Structure-activity relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase

To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2′- and/or 4′-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2′-OH with 2′-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2′-fluorination, the introduction of fluorine at the ribose 4′-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2′-C-ethynyl-4′-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.     

A multifunctional turnip crinkle virus replication enhancer revealed by in vivo functional SELEX

The motif1-hairpin (M1H), located on (-)-strands of Turnip Crinkle Virus (TCV)-associated satellite RNA C (satC), is a replication enhancer and recombination hotspot. Results of in vivo genetic selection (SELEX: systematic evolution of ligands by exponential enrichment), where 28 bases of the M1H were randomized and then subjected to selection in plants, revealed that most winners contained one to three short motifs, many of which in their (-)-sense orientation are found in TCV and satC (-)-strand promoter elements. Ability to replicate in protoplasts correlated with fitness to accumulate in plants with one significant exception. Winner UC, containing only a seven-base replacement sequence, was the second most fit winner, yet replicated no better than a 28-base random replacement sequence. Fitness of satC containing different M1H replacement sequences could be due to enhanced satC replication or enhanced ability to affect TCV movement, since satC interferes with TCV virion accumulation, which is correlated with enhanced movement to younger tissue. Cells inoculated with TCV and UC accumulated fewer virions when compared to other winners that replicated better in protoplasts but were less fit in plants. UC, and other first and second round winners, contained structures that were on average 33% more stable in their (+)-strand orientation, and most formed hairpins with a A-rich sequence at the base. These results suggest that M1H replacement sequences contribute to the fitness of satC by either containing (-)-strand elements that enhance satRNA replication and/or a (+)-strand hairpin flanked with single-stranded sequence that enhances TCV movement.     

The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

There is limited knowledge regarding how the RNA-dependent RNA polymerases of the nonsegmented negative-strand RNA viruses initiate genome replication. In a previous study of respiratory syncytial virus (RSV) RNA replication, we found evidence that the polymerase could select the 5'-ATP residue of the genome RNA independently of the 3' nucleotide of the template. To investigate if a similar mechanism is used during antigenome synthesis, a study of initiation from the RSV leader (Le) promoter was performed using an intracellular minigenome assay in which RNA replication was restricted to a single step, so that the products examined were derived only from input mutant templates. Templates in which Le nucleotides 1U, or 1U and 2G, were deleted directed efficient replication, and in both cases, the replication products were initiated at the wild-type position, at position -1 or -2 relative to the template, respectively. Sequence analysis of the RNA products showed that they contained ATP and CTP at the -1 and -2 positions, respectively, thus restoring the mini-antigenome RNA to wild-type sequence. These data indicate that the RSV polymerase is able to select the first two nucleotides of the antigenome and initiate at the correct position, even if the 3'-terminal two nucleotides of the template are missing. Substitution of positions +1 and +2 of the template reduced RNA replication and resulted in increased initiation at positions +3 and +5. Together these data suggest a model for how the RSV polymerase initiates antigenome synthesis.     

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM

By dissecting and reconstituting a cell-free influenza virus genome replication system, we have purified and identified the minichromosome maintenance (MCM) complex, which is thought to be a DNA replicative helicase, as one of the host factors that regulate the virus genome replication. MCM interacted with the PA subunit of the viral RNA-dependent RNA polymerase that is found to be involved in the replication genetically. The virus genome replication was decreased in MCM2 knockdown cells. The viral polymerase appeared to be a nonproductive complex, that is, it was capable of initiating replication but produced only abortive short RNA chains. MCM stimulated de novo-initiated replication reaction by stabilizing a replication complex during its transition from initiation to elongation. Based on the findings, including the result that the MCM-mediated RNA replication reaction was competed with exogenously added RNA, we propose that MCM functions as a scaffold between the nascent RNA chains and the viral polymerase.     

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp,nonstructural proteins p16 and p23, playing a role in virus replication

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.     

Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue

NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM. The induced cultures were then grown for 20 h at 18 °C and cells were harvested by centrifugation at 6000 x g for 15 min at 4 °C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems.     

An E3 ubiquitin ligase from Nicotiana benthamiana targets the replicase of Bamboo mosaic virus and restricts its replication

One up-regulated host gene identified previously was found involved in the infection process of Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. The full length cDNA of this gene was cloned by 5′ and 3′-rapid amplification of cDNA ends and found to encode a polypeptide containing a conserved really interesting new gene (RING) domain and a transmembrane domain. The gene might function as an ubiquitin E3 ligase. We designated this protein in Nicotiana benthamiana as ubiquitin E3 ligase containing RING domain 1 (NbUbE3R1). Further characterization by using Tobacco rattle virus-based virus-induced gene silencing (loss-of-function) revealed that increased BaMV accumulation was in both knockdown plants and protoplasts. The gene might have a defensive role in the replication step of BaMV infection. To further inspect the functional role of NbUbE3R1 in BaMV accumulation, NbUbE3R1 was expressed in N. benthamiana plants. The wild-type NbUbE3R1-orange fluorescent protein (NbUbE3R1-OFP), NbUbE3R1/△TM-OFP (removal of the transmembrane domain) and NbUbE3R1/mRING-OFP (mutation at the RING domain, the E2 interaction site) were transiently expressed in plants. NbUbE3R1 and its derivatives all functioned in restricting the accumulation of BaMV. The common feature of these constructs was the intact substrate-interacting domain. Yeast two-hybrid and co-immunoprecipitation experiments used to determine the possible viral-encoded substrate of NbUbE3R1 revealed the replicase of BaMV as the possible substrate. In conclusion, we identified an up-regulated gene, NbUbE3R1 that plays a role in BaMV replication.     

Localization of the replicase recognition site within brome mosaic virus RNA by hybrid-arrested RNA synthesis

Summary 3 terminal fragments of BMV RNA as short as 153 bases in length serve as efficient templates in vitro for BMV-specific RNA polymerase. Template activity of such fragments or of native BMV RNA is abolished when cDNA fragments as short as 39 bases are hybridized to their 3 termini. Hybridization of cDNa fragments to regions of BMV RNA 200 or more bases distal to the 3 end has no discernible effect on initiation and little effect on elongation. We conclude that BMV RNA polymerase initiates binding with an RNA template through a mechanism mediated by the tRNA-like 3 end of BMV RNA, requiring at least some of the last 39, but no more than the last 153 bases.     

Replication of double-stranded RNA in mycovirus from the plant pathogenic fungus, Fusarium solani

Abstract A mycovirus (named FusoV) from the plant pathogenic fungus Fusarium solani possessed two types of double-stranded (ds) RNA genome, designated Ml and M2. RNA-dependent RNA polymerase activity was detected in FusoV particle fractions. An in vitro RNA polymerase reaction using purified FusoV particles that was supplemented with NTPs revealed the synthesis of single-stranded (ss) RNA species and a subsequent formation of dsRNAs having the same size as Ml and M2. The ssRNA species synthesized in the first stage were proved to be of positive polarity (coding strand) for both M1 and M2 by dot blot hybridization analysis. These results suggest that FusoV genomic dsRNA replicates in a conservative manner.     

HCV 1a／1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a／1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Westem blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV) 1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达。结果证明,转染细胞中检测到分子量约70kDa的HCV NS3蛋白,转染细胞连续传20代,仍能检测到HCV正、负链RNA。表明该HCV嵌合体可以在细胞中复制和表达,HCV1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制。HCV5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合。3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失“A”,9409,9410位及9495,9496,9497位分别插入“TT”和“AAT”可不影响RdRp的生物活性。本研究对阐明HCV复制和翻译机制有重要意义。     

丙型肝炎病毒依赖于RNA的RNA聚合酶(RdRp)研究进展

由于缺乏合适的HCV感染细胞模型,严重制约了HCV复制,特别是HCV复制的关键因子依赖于RNA的RNA聚合酶(RdRp)的研究.对HCV序列比较分析并通过异源表达证明NS5B是HCV复制的RdRp.NS5B C端疏水性氨基酸区域以及NS5B与细胞膜形成复合体等影响NS5B溶解性.在合适的反应条件下NS5B可以多种RNA分子为模板催化RNA复制,特别是能有效复制HCV全长(+)RNA.高浓度GTP激活HCV RdRp活性.NS5B N/C端缺失突变和保守性A、B、C区中的点突变影响RdRp活性,但D区345位精氨酸突变为赖氨酸时RdRp活性明显升高.HCV RdRp的发现及其功能研究为HCV药物研究提供了新型靶标.     

Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase

Understanding how the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) interacts with nascent RNA would provide valuable insight into the virus's mechanism for RNA synthesis. Using a peptide mass fingerprinting method and affinity capture of peptides reversibly cross-linked to an alkyn-labeled nascent RNA, we identified a region below the Δ1 loop in the fingers domain of the HCV RdRp that contacts the nascent RNA. A modification protection assay was used to confirm the assignment. Several mutations within the putative nascent RNA binding region were generated and analyzed for RNA synthesis in vitro and in the HCV subgenomic replicon. All mutations tested within this region showed a decrease in primer-dependent RNA synthesis and decreased stabilization of the ternary complex. The results from this study advance our understanding of the structure and function of the HCV RdRp and the requirements for HCV RNA synthesis. In addition, a model of nascent RNA interaction is compared with results from structural studies.     

Novel inhibitors of hepatitis C virus RNA-dependent RNA polymerases

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide-and is the main cause of adult liver transplants in developed nations. We have identified a class of novel and specific inhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) activity in vitro. Characterization of two such inhibitors, COMPOUND1 (5-(4-chlorophenylmethylene)-3-(benzenesulfonylamino)-4-oxxo-2-thionothiazolidine) and COMPOUND2 (5-(4-bromophenylmethylene)-3-(benzenesulfonylamino)-4-oxxo-2-thionothiazolidine), is reported here. With IC(50) values of 0.54muM and 0.44muM, respectively, they are reversible and non-competitive with nucleotides. Biochemical and structural studies have suggested that these compounds can inhibit the initiation of the RdRp reaction. Interestingly, these inhibitors appear to form a reversible covalent bond with the NS5B cysteine 366, a residue that is not only conserved among all HCV genotypes and a large family of viruses but also required for full NS5B RdRp activity. This may reduce the potential resistance of the viruses to this class of inhibitors.     

Molecular mechanisms of transcription and replication of the influenza A virus genome

Influenza A virus is one of the major pathogens that pose a large threat to human health worldwide and has caused pandemics.Influenza A virus is the Orthomyxoviridae prototype,and has 8 segmented negat...     

Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.