Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication.

UNLABELLED: Dengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, and trans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites. In vivo protein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex. IMPORTANCE: This is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

Maintenance of dimer conformation by the dengue virus core protein α4-α4' helix pair is critical for nucleocapsid formation and virus production.

The virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal α2 and α4 helices of each monomer. Pairing of α2-α2' creates a continuous hydrophobic surface, while the α4-α4' helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the α4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. The in vitro characterization assays further highlighted that the α4-α4' helix pair conformation is critical in preserving the overall α-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improves in vitro nucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formation in vivo. This study provides the first direct link between the α4-α4' helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production. Importance: Structure-based mutagenesis study of the dengue virus core protein (CP) reveals that the α4-α4' helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus translation and replication were detected. In vitro inefficiency and size of nucleocapsid-like particle (NLP) formation offer a possible explanation for in vivo virus production inefficiency upon CP mutation. Further, the transition of NLP morphology from an incomplete state to an intact particle shown by α4-α4' helix pair mutants in the presence of a nonionic detergent suggests the regulatory role of the intracellular lipid droplet (LD) in CP-LD interaction and in promoting nucleocapsid formation. This study provides the first direct link between the α4-α4' helix pair interaction and CP dimer conformation that is the fundamental requirement of CP function, particularly in nucleocapsid formation during virion production.

Nucleic acid chaperone activity associated with the arginine-rich domain of human hepatitis B virus core protein.

UNLABELLED: Hepatitis B virus (HBV) DNA replication occurs within the HBV icosahedral core particles. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its carboxyl terminus. This ARD domain of HBc 149-183 is known to be important for viral replication but not known to have a structure. Recently, nucleocapsid proteins of several viruses have been shown to contain nucleic acid chaperone activity, which can facilitate structural rearrangement of viral genome. Major features of nucleic acid chaperones include highly basic amino acid residues and flexible protein structure. To test the nucleic acid chaperone hypothesis for HBc ARD, we first used the disassembled full-length HBc from Escherichia coli to analyze the nucleic acid annealing and strand displacement activities. To exclude the potential contamination of chaperones from E. coli, we designed synthetic HBc ARD peptides with different lengths and serine phosphorylations. We demonstrated that HBc ARD peptide can behave like a bona fide nucleic acid chaperone and that the chaperone activity depends on basic residues of the ARD domain. The loss of chaperone activity by arginine-to-alanine substitutions in the ARD can be rescued by restoring basic residues in the ARD. Furthermore, the chaperone activity is subject to regulation by phosphorylation and dephosphorylation at the HBc ARD. Interestingly, the HBc ARD can enhance in vitro cleavage activity of RNA substrate by a hammerhead ribozyme. We discuss here the potential significance of the HBc ARD chaperone activity in the context of viral DNA replication, in particular, at the steps of primer translocations and circularization of linear replicative intermediates. IMPORTANCE: Hepatitis B virus is a major human pathogen. At present, no effective treatment can completely eradicate the virus from patients with chronic hepatitis B. We report here a novel chaperone activity associated with the viral core protein. Our discovery could lead to a new drug design for more effective treatment against hepatitis B virus in the future.

A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease.

Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC50) of 1.03 ± 0.09 µM. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future.

Identification of a small-molecule inhibitor of dengue virus using a replicon system.

Dengue virus (DENV) is a mosquito-borne human pathogen that causes a serious public-health threat in tropical and subtropical regions of the world. Neither a vaccine to prevent nor an effective therapeutic agent to treat DENV infection is currently available. We established a stable cell line harboring a luciferase-reporting DENV subgenomic replicon to screen for inhibitors of DENV. A total of 14,400 small-molecule (MW < 500 Da) chemicals were evaluated for their ability to reduce luciferase reporter activity in cell lysates. One effective compound was identified from the screening. This compound was found to reduce virus production but did not block virus entry in virus-based assay. Mode-of-action analysis revealed that this inhibitor suppressed viral RNA replication but did not affect replicon translation. This compound potentially could be developed as an anti-DENV agent and might be useful for dissecting the molecular mechanism of DENV replication.

Identification of the phytobioactive Polygonum cuspidatum as an antiviral source for restricting dengue virus entry.

Dengue virus (DENV) is a mosquito-borne pathogen that is becoming a serious global threat, owing to its rising incidence in inter-tropical regions that yield over 50 million annual infections. There are currently no approved antiviral agents for the management of dengue, and recent shortcomings in its immunization called for immediate action to develop effective drugs with prophylactic ability to better manage its infection. In an attempt to discover novel antiviral sources, we identified the medicinal herb Polygonum cuspidatum (PC) as a bioactive botanical material against DENV infectivity. Specifically, the methanolic extract from PC rhizomes (PCME) potently inhibited DENV infection without causing significant cytotoxicity. Further examination on the viral life cycle demonstrated that PCME particularly targeted the initial stages of DENV infection, while pre- and post-infection treatments had no effect. More importantly, the PCME could efficiently inactivate DENV free virus particles and block the viral attachment and entry/fusion events without apparently influencing viral replication, egress, and cell-to-cell spread. The antiviral effect of PCME was also recapitulated in infection analysis using DENV pseudoparticles displaying viral structural proteins that mediate DENV particle entry. Besides, PCME treatment also inhibited direct DENV entry into several cell types relevant to its infection and reduced viral infectivity of other members of the Flaviviridae family, including the hepatitis C virus (HCV) and Zika virus (ZIKV). Due to its potency against DENV entry, we suggest that the phytobioactive extract from PC is an excellent starting point as an antiviral source material for further development of therapeutic strategies in the prophylactic management of DENV infection.

Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode.

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 A resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB-dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB-ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L45 plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA-PriB-ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.

Insights into the coordinated interplay of the sHP hairpin and its co-existing and mutually-exclusive dengue virus terminal RNA elements for viral replication.

Terminal RNA elements of the dengue virus (DENV) genome are necessary for balanced stability of linear and circular conformations during replication. We examined the small hairpin (sHP) and co-existing and mutually-exclusive terminal RNA elements by mutagenesis analysis, compensatory mutation screening, and by probing with RNA fragments to explore localized RNA folding and long-range RNA interactions. We found that the first base pair of the sHP and the stability of SLB and the 3'SL bottom stem affected circularization; sHPgc/C10631G+G10644C prohibited circularization, sHPuG accelerated and stabilized 5'-to-3' RNA hybridization, while C94A and A97G and C10649 mutations loosened SLB and 3'SL, respectively, for circularization. sHPuG+C10649G induced circularization and impeded replication, whereas point mutations that loosened the UAR or DAR ds region, strengthened the sHP, or reinforced the 3'SL bottom stem, rescued the replication deficiency. Overall, we reveal structural and sequence features and interplay of DENV genome terminal RNA elements essential to viral replication.

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen.

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid-NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA-protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA-protein complex, reduce the nucleic acid chaperone activity of NdAg.

The role of surface basic amino acids of dengue virus NS3 helicase in viral RNA replication and enzyme activities.

Structure-based mutagenesis analysis on selected conserved surface basic residues of DENV NS3 helicase was performed using a selectable replicon and recombinant protein. We found a requirement for basic side chains of NS3 residues #225, #268, and #538 to activate viral RNA replication and ensure RNA-stimulated ATPase activity, and a critical role for R560 and R599 residues in maintaining NS3 helicase structure, linked to its biological function and catalytic activity. Three screened NS3 second-site mutations for R225A and R268A/E mutations elevated the functional RNA binding of NS3 helicase and compensated the replication defect of the original NS3 mutant replicons.

Characterization of the activity of 2'-C-methylcytidine against dengue virus replication.

Dengue virus (DENV) is a severe mosquito-borne viral pathogen. Neither vaccines nor antiviral therapy is currently available to treat DENV infection. Nucleoside inhibitors targeting viral polymerase have proved promising for the development of drugs against viruses. In this study, we report a nucleoside analog, 2'-C-methylcytidine (2CMC), which exerts potent anti-DENV activity in DENV subgenomic RNA replicon and infectious systems, with an IC50 value of 11.2±0.3µM. This study utilized both cell-based and cell-free reporter assay systems to reveal the specific anti-DENV RNA polymerase activity of 2CMC. In addition, both xenograft bioluminescence-based DENV replicon and DENV-infected Institute of Cancer Research (ICR) suckling mice models evaluated the anti-DENV replication activity of 2CMC in vivo. Collectively, these findings provide a promising compound for the development of direct-acting antivirals against DENV infection.

Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection.

Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV) infection. Honokiol, a lignan biphenol derived from the Magnolia tree, is commonly used in Eastern medicine. Here we report that honokiol has profound antiviral activity against serotype 2 DENV (DENV-2). In addition to inhibiting the intracellular DENV-2 replicon, honokiol was shown to suppress the replication of DENV-2 in baby hamster kidney (BHK) and human hepatocarcinoma Huh7 cells. At the maximum non-toxic dose of honokiol treatment, the production of infectious DENV particles was reduced >90% in BHK and Huh7 cells. The underlying mechanisms revealed that the expression of DENV-2 nonstructural protein NS1/NS3 and its replicating intermediate, double-strand RNA, was dramatically reduced by honokiol treatment. Honokiol has no effect on the expression of DENV putative receptors, but may interfere with the endocytosis of DENV-2 by abrogating the co-localization of DENV envelope glycoprotein and the early endosomes. These results indicate that honokiol inhibits the replication, viral gene expression, and endocytotic process of DENV-2, making it a promising agent for chemotherapy of DENV infection.

Characterization and application of the selective strand annealing activity of the N terminal domain of hepatitis delta antigen.

We used synthetic DNA oligos to investigate the nucleic acid chaperone properties of the N terminal domain of hepatitis delta antigen (NdAg). We found that NdAg possessed a bona fide chaperone activity. NdAg could distinguish subtle differences in the thermal stability of the base pairing region, and enabled DNA oligos to form a more stable duplex among competing sequences through facilitating strand annealing selectively, stimulating duplex conversion selectively, and stabilizing the more stable duplex. The property of NdAg identified in this study could be applied to improve the efficiency and specificity of dot blot hybridization under conditions of low stringency.

Synthesis, antiproliferative and anti-dengue virus evaluations of 2-aroyl-3-arylquinoline derivatives.

A number of 2-aroyl-3-arylquinoline derivatives was synthesized and evaluated for their anti-Dengue virus activity. Both 2-(hydroxyphenylmethyl)-3-(4-methoxyphenyl)quinoline (13a) and 2-(4-hydroxybenzoyl)-3-(4-hydroxyphenyl)quinoline (17) were found to significantly inhibit the DENV2 RNA expression in Huh-7-DV-Fluc cells with a potency approximately equal to that of ribavirin and the inhibition is in a dose-dependent manner. Compounds 13a and 17 reduced DENV replication in both viral protein and mRNA levels, and no significant cell cytotoxicity was detected, with greater than 50% viability of Huh-7-DV-Fluc cells at a concentration of 100 µM. However, significant cytotoxicity was detected for the positive ribavirin. In addition, we performed infectious assay to further verify the inhibitory activity of 13a and 17 on DENV replication in protein and RNA levels. On the other hand, compounds 19a-19c exhibited IC50 values ranged from 4.47 to 8.68 µM against A549, H1299, MCF-7, and Huh-7 which were approximately equal potent to the positive topotecan. Structural optimization of lead compounds, 13a and 17, and their detailed molecular mechanism of action are ongoing.

RNA binding property and RNA chaperone activity of dengue virus core protein and other viral RNA-interacting proteins.

In this study we showed that the dengue virus (DENV) core protein forms a dimer with an α-helix-rich structure, binds RNA and facilitates the strand annealing process. To assess the RNA chaperone activity of this core protein and other dengue viral RNA-interacting proteins, such as NS3 helicase and NS5 proteins, we engineered cis- and trans-cleavage hammerhead ribozyme constructs carrying DENV genomic RNA elements. Our results indicate that DENV core protein facilitates typical hammerhead structure formation by acting as an RNA chaperone and DENV NS5 has a weak RNA chaperone activity, while DENV NS3 helicase failed to refold RNA with a complex secondary structure.

HCV NS3 protein helicase domain assists RNA structure conversion.

NS3H, the helicase domain of HCV NS3, possesses RNA-stimulated ATPase and ATP hydrolysis-dependent dsRNA unwinding activities. Here, the ability of NS3H to facilitate RNA structural rearrangement is studied using relatively long RNA strands as the model substrates. NS3H promotes intermolecular annealing, resolves three-stranded RNA duplexes, and assists dsRNA and ssRNA inter-conversions to establish a steady state among RNA structures. NS3H facilitates RNA structure conversions in a mode distinct from an ATP-independent RNA chaperone. These findings expand the known function of HCV NS3 helicase and reveal a role for viral helicase in assisting RNA structure conversions during virus life cycle.

Analysis of the nucleoside triphosphatase, RNA triphosphatase, and unwinding activities of the helicase domain of dengue virus NS3 protein.

The helicase domain of dengue virus NS3 protein (DENV NS3H) contains RNA-stimulated nucleoside triphosphatase (NTPase), ATPase/helicase, and RNA 5'-triphosphatase (RTPase) activities that are essential for viral RNA replication and capping. Here, we show that DENV NS3H unwinds 3'-tailed duplex with an RNA but not a DNA loading strand, and the helicase activity is poorly processive. The substrate of the divalent cation-dependent RTPase activity is not restricted to viral RNA 5'-terminus, a protruding 5'-terminus made the RNA 5'-triphosphate readily accessible to DENV NS3H. DENV NS3H preferentially binds RNA to DNA, and the functional interaction with RNA is sensitive to ionic strength.

Dengue virus infection induces passive release of high mobility group box 1 protein by epithelial cells.

OBJECTIVES: Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of secondary dengue virus (DV) infection. In the current study, we provide the first evidence of induction of cellular necrosis by DV type 2 (DV-2). METHODS AND RESULTS: The epithelial cell line A549 can support replication of dengue virus as demonstrated by expression of viral NS1 antigen and virus plaque assay. DV-2 infection of cells induced cell death in approximately half of the cells that were actively infected. Using sodium 3'-[1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate [XTT]-based cell viability assays, we found that DV-2 infection at a multiplicity of infection (MOI) of 10 resulted in significant death of cells as well as high extracellular lactate dehydrogenase (LDH) activity and leakage of the high mobility group 1 (HMGB1) protein into the extracellular space. CONCLUSIONS: These results suggest that HMGB1 may be a signal of tissue or cellular injury by DV-2, which in turn is likely to induce and/or enhance an immune reaction.

Selective strand annealing and selective strand exchange promoted by the N-terminal domain of hepatitis delta antigen.

We have previously shown that the N-terminal domain of hepatitis delta virus (NdAg) has an RNA chaperone activity in vitro (Huang, Z. S., and Wu, H. N. (1998) J. Biol. Chem. 273, 26455-26461). Here we investigate further the basis of the stimulatory effect of NdAg on RNA structural rearrangement: mainly the formation and breakage of base pairs. Duplex dissociation, strand annealing, and exchange of complementary RNA oligonucleotides; the hybridization of yeast U4 and U6 small nuclear RNAs and of hammerhead ribozymes and cognate substrates; and the cis-cleavage reaction of hepatitis delta ribozymes were used to determine directly the role of NdAg in RNA-mediated processes. The results showed that NdAg could accelerate the annealing of complementary sequences in a selective fashion and promote strand exchange for the formation of a more extended duplex. These activities would prohibit NdAg from modifying the structure of a stable RNA, but allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a more extensively matched target in cognate substrate. These and other results suggest that hepatitis delta antigen may have a biological role as an RNA chaperone, modulating the folding of viral RNA for replication and transcription.

Crystal structure of PriB, a primosomal DNA replication protein of Escherichia coli.

PriB is one of the Escherichia coli varphiX-type primosome proteins that are required for assembly of the primosome, a mobile multi-enzyme complex responsible for the initiation of DNA replication. Here we report the crystal structure of the E. coli PriB at 2.1 A resolution by multi-wavelength anomalous diffraction using a mercury derivative. The polypeptide chain of PriB is structurally similar to that of single-stranded DNA-binding protein (SSB). However, the biological unit of PriB is a dimer, not a homotetramer like SSB. Electrophoretic mobility shift assays demonstrated that PriB binds single-stranded DNA and single-stranded RNA with comparable affinity. We also show that PriB binds single-stranded DNA with certain base preferences. Based on the PriB structural information and biochemical studies, we propose that the potential tetramer formation surface and several other regions of PriB may participate in protein-protein interaction during DNA replication. These findings may illuminate the role of PriB in varphiX-type primosome assembly.