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Thieno[3,2-b]pyrroles are a novel class of allosteric inhibitors of HCV NS5B RNA-dependent RNA polymerase which show potent affinity for the NS5B enzyme. Introduction of a polar substituent in the position N1 led to a compound that efficiently blocks subgenomic HCV RNA replication in HUH-7 cells with an EC50 of 2.9 microM.
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A series of 1-cycloalkyl-2-phenyl-1H-benzimidazole-5-carboxylic acid derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase (RdRp). A SAR study was performed and led to identify the 2-[(4-diarylmethoxy)phenyl]-benzimidazoles as potent inhibitors. They inhibit subgenomic HCV RNA replication in the replicon cells at low micromolar concentrations (EC(50) as low as 1.1microM). They are selective against DNA polymerases (IC(50)>10microM) and exhibit low cytotoxicity.
Naturally occurring defective interfering RNAs (DI-RNAs) and satellite RNAs greatly reduce the accumulation of their helper virus in vivo, but often modulate symptom expression in an unpredictable manner. Deletion mutants Nc/S, Na/M and Sa/Nc + M/S, derived from brome mosaic virus (BMV) RNA-2, failed to replicate when co-inoculated with BMV RNAs-1 and -2 to barley protoplasts. However, the inoculum RNA corresponding to these deletion mutants was extremely stable and could have been mistaken for plus-strand progeny had minus-strand progeny analysis been omitted. These results accentuate the need for such tests in evaluating the ability of mutant viral sequences to replicate. One of the mutants, Nc/S, effectively interfered with the accumulation of BMV RNAs-1 and -2 in barley protoplasts. This non-replicating interfering RNA was termed NRI RNA-2 Nc/S. When present with RNAs-1 and -2 at low inoculum amounts (1 microgram), NRI RNA-2 Nc/S reduced replication of RNA-2, the parental RNA, by 63% and preferentially interfered with minus-strand RNA accumulation. At higher levels (4 micrograms), it completely displaced replication of both RNAs-1 and -2. Mutations eliminating translation of a truncated p2a protein from NRI RNA-2 Nc/S did not alleviate the interference effect, demonstrating that a defective replicase protein was not responsible for the decreased accumulation of genomic RNA. At an NRI RNA: genomic RNA inoculum molar ratio of 1:1, NRI RNA-2 Nc/S reduced the accumulation of all helper virus RNAs by 55%. Since this reduction was seen for both wild-type RNA-3 and delta SGP RNA-3, a deletion mutant of RNA-3 that lacks the subgenomic promoter necessary for coat protein expression, it was evident that the effective interference mediated by NRI RNA-2 Nc/S was not mitigated by encapsidation. The ability of the NRI RNAs to mimic satellite DI RNAs in depressing helper virus replication suggests that their expression in transgenic plants may provide a new and widely applicable approach for inducing resistance to viral infection.
Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures of the swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.
When the small form of the delta antigen (deltaAg-S) was expressed from a cDNA expression plasmid and subsequently detected by immunofluorescence, it was found localized to the nucleoli. However, if the cDNA was cotransfected with a cDNA expressing a mutated hepatitis delta virus (HDV) genome that could only replicate by using the deltaAg-S provided by the first plasmid, then most of the deltaAg-S was redistributed to the nucleoplasm, largely to specific discrete nucleoplasmic sites or speckles; this pattern was stable for at least 50 days after transfection. These speckles coincided with those detected with an antibody to SC35, an essential non-small nuclear ribonucleoprotein splicing factor. Others have shown that SC35 speckles correspond to active sites of DNA-directed transcription by RNA polymerase II and also of RNA processing. We also found, in contrast to the cotransfections with the mutant HDV and the deltaAg-S provided in trans, that cells transfected with wild-type HDV showed a variable pattern of staining. The SC35-like speckle pattern of accumulation of delta antigen deltaAg was maintained for only 6 days, after which the pattern began to change. By 18 days posttransfection, a variety of different deltaAg staining patterns were observed. This pattern of change occurs at a time when the large form of the delta antigen deltaAg-L appears and HDV RNA synthesis begins to shut down. Our studies therefore support the interpretation that HDV RNA and deltaAg-S accumulate at SC35 speckle sites in the nucleoplasm. We speculate that these may be the sites at which HDV RNA is transcribed by RNA polymerase II and/or sites of HDV RNA processing. Furthermore, when deltaAg-L, as well as other mutant deltaAg accumulate, the speckle association is disrupted, thereby stopping HDV RNA replication.
We have analysed the cDNA coding for the envelope glycoprotein (E1) gene and the terminal non-translated regions (NTRs) of the avirulent Semliki Forest virus (SFV) A774 (A7) variant. The E1 gene exhibited 98.5% identify to the SFV prototype strain L10 (WT) sequence at the nucleotide level. Of the 34 single base substitutions, six led to a change in the deduced amino acid sequence. The 3' NTR of A7 consisted of a 101 nucleotide sequence, not found in WT, followed by five tandemly arranged sequence motifs, two of which were truncated forms of the others. One full-length and one truncated repeat are found at the 3' NTR of WT. The repeats of A7 were followed by a non-repeating sequence, very similar to the equivalent region in WT. Owing to the unique sequence motif and the tandem repeats, the 3' NTR of A7 is 334 nucleotides longer than that of WT. Each of the repeats had an internal 12 nucleotide motif complementary to a conserved sequence in the 5'-terminal non-structural protein 1-encoding region, thought to be important in alphavirus RNA replication. In the 5' NTR, three point mutations were found. The conserved sequence binding to the repeated 3' motifs was identical in A7 and WT.
The synthetic base analogue, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), can efficiently base pair with A and G. We have previously demonstrated that the deoxyribonucleoside of P (dP) is highly mutagenic and that this is due to the ambiguous base pairing ability of P. In this work, we have shown that the ribonucleoside triphosphate of P (rPTP) induces C to U mutation on an in vitro model of retroviral genomic RNA replication pathway. This mutation induction by rPTP may be specific to retroviruses, since host genomic DNA should not be affected by such a ribonucleotide analogue, although temporary transcription-translation errors may occur.
The nucleotide sequence of Apple chlorotic leaf spot closterovirus (ACLSV) genomic RNA has been determined from cDNA clones. It is 7555 nucleotides in length excluding the 3' terminal poly(A) tail and contains three putative open reading frames capable of encoding proteins of 216.5, 50, and 28 kDa. ACLSV RNA has untranslated regions of 151 and 190 nucleotides at its 5' and 3' termini, respectively. The 216.5-kDa ORF encodes a protein which contains the conserved "signature" sequences and has significant homology with the proteins suspected to be involved in viral RNA replication of members of the "Sindbis-like" supergroup of viruses. On the basis of distant homologies with viral movement proteins (M proteins), the 50-kDa ORF is suspected to encode a protein responsible for virus cell-to-cell spread. The 28-kDa ORF contains, in frame, a smaller 21.5-kDa ORF encoding the coat protein of ACLSV. These results show that ACLSV and probably at least the subgroup A of closteroviruses should be regarded as members of the "Sindbis-like" supergroup of RNA viruses.
The linear, positive-stranded RNA genome of flaviviruses is thought to adopt a circularized conformation via interactions of short complementary sequence elements located within its terminal regions. This process of RNA cyclization is a crucial precondition for RNA replication. In the case of mosquito-borne flaviviruses, highly conserved cyclization sequences (CS) have been identified, and their functionality has been experimentally confirmed. Here, we provide an experimental identification of CS elements of tick-borne encephalitis virus (TBEV). These elements, termed 5'-CS-A and 3'-CS-A, are conserved among various tick-borne flaviviruses, but they are unrelated to the mosquito-borne CS elements and are located at different genomic positions. The 5'-CS-A element is situated upstream rather than downstream of the AUG start codon and, in contrast to mosquito-borne flaviviruses, it was found that the entire protein C coding region is not essential for TBEV replication. The complementary 3'-CS-A element is located within the bottom stem rather than upstream of the characteristic 3'-terminal stem-loop structure, implying that this part of the proposed structure cannot be formed when the genome is in its circularized conformation. Finally, we demonstrate that the CS-A elements can also mediate their function when the 5'-CS-A element is moved from its natural position to one corresponding to the mosquito-borne CS. The recognition of essential RNA elements and their differences between mosquito-borne and tick-borne flaviviruses has practical implications for the design of replicons in vaccine and vector development.
Strains of the budding yeast, Saccharomyces cerevisiae, may contain one or more cytoplasmic viruses with double-stranded RNA (dsRNA) genomes. The killer phenomenon in yeast, in which one cell secretes a killer toxin that is lethal to another cell, is dependent upon the presence of the L-A and M1 dsRNA viruses. The L-A viral genome encodes proteins for the viral capsid, and for synthesis and encapsidation of single-stranded RNA replication cycle intermediates. The M1 virus depends upon the L-A-encoded proteins for its capsid and for the replication of its killer-toxin-encoding genome. A full-length cDNA clone of an M genome has been made from a single dsRNA molecule and shown to encode functional killer and killer-immunity functions. The sequence of the clone indicates minor differences from previously published sequences of parts of the M1 genome and of the complete genome of S14 (an internal deletion derivative of M1) but no unreported amino acid variants and no changes in putative secondary structures of the single-stranded RNA. A 118-nucleotide contiguous segment of the M1 genome has not previously been reported; 92 of those nucleotides comprise a segment of A nucleotides in the AU-rich bubble that follows the toxin-encoding reading frame.
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the membrane anchor domain of NS5A as determined by NMR spectroscopy. An alpha-helix extending from amino acid residue 5 to 25 was observed in the presence of different membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A membrane anchor domain forms an in-plane amphipathic alpha-helix embedded in the cytosolic leaflet of the membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the membrane association of NS5A. In conclusion, the NS5A membrane anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.
Current concerns over insertional mutagenesis by retroviral vectors mitigate investigations into alternative, potentially persistent gene therapy vector systems not dependent on genomic integration, such as Sendai virus vectors (SeVV). Prenatal gene therapy requires efficient gene delivery to several tissues, which may not be achievable by somatic gene transfer to the adult. Initially, to test the potential and tropism of the SeVV for gene delivery to fetal tissues, first-generation (replication- and propagation-competent) recombinant SeVV, expressing beta-galactosidase was introduced into late gestation immunocompetent mice via the amniotic and peritoneal cavities and the yolk sac vessels. At 2 days, this resulted in very high levels of expression particularly in the airway epithelium, mesothelium and vascular endothelium, respectively. However, as expected, substantial vector toxicity was observed. The efficiency of gene transfer and the level of gene expression were then examined using a second-generation SeVV. The second generation was developed to be still capable of cytoplasmic RNA replication and therefore high-level gene expression, but incapable of vector spread due to lack of the gene for viral F-protein. Vector was introduced into the fetal amniotic and peritoneal cavities, intravascularly, intramuscularly and intraspinally; at 2 days, expression was observed in the airway epithelia, peritoneal mesothelia, unidentified cells in the gut wall, locally at the site of muscle injection and in the dorsal root ganglia, respectively. Mortality was dramatically diminished compared with the first-generation vector.
The largest extant RNA genomes are found in two diverse families of positive-strand RNA viruses, the animal Coronaviridae and the plant Closteroviridae. Comparative analysis of the viruses from the latter family reveals three levels of gene conservation. The most conserved gene module defines RNA replication and is shared with plant and animal viruses in the alphavirus-like superfamily. A module of five genes that function in particle assembly and transport is a hallmark of the family Closteroviridae and was likely present in the ancestor of all three closterovirus genera. This module includes a homologue of Hsp70 molecular chaperones and three diverged copies of the capsid protein gene. The remaining genes show dramatic variation in their numbers, functions, and origins among closteroviruses within and between the genera. Proteins encoded by these genes include suppressors of RNA silencing, RNAse III, papain-like proteases, the AlkB domain implicated in RNA repair, Zn-ribbon-containing protein, and a variety of proteins with no detectable homologues in the current databases. The evolutionary processes that have shaped the complex and fluid genomes of the large RNA viruses might be similar to those that have been involved in evolution of genomic complexity in other divisions of life.
Benzimidazole-based allosteric inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified to a variety of topologically related scaffolds. Replacement of the polar benzimidazole core by lipophilic indoles led to inhibitors with improved potency in the cell-based subgenomic HCV replicon system. Transposing the indole scaffold into a previously described series of benzimidazole-tryptophan amides generated the most potent inhibitors of HCV RNA replication in cell culture reported to date in this series (EC(50) approximately 50 nM).
Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids.
Interferon alfa (IFN-alpha)-based treatment is the only therapeutic option for chronic hepatitis C viral infection. However, the molecular mechanisms of IFN-alpha antiviral activity are not completely understood. The recent development of an HCV replicon cell culture system provides a feasible experimental model to investigate the molecular details of IFN-induced direct antiviral activity in hepatocytes. In this report, we show that IFN-alpha can effectively inhibit HCV subgenomic RNA replication and suppress viral nonstructural protein synthesis. Using cDNA microarray analysis, we also show that the replicon cells have different gene expression profile compared with the parental hepatoma cells (Huh7). IFN-alpha can induce a number of responsive genes in the replicon cells. One of the genes, 6-16 (G1P3), can enhance IFN-alpha antiviral efficacy. In addition, we demonstrate that IFN-alpha can significantly activate STAT3 in hepatoma cells, suggesting that this pathway plays a role in IFN-alpha signaling. In conclusion, our results indicate that IFN-alpha antiviral activity is associated with activation of STAT3-signaling pathway and intracellular gene activation. Our results also suggest that IFN-alpha-induced target genes may play an important role in IFN-alpha anti-HCV activity.