Kyung H. Choi

Poly(ADP-ribosyl)ation of Histone H1 Correlates with Internucleosomal DNA Fragmentation during Apoptosis*

The biochemical role of poly(ADP-ribosyl)ation on internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells. It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C, and cisplatin increased poly(ADP-ribosyl)ation of nuclear proteins, particularly histone H1. A poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 poly(ADP-ribosyl)ation in cells treated with the apoptosis inducers. When nuclear chromatin was made accessible to the exogenous nuclease in a permeabilized cell system, chromatin of UV-treated cells was more susceptible to micrococcal nuclease than the chromatin of control cells. Suppression of histone H1 poly(ADP-ribosyl)ation by 3-aminobenzamide reduced the micrococcal nuclease digestibility of internucleosomal chromatin in UV-treated cells. These results suggest that the poly(ADP-ribosyl)ation of histone H1 correlates with the internucleosomal DNA fragmentation during apoptosis mediated by DNA damaging agents. This suggestion is supported by the finding that xeroderma pigmentosum cells which are defective in introducing incision at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 poly(ADP-ribosyl)ation after UV irradiation. We propose that poly(ADP-ribosyl)ation of histone H1 protein in the early stage of apoptosis facilitates internucleosomal DNA fragmentation by increasing the susceptibility of chromatin to cellular endonuclease.

Classical Swine Fever Virus Can Remain Virulent after Specific Elimination of the Interferon Regulatory Factor 3-Degrading Function of Npro

ABSTRACT Pestiviruses prevent alpha/beta interferon (IFN-α/β) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral Npro nonstructural protein. Npro is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire Npro gene resulted in attenuation in pigs. In order to elaborate on the role of the Npro-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in Npro that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of Npro. We describe two mutations in Npro that eliminate Npro-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for Npro to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-α/β induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-α/β induction is not a prerequisite for CSFV virulence.

Insight into DNA and Protein Transport in Double-stranded DNA Viruses: The Structure of Bacteriophage N4

Bacteriophage N4 encapsidates a 3500-aa-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryoelectron microscopy. The virion has an icosahedral capsid with T=9 quasi-symmetry that encapsidates well-organized double-stranded DNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, 12 appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insight into its assembly and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system.

RNA-dependent RNA polymerases from Flaviviridae

Viral genome replication in Flaviviridae is carried out by a virally encoded RNA-dependent RNA polymerase (RdRp). These viruses initiate the RNA synthesis via a de novo mechanism that differs from the primer-dependent mechanism used by Picornaviridae. Like all polymerases, the structure of Flaviviridae RdRps resembles a right hand with characteristic fingers, palm, and thumb domains. Structural features that distinguish Flaviviridae RdRps from other polymerases are a large thumb domain and a C-terminal motif that encircles the active site. This domain arrangement restricts the volume of the template-binding channel, allowing only single-stranded RNA to enter the active site. While this closed form of the polymerase is ideal to stabilize a de novo initiation complex, significant conformational changes are expected to accommodate the elongation complex containing the RNA duplex product.

Zinc Binding in Pestivirus Npro Is Required for Interferon Regulatory Factor 3 Interaction and Degradation

Pestiviruses, such as bovine viral diarrhea virus and classical swine fever virus (CSFV), use the viral protein N(pro) to subvert host cell antiviral responses. N(pro) is the first protein encoded by the single large open reading frame of the pestivirus positive-sense RNA genome and has an autoproteolytic activity, cleaving itself off from the polyprotein. N(pro) also targets interferon regulatory factor 3 (IRF3), a transcription factor for alpha/beta interferon genes, and promotes its proteasomal degradation, a process that is independent of the proteolytic activity of N(pro). We determined that N(pro) contains a novel metal-binding TRASH motif consisting of Cys-X(21)-Cys-X(3)-Cys (where X is any amino acid) at its C-terminus. We also found that N(pro) coordinates a single zinc atom as determined by graphite furnace-atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry. Mutational and biochemical analyses show that the cysteine residues in the TRASH motif are required for zinc binding and protein stability. Individual substitutions of the cysteines in the TRASH motif of CSFV N(pro) abolished the interaction of N(pro) with IRF3 and resulted in the loss of virus-mediated IRF3 degradation in CSFV-infected cells. Thus, the zinc-binding ability of N(pro) in pestiviruses appears to be essential for the virus-mediated degradation of IRF3.

Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface.

Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.

Flaviviral Replication Complex: Coordination between RNA Synthesis and 5’-RNA Capping

Genome replication in flavivirus requires (−) strand RNA synthesis, (+) strand RNA synthesis, and 5′-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (−) RNA synthesis, (+) RNA synthesis, and 5′-RNA capping and methylation.

The Structure of Classical Swine Fever Virus Npro: A Novel Cysteine Autoprotease and Zinc-Binding Protein Involved in Subversion of Type I Interferon Induction

Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (Npro) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, Npro is no longer active as a protease. The released Npro suppresses the induction of the hosts type-I interferon-α/β (IFN-α/β) response. Npro binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus Npro. Npro is structurally distinct from other known cysteine proteases and has a novel “clam shell” fold consisting of a protease domain and a zinc-binding domain. The unique fold of Npro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of Npro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of Npros interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of Npro, and its role in subversion of host immune response.

Physicochemical property consensus sequences for functional analysis, design of multivalent antigens and targeted antivirals

BackgroundAnalysis of large sets of biological sequence data from related strains or organisms is complicated by superficial redundancy in the set, which may contain many members that are identical except at one or two positions. Thus a new method, based on deriving physicochemical property (PCP)-consensus sequences, was tested for its ability to generate reference sequences and distinguish functionally significant changes from background variability.MethodsThe PCP consensus program was used to automatically derive consensus sequences starting from sequence alignments of proteins from Flaviviruses (from the Flavitrack database) and human enteroviruses, using a five dimensional set of Eigenvectors that summarize over 200 different scalar values for the PCPs of the amino acids. A PCP-consensus protein of a Dengue virus envelope protein was produced recombinantly and tested for its ability to bind antibodies to strains using ELISA.ResultsPCP-consensus sequences of the flavivirus family could be used to classify them into five discrete groups and distinguish areas of the envelope proteins that correlate with host specificity and disease type. A multivalent Dengue virus antigen was designed and shown to bind antibodies against all four DENV types. A consensus enteroviral VPg protein had the same distinctive high pKa as wild type proteins and was recognized by two different polymerases.ConclusionsThe process for deriving PCP-consensus sequences for any group of aligned similar sequences, has been validated for sequences with up to 50% diversity. Ongoing projects have shown that the method identifies residues that significantly alter PCPs at a given position, and might thus cause changes in function or immunogenicity. Other potential applications include deriving target proteins for drug design and diagnostic kits.

Design, expression, and purification of a Flaviviridae polymerase using a high‐throughput approach to facilitate crystal structure determination

Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA‐dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695‐residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical “core,” although overall sequence identity is low. Eighty‐four expression clones of the BVDV polymerase were designed by fine‐sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high‐throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96‐well format, and two of these proteins were successfully crystallized.