Nrf2-dependent induction of innate host defense via heme oxygenase-1 inhibits Zika virus replication.

We identified primary human monocyte-derived macrophages (MDM) as vulnerable target cells for Zika virus (ZIKV) infection. We demonstrate dramatic effects of hemin, the natural inducer of the heme catabolic enzyme heme oxygenase-1 (HO-1), in the reduction of ZIKV replication in vitro. Both LLC-MK2 monkey kidney cells and primary MDM exhibited hemin-induced HO-1 expression with major reductions of >90% in ZIKV replication, with little toxicity to infected cells.

Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication.

Using the massively parallel genetic algorithm for RNA folding, we show that the core region of the 3'-untranslated region of the dengue virus (DENV) RNA can form two dumbbell structures (5'- and 3'-DBs) of unequal frequencies of occurrence. These structures have the propensity to form two potential pseudoknots between identical five-nucleotide terminal loops 1 and 2 (TL1 and TL2) and their complementary pseudoknot motifs, PK2 and PK1. Mutagenesis using a DENV2 replicon RNA encoding the Renilla luciferase reporter indicated that all four motifs and the conserved sequence 2 (CS2) element within the 3'-DB are important for replication.

Evolution of attenuating mutations in dengue-2 strain S16803 PDK50 vaccine and comparison of growth kinetics with parent virus.

A live-attenuated dengue-2 virus strain S16803 vaccine candidate that is immunogenic and safe in humans was derived by 50 passages in primary dog kidney (PDK) cells. To identify mutations associated with attenuation of the dengue-2 PDK50 vaccine strain, we determined the nucleotide changes that arose during PDK passage of the dengue-2 virus. Thirteen mutations distinguished the PDK50 virus from low-passage parent resulting in amino acid substitutions in the premembrane (E89G), envelope (E202K, N203D), nonstructural proteins NS1 (A43T), NS2A (L181F), NS2B (I26V), and NS4B (I/T108T, L112F).

Identification of mutations in a candidate dengue 4 vaccine strain 341750 PDK20 and construction of a full-length cDNA clone of the PDK20 vaccine candidate.

Dengue 4 virus strain 341750 serially passaged 20 times in primary dog kidney (PDK) cells was shown to have reduced infectivity for rhesus monkeys but was immunogenic and protected the monkeys from challenge with low passage parent dengue 4 virus. The dengue 4 PDK20 virus was also shown to be attenuated for human volunteers. We compared the genomic nucleotide sequences of low passage parent and PDK20 attenuated vaccine strains and identified 11 nucleotide (nt) substitutions in the PDK20 genome.

Genome 3'-end repair in dengue virus type 2.

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3'-end repair in dengue virus-2 in mammalian cells, a series of 3'-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis.

Multiple enzyme activities of flavivirus proteins.

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing.

Triaryl pyrazoline compound inhibits flavivirus RNA replication.

Triaryl pyrazoline {[5-(4-chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-1-yl]-phenyl-methanone} inhibits flavivirus infection in cell culture. The inhibitor was identified through high-throughput screening of a compound library using a luciferase-expressing West Nile (WN) virus infection assay. The compound inhibited an epidemic strain of WN virus without detectable cytotoxicity (a 50% effective concentration of 28 microM and a compound concentration of >or=300 microM required to reduce 50% cell viability).

A live, attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and immunogenic in monkeys.

The Delta30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3' untranslated region (3' UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1Delta30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4Delta30 vaccine candidate. rDEN1Delta30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3' UTR, and both vaccines were highly protective against challenge with wt DEN1.

Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference.

Mosquitoes transmit numerous viral pathogens to humans including dengue virus which affects approximately 50 million individuals per year. Inhibition of viral gene expression within an insect host could be used to block virus replication and subsequent transmission of the pathogen to humans. A naturally occurring gene silencing mechanism triggered by double-stranded RNA (dsRNA), RNA interference (RNAi), has recently been described in a number of species including Drosophila.

Derivation and characterization of a dengue type 1 host range-restricted mutant virus that is attenuated and highly immunogenic in monkeys.

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-SL) that is conserved among all flavivirus species. The 3'-SL constitutes the downstream terminal similar95 nucleotides of the 3' noncoding region in flavivirus RNA.

In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure.

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs.

Construction of a full length infectious clone for dengue-1 virus Western Pacific,74 strain.

The flavivirus dengue 1 Western Pacific,74 (DEN1 WP) virus has a positive-stranded RNA genome of 10,735 nucleotides. DEN1 WP genomic RNA was amplified into three overlapping fragments by RT-PCR. These fragments were assembled into a full-length cDNA clone in the yeast-E.

Identification of specific nucleotide sequences within the conserved 3'-SL in the dengue type 2 virus genome required for replication.

The flavivirus genome is a positive-stranded approximately 11-kb RNA including 5' and 3' noncoding regions (NCR) of approximately 100 and 400 to 600 nucleotides (nt), respectively. The 3' NCR contains adjacent, thermodynamically stable, conserved short and long stem-and-loop structures (the 3'-SL), formed by the 3'-terminal approximately 100 nt. The nucleotide sequences within the 3'-SL are not well conserved among species.

Mutagenesis of the NS3 protease of dengue virus type 2.

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases.

Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E.

A conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein.

The mature flavivirus capsid protein (virion C) is commonly thought to be free in the cytoplasm of infected cells and to form a nucleocapsid-like complex with genomic RNA in mature virus particles. There is little sequence conservation among flavivirus virion C proteins, but they are similar in size (e.g.

Evidence that flavivirus NS1-NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum.

Previous deletion mutagenesis studies have shown that the flavivirus NS1-NS2A clevage requires the eight C-terminal residues of NS1, constituting the cleavage recognition sequence, and sequences in NS2A far downstream of the cleavage site. We now demonstrate that replacement of all of NS1 upstream of the cleavage recognition sequence with prM sequences still allows cleavage in vivo. Thus, other than the eight C-terminal residues, NS1 is dispensable for NS1-NS2A cleavage.

Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and heterologous proteases.

Processing of Japanese encephalitis (JE) virus non-structural (NS) proteins expressed by recombinant vaccinia viruses was analysed to characterize the responsible viral protease. Analysis of the processing of polyprotein NS2A-2B-3' containing the N-terminal 322 amino acids of NS3 revealed products consistent with cleavages at the predicted intergenic junctions as well as at one or possibly two sites within NS2A. Cleavage at the alternate site(s) containing the cleavage sequence motif within NS2A could possibly explain the production of the NS1' protein in JE virus-infected cells.

Processing of flavivirus structural glycoproteins: stable membrane insertion of premembrane requires the envelope signal peptide.

The flavivirus structural proteins capsid (C), premembrane (prM), and envelope (E) are cleaved in that order from the N-terminus of the polyprotein by the ER intralumenal enzyme signal peptidase. The prM-E and E-NS1 junctions contain hydrophobic domains with both transmembrane and signal function. These domains reside at the C-termini of prM and E, respectively, after cleavage.

Processing of dengue type 4 and other flavivirus nonstructural proteins.

Dengue type 4 (DEN4) and other flaviviruses employ host and viral proteases for polyprotein processing. Most proteolytic cleavages in the DEN4 nonstructural protein (NS) region are mediated by the viral NS2B-NS3 protease. The N-terminal third of NS3, containing sequences homologous to serine protease active sites, is the protease domain.

Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity.

Most proteolytic cleavages in the nonstructural protein (NS) region of the flavivirus polyprotein are effected by a virus-encoded protease composed of two viral proteins, NS2B and NS3. The N-terminal 180-amino-acid-region of NS3 includes sequences with homology to the active sites of serine proteases, and there is evidence that this portion of NS3 can mediate proteolytic cleavages. In contrast, nothing is known about required sequences in NS2B.

Dengue virus protein recognition by virus-specific murine CD8+ cytotoxic T lymphocytes.

The identification of the protein targets for dengue virus-specific T lymphocytes may be useful for planning the development of subunit vaccines against dengue. We studied the recognition by murine dengue virus-specific major histocompatibility complex class I-restricted, CD8+ cytotoxic T lymphocytes (CTL) of dengue virus proteins using recombinant vaccinia viruses containing segments of the dengue virus genome. CTL from H-2k mice recognized a single serotype-cross-reactive epitope on the nonstructural (NS) protein NS3.

Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus.

We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala.

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation.

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins.

The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages.