Discovery of Potent Degraders of the Dengue Virus Envelope Protein.

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here we report the development of small molecule degraders of the envelope (E) protein of dengue virus. We developed two classes of bivalent E-degraders, linking two previously reported E-binding small molecules, GNF-2 and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E degrader with ABL inhibition while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof-of-concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class antiviral drugs.

Modulation of Capsid Dynamics in Bromoviruses by the Host and Heterologous Viral Replicase.

The three genomic and a single subgenomic RNA of Cowpea chlorotic mottle virus (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1V and C2V, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4V, copackages genomic RNA3 and its subgenomic RNA (RNA4). In this study, we sought to evaluate how the alteration of native capsid dynamics by the host and viral replicase modulate the general biology of the virus. The application of a series of biochemical, molecular, and biological assays revealed the following. (i) Proteolytic analysis of the three virion types of CCMV assembled individually in planta revealed that, while retaining the structural integrity, C1V and C2V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4V virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. (ii) The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from Nicotiana benthamiana remained resistant, and (iii) finally, the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis evaluated the relative dynamics of C3+4V and B3+4V virions assembled under the control of the homologous versus heterologous replicase. The role of viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4V and C3+4V virions assembled under the homologous versus heterologous replicase. Our results collectively conclude that constant modulation of capsid dynamics by the host and viral replicase is obligatory for successful infection. IMPORTANCE Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. In viruses, conformational changes are essential for establishing infection and evolution. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. The primary focus of this study is to address how capsid dynamics of plant-pathogenic RNA viruses, namely, Cowpea chlorotic mottle (CCMV) and Brome mosaic virus (BMV), are modulated by the host and viral replicase. The results presented have improved and transformed our understanding of the functional relationship between capsid dynamics and the general biology of the virus. They are likely to provide stimulus to extend similar studies to viruses pathogenic to eukaryotic organisms.

Targeted protein degradation as an antiviral approach.

Targeted protein degradation (TPD) has emerged as a new modality in drug discovery. In this approach, small molecules are used to drive degradation of the target protein of interest. Whereas most direct-acting antivirals (DAAs) inhibit or derange the activity of their viral protein targets and have occupancy-driven pharmacology, small molecules with a TPD-based mechanism have event-driven pharmacology exerted through their ability to induce target degradation. These contrasting mechanisms can result in significant differences in drug efficacy and pharmacodynamics that may be useful in the development of new classes of antivirals. While now being widely pursued in cancer biology and autoimmune disease, TPD has not yet been widely applied as an antiviral strategy. Here, we briefly review TPD pharmacology along with the current status of tools available for developing small molecules that achieve antiviral activity through a TPD mechanism. We also highlight aspects of TPD that may be especially useful in the development of antivirals and that we hope will motivate pursuit of TPD-based antivirals by the antivirals research community.

Zebrafish twist2/dermo1 regulates scale shape and scale organization during skin development and regeneration.

Scales are skin appendages in fishes that evolutionarily predate feathers in birds and hair in mammals. Zebrafish scales are dermal in origin and develop during metamorphosis. Understanding regulation of scale development in zebrafish offers an exciting possibility of unraveling how the mechanisms of skin appendage formation evolved in lower vertebrates and whether these mechanisms remained conserved in birds and mammals. Here we have investigated the expression and function of twist 2/dermo1 gene - known for its function in feather and hair formation - in scale development and regeneration. We show that of the four zebrafish twist paralogues, twist2/dermo1 and twist3 are expressed in the scale forming cells during scale development. Their expression is also upregulated during scale regeneration. Our knockout analysis reveals that twist2/dermo1 gene functions in the maintenance of the scale shape and organization during development as well as regeneration. We further show that the expression of twist2/dermo1 and twist3 is regulated by Wnt signaling. Our results demonstrate that the function of twist2/dermo1 in skin appendage formation, presumably under regulation of Wnt signaling, originated during evolution of basal vertebrates.

The interplay between capsid dynamics and pathogenesis in tripartite bromoviruses.

Infectious virus capsids or virions are considered static structures and undergo various conformational transitions to replicate and infect a wide range of eukaryotic cells. Therefore, virus capsids must be stable enough to overcome the physicochemical environment and flexible enough to reorganize their biologically relevant surface peptides for optimal interaction with the host machinery. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. However, more recent attempts have been made in characterizing the capsid dynamics in the plant virus genus bromovirus characterized by having a tripartite, positive-sense RNA genome. Using the available research data on the genus bromovirus members, this review is focused on updating the readers on the interrelationships between the viral capsid dynamics and host-pathogen interactions.

Genome organization and interaction with capsid protein in a multipartite RNA virus.

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content-specifically, RNAs 3 and 4-assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qß for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific "packaging signals" throughout the viral RNA to package their monopartite genomes.

Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo.

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids.IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.