Dae-Gyun Ahn

New Thermophilic and Thermostable Esterase with Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned from a Metagenomic Library

ABSTRACT A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30°C up to 95°C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C4 to C16) examined was p-nitrophenyl caproate (C6), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.

Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication.

Abstract The programmed −1 ribosomal frameshifting (−1 PRF) utilized by eukaryotic RNA viruses plays a crucial role for the controlled, limited synthesis of viral RNA replicase polyproteins required for genome replication. The viral RNA replicase polyproteins of severe acute respiratory syndrome coronavirus (SARS-CoV) are encoded by the two overlapping open reading frames 1a and 1b, which are connected by a −1 PRF signal. We evaluated the antiviral effects of antisense peptide nucleic acids (PNAs) targeting a highly conserved RNA sequence on the – PRF signal. The ribosomal frameshifting was inhibited by the PNA, which bound sequence-specifically a pseudoknot structure in the −1 PRF signal, in cell lines as assessed using a dual luciferase-based reporter plasmid containing the −1 PRF signal. Treatment of cells, which were transfected with a SARS-CoV-replicon expressing firefly luciferase, with the PNA fused to a cell-penetrating peptide (CPP) resulted in suppression of the replication of the SARS-CoV replicon, with a 50% inhibitory concentration of 4.4μM. There was no induction of type I interferon responses by PNA treatment, suggesting that the effect of PNA is not due to innate immune responses. Our results demonstrate that −1 PRF, critical for SARS-CoV viral replication, can be inhibited by CPP-PNA, providing an effective antisense strategy for blocking −1 PRF signals.

Interference of hepatitis C virus replication in cell culture by antisense peptide nucleic acids targeting the X-RNA

Summary.  The RNA‐dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is the essential catalytic enzyme for viral genome replication. It initiates minus‐strand RNA synthesis from a highly conserved 98‐nt sequence, called the X‐RNA, at the 3′‐end of the plus‐strand viral genome. In this study, we evaluated the antiviral effects of peptide nucleic acids (PNAs) targeting the X‐RNA. Our in vitro RdRp assay results showed that PNAs targeting the three major stem‐loop (SL) domains of X‐RNA can inhibit RNA synthesis initiation. Delivery of X‐RNA‐targeted PNAs by fusing the PNAs to cell‐penetrating peptides (CPPs) into HCV‐replicating cells effectively suppressed HCV replication. Electrophoretic mobility shift assays revealed that the PNA targeting the SL3 region at the 5′‐end of X‐RNA dissociated the viral RdRp from the X‐RNA. Furthermore, delivery of the SL3‐targeted PNA into HCV‐infected cells resulted in the suppression of HCV RNA replication without activation of interferon β expression. Collectively, our results indicate that the HCV X‐RNA can be effectively targeted by CPP‐fused PNAs to block RNA–protein and/or RNA–RNA interactions essential for viral RNA replication and identify X‐RNA SL3 as an RdRp binding site crucial for HCV replication. In addition, the ability to inhibit RNA synthesis initiation by targeting HCV X‐RNA using antisense PNAs suggests their promising therapeutic potential against HCV infection.

Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates.

The severe acute respiratory syndrome coronavirus (SARS-CoV) RNA genome is replicated by a virus-encoded RNA replicase, the key component of which is the nonstructural protein 12 (nsp12). In this report, we describe the biochemical properties of a full-length recombinant SARS-CoV nsp12 RNA-dependent RNA polymerase (RdRp) capable of copying viral RNA templates. The purified SARS-CoV nsp12 showed both primer-dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates. The RdRp activity was strictly dependent on Mn2+. The nsp12 preferentially copied homopolymeric pyrimidine RNA templates in the absence of an added oligonucleotide primer. It was also able to initiate de novo RNA synthesis from the 3’-ends of both the plus- and minus-strand genome of SARS-CoV, using the 3’-terminal 36- and 37-nt RNA, respectively. The in vitro RdRp assay system established with a full-length nsp12 will be useful for understanding the mechanisms of coronavirus replication and for the development of anti-SARS-CoV agents.

Analysis of the Thermostability Determinants of Hyperthermophilic Esterase EstE1 Based on Its Predicted Three-Dimensional Structure

ABSTRACT The three-dimensional (3D) structure of the hyperthermophilic esterase EstE1 was constructed by homology modeling using Archaeoglobus fulgidus esterase as a reference, and the thermostability-structure relationship was analyzed. Our results verified the predicted 3D structure of EstE1 and identified the ion pair networks and hydrophobic interactions that are critical determinants for the thermostability of EstE1.

Ginsenoside Rg3 restores hepatitis C virus–induced aberrant mitochondrial dynamics and inhibits virus propagation

Hepatitis C virus (HCV) alters mitochondrial dynamics associated with persistent viral infection and suppression of innate immunity. Mitochondrial dysfunction is also a pathologic feature of direct‐acting antiviral (DAA) treatment. Despite the high efficacy of DAAs, their use in treating patients with chronic hepatitis C in interferon‐sparing regimens occasionally produces undesirable side effects such as fatigue, migraine, and other conditions, which may be linked to mitochondrial dysfunction. Here, we show that clinically prescribed DAAs, including sofosbuvir, affect mitochondrial dynamics. To counter these adverse effects, we examined HCV‐induced and DAA‐induced aberrant mitochondrial dynamics modulated by ginsenoside, which is known to support healthy mitochondrial physiology and the innate immune system. We screened several ginsenoside compounds showing antiviral activity using a robust HCV cell culture system. We investigated the role of ginsenosides in antiviral efficacy, alteration of mitochondrial transmembrane potential, abnormal mitochondrial fission, its upstream signaling, and mitophagic process caused by HCV infection or DAA treatment. Only one of the compounds, ginsenoside Rg3 (G‐Rg3), exhibited notable and promising anti‐HCV potential. Treatment of HCV‐infected cells with G‐Rg3 increased HCV core protein–mediated reduction in the expression level of cytosolic p21, required for increasing cyclin‐dependent kinase 1 activity, which catalyzes Ser616 phosphorylation of dynamin‐related protein 1. The HCV‐induced mitophagy, which follows mitochondrial fission, was also rescued by G‐Rg3 treatment. Conclusion: G‐Rg3 inhibits HCV propagation. Its antiviral mechanism involves restoring the HCV‐induced dynamin‐related protein 1–mediated aberrant mitochondrial fission process, thereby resulting in suppression of persistent HCV infection. (Hepatology 2017;66:758–771)

The essential role of mitochondrial dynamics in antiviral immunity

Abstract Viruses alter cellular physiology and function to establish cellular environment conducive for viral proliferation. Viral immune evasion is an essential aspect of viral persistence and proliferation. The multifaceted mitochondria play a central role in many cellular events such as metabolism, bioenergetics, cell death, and innate immune signaling. Recent findings accentuate that viruses regulate mitochondrial function and dynamics to facilitate viral proliferation. In this review, we will discuss how viruses exploit mitochondrial dynamics to modulate mitochondria-mediated antiviral innate immune response during infection. This review will provide new insight to understanding the virus-mediated alteration of mitochondrial dynamics and functions to perturb host antiviral immune signaling.