the past decade it has become increasingly apparent that in cells, the pathway from Gag synthesis to assembled capsids is coordinated and facilitated by host factors. These cellular factors likely direct the trafficking, membrane targeting, and multimerization of Gag, and could also assist with encapsidation of viral RNA.

T cells contain an RNase-insensitive inhibitor of APOBEC3G deaminase activity.

The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected.

Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production.

In primate cells, assembly of a single HIV-1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV-1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation.

Basic residues in the nucleocapsid domain of Gag are required for interaction of HIV-1 gag with ABCE1 (HP68), a cellular protein important for HIV-1 capsid assembly.

During human immunodeficiency virus, type 1 (HIV-1) assembly, Gag polypeptides multimerize into immature HIV-1 capsids. The cellular ATP-binding protein ABCE1 (also called HP68 or RNase L inhibitor) appears to be critical for proper assembly of the HIV-1 capsid. In primate cells, ABCE1 associates with Gag polypeptides present in immature capsid assembly intermediates.

Recent insights into biological regulation from cell-free protein-synthesizing systems.

We review the important role that cell-free protein-synthesizing systems (CFPSS) have played in the history of modern biology, and highlight two recent applications that illustrate their continued utility for the exploration of otherwise intractable aspects of gene expression and its regulation. Viral capsid assembly recreated in CFPSS reveals a catalyzed biochemical pathway involving transient, energy-dependent action of host proteins and discrete assembly intermediates, rather than the classical notion of self-assembly that was expected for capsid formation. Study of prion protein biogenesis reveals a new conformation critical for disease pathogenesis and advances the paradigm of protein bioconformatics, by which cells may productively regulate the folding of various proteins.

Identification of residues in the hepatitis C virus core protein that are critical for capsid assembly in a cell-free system.

Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria.

Comparing capsid assembly of primate lentiviruses and hepatitis B virus using cell-free systems.

Many viruses that assemble their capsids in the eukaryotic cytoplasm require a threshold concentration of capsid protein to achieve capsid assembly. Strategies for achieving this include maintaining high levels of capsid protein synthesis and targeting to specific sites to raise the effective concentration of capsid polypeptides. To understand how different viruses achieve the threshold capsid protein concentration required for assembly, we used cell-free systems to compare capsid assembly of hepatitis B virus (HBV) and three primate lentiviruses.

Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity.

The antiretroviral activity of the cellular enzyme APOBEC3G has been attributed to the excessive deamination of cytidine (C) to uridine (U) in minus strand reverse transcripts, a process resulting in guanosine (G) to adenosine (A) hypermutation of plus strand DNAs. The HIV-1 Vif protein counteracts APOBEC3G by inducing proteasomal degradation and exclusion from virions through recruitment of a cullin5 ECS E3 ubiquitin ligase complex. APOBEC3G belongs to the APOBEC protein family, members of which possess consensus (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C cytidine deaminase motifs.