Potential Dual Role of West Nile Virus NS2B in Orchestrating NS3 Enzymatic Activity in Viral Replication.

West Nile virus (WNV) nonstructural protein 3 (NS3) harbors the viral triphosphatase and helicase for viral RNA synthesis and, together with NS2B, constitutes the protease responsible for polyprotein processing. NS3 is a soluble protein, but it is localized to specialized compartments at the rough endoplasmic reticulum (RER), where its enzymatic functions are essential for virus replication. However, the mechanistic details behind the recruitment of NS3 from the cytoplasm to the RER have not yet been fully elucidated. In this study, we employed immunofluorescence and biochemical assays to demonstrate that NS3, when expressed individually and when cleaved from the viral polyprotein, is localized exclusively to the cytoplasm. Furthermore, NS3 appeared to be peripherally recruited to the RER and proteolytically active when NS2B was provided in trans. Thus, we provide evidence for a potential additional role for NS2B in not only serving as the cofactor for the NS3 protease, but also in recruiting NS3 from the cytoplasm to the RER for proper enzymatic activity. Results from our study suggest that targeting the interaction between NS2B and NS3 in disrupting the NS3 ER localization may be an attractive avenue for antiviral drug discovery.

Selective Reactivity of Anti-Japanese Encephalitis Virus NS4B Antibody Towards Different Flaviviruses.

Studies investigating West Nile virus (WNV) NS4B protein function are hindered by the lack of an antibody recognizing WNV NS4B protein. Few laboratories have produced WNV NS4B antibodies, and none have been shown to work consistently. In this report, we describe a NS4B antibody against Japanese encephalitis virus (JEV) NS4B protein that cross-reacts with the NS4B protein of WNV but not of dengue virus (DENV). This JEV NS4B antibody not only recognizes WNV NS4B in infected cells, but also recognizes the NS4B protein expressed using transfection. It is evident from this data that the JEV NS4B antibody is specific to NS4B of WNV but not to NS4B of the four DENV serotypes. The specificity of this antibody may be due to the notable differences that exist between the amino acid sequence identity and antigenic relationships within the NS4B protein of the WNV, DENV, and JEV.

Functional Analysis of West Nile Virus Proteins in Human Cells.

West Nile Virus (WNV) lineage 2 strains have been responsible for large outbreaks of neuroinvasive disease in the United States and Europe between 1999 and 2012. Different strains in this lineage have previously been shown to produce either severe or mild neuroinvasive disease in mice. Phylogenetic and amino acid comparisons between highly or less virulent lineage 2 strains have demonstrated that the nonstructural (NS) gene(s) were most variable. However, the roles of some of the NS proteins in virus life cycle are unknown. The aim of this chapter is to describe simple computational and experimental approaches that can be used to: (1) explore the possible roles of the NS proteins in virus life cycle and (2) test whether the subtle amino acid changes in WNV NS gene products contributed to the evolution of more virulent strains. The computational approaches include methods based on: (1) sequence similarity, (2) sequence motifs, and (3) protein membrane topology predictions. Highlighted experimental procedures include: (1) isolation of viral RNA from WNV-infected cells, (2) cDNA synthesis and PCR amplification of WNV genes, (3) cloning into GFP expression vector, (4) bacterial transformation, (5) plasmid isolation and purification, (6) transfection using activated dendrimers (Polyfect), and (7) immunofluorescence staining of transfected mammalian cells.

Induction of endoplasmic reticulum-derived replication-competent membrane structures by West Nile virus non-structural protein 4B.

Replication of flaviviruses (family Flaviviridae) occurs in specialized virus-induced membrane structures (IMS). The cellular composition of these IMS varies for different flaviviruses implying different organelle origins for IMS biogenesis. The role of flavivirus non-structural (NS) proteins for the alteration of IMS remains controversial. In this report, we demonstrate that West Nile virus strain New York 99 (WNVNY99) remodels the endoplasmic reticulum (ER) membrane to generate specialized IMS. Within these structures, we observed an element of the cis-Golgi, viral double-stranded RNA, and viral-envelope, NS1, NS4A and NS4B proteins using confocal immunofluorescence microscopy. Biochemical analysis and microscopy revealed that NS4B lacking the 2K-signal peptide associates with the ER membrane where it initiates IMS formation in WNV-infected cells. Co-transfection studies indicated that NS4A and NS4B always remain co-localized in the IMS and are associated with the same membrane fractions, suggesting that these proteins function cooperatively in virus replication and may be an ideal target for antiviral drug discovery.

Stimulation of unprimed macrophages with immune complexes triggers a low output of nitric oxide by calcium-dependent neuronal nitric-oxide synthase.

Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.

Dengue hemorrhagic fever-associated immunomediators induced via maturation of dengue virus nonstructural 4B protein in monocytes modulate endothelial cell adhesion molecules and human microvascular endothelial cells permeability.

We previously demonstrated that dengue virus (DENV) nonstructural 4B protein (NS4B) induced dengue hemorrhagic fever (DHF)-associated immunomediators in THP-1 monocytes. Moreover, cleavage of NS4AB polyprotein by the NS2B3 protease, significantly increased immunomediator production to levels found after DENV infection. In this report using primary human microvascular endothelial cells (HMVEC) transwell permeability model and HMVEC monolayer, we demonstrate that the immunomediators secreted in the supernatants of DENV-infected monocytes increase HMVEC permeability and expression of ICAM-1, VCAM-1 and E-selectin. Moreover, maturation of NS4B via cleavage of 2KNS4B is sufficient to induce immunomediators that cause HMVEC phenotypic changes, which appear to be synergistically induced by TNFα and IL-8. These data suggest that therapies targeting the maturation steps of NS4B, particularly 2KNS4B processing, may reduce overall DHF-associated immunomediator levels, thereby reducing DHF-associated morbidity and mortality. Alternatively, TNFα inhibitors may be a valid intervention strategy during the later stages of infection to prevent DHF progression.

Maturation of dengue virus nonstructural protein 4B in monocytes enhances production of dengue hemorrhagic fever-associated chemokines and cytokines.

High levels of viremia and chemokines and cytokines underlie the progression of severe dengue disease. Dengue virus (DENV) preferentially infects peripheral blood monocytes, which secrete elevated levels of immunomediators in patients with severe disease. Further, DENV nonstructural proteins (NS) are capable of modifying intracellular signaling, including interferon inhibition. We demonstrate that peak secretions of immunomediators such as IL-6, IL-8, IP-10, TNFα or IFNγ in DENV-infected monocytes correlate with maximum virus production and NS4B and NS5 are primarily responsible for the induction of immunomediators. Furthermore, we demonstrate that sequential NS4AB processing initiated by the viral protease NS2B3(pro) and via the intermediate 2KNS4B significantly enhances immunomediator induction. While the 2K-signal peptide is not essential for immunomediator induction, it plays a synergistic role with NS4B. These data suggest that NS4B maturation is important during innate immune signaling in DENV-infected monocytes. Given similar NS4B topologies and polyprotein processing across flaviviruses, NS4B may be an attractive target for developing Flavivirus-wide therapeutic interventions.

Serotonin receptor 2A blocker (risperidone) has no effect on human polyomavirus JC infection of primary human fetal glial cells.

A recent report demonstrated that JC virus (JCV) employs serotonin receptor 2A (5HT(2A)R) to infect the glial cells. To assess the ability of a potent 5HT(2A)R blocker, risperidone, to inhibit JCV infection, the authors treated primary human fetal glial (PHFG) cells in vitro with risperidone for 24 h and inoculated with JCV(Mad1). There was no significant difference in JCV genome copies or mRNA transcripts and protein expression in treatment-naive and risperidone-treated PHFG cells. These data indicate that risperidone does not inhibit JCV(Mad1) attachment, internalisation, and replication in PHFG cells, and 5HT(2A)R blockers may not be effective in treating progressive multifocal leukoencephalopathy (PML).

Traditional kava beverage consumption and liver function tests in a predominantly Tongan population in Hawaii.

PURPOSE: To determine the effects of traditionally prepared kava beverages on the liver function tests of regular kava beverage consumers in a population of Tongan and non-Tongan residents of Hawaii (Oahu). METHODS: The liver function tests of 31 healthy adult kava drinkers were compared against a control group of 31 healthy adult non-kava drinkers. Subjects were recruited from the general population, a kava bar, and Tongan kava drinking circles. The liver function profile included AST, ALT, ALP, GGT, and bilirubin (total and direct). Other tests included total protein, albumin, and screens for viral hepatitis and hemochromatosis when indicated. RESULTS: Chronic kava beverage consumption was associated with elevation of GGT in 65% of the kava drinkers versus 26% in the controls (P = .005). ALP was elevated in 23% of kava drinkers versus 3% in the controls (P = .053). CONCLUSION: Heavy kava beverage consumption was associated with significantly elevated GGT levels.

Determination of kavalactones in dried kava (Piper methysticum) powder using near-infrared reflectance spectroscopy and partial least-squares regression.

Kava (Piper methysticum Forst F.), or àwa in the Hawaiian language, has been used for thousands of years by the people of the South Pacific Islands, in particular Fiji, Vanuatu, Tonga, and Samoa, for social and ceremonial occasions. Kava has the unique ability to promote a state of relaxation without the loss of mental alertness. Kava recently became part of the herbal pharmacopoeia throughout the United States and Europe because of its anxiolytic properties. The active compounds are collectively called kavalactones (or kava pyrones). The need for a less time-consuming and costly method to determine the concentration of kavalactones in dried kava is urgent. The combination of near-infrared reflectance spectroscopy (NIRS) and partial least-squares (PLS) methods has been found to be a convenient, versatile, and rapid analytical tool for determination of kavalactones in dried kava powder. Calibration equations were developed based on the analyses of 110 samples with variable physical and chemical properties collected over time from Hawaii kava growers and validated by analyses of a set of 12 samples with unknown kavalactones concentration. All six major kavalactones and the total kavalactones were measured using NIRS with accuracy acceptable for commercial use. The NIRS measurements are reproducible and have a repeatability on a par with HPLC methods.

Regulation of exopolysaccharide synthesis in Rhizobium sp. strain TAL1145 involves an alternative sigma factor gene, rpoH2.

Exopolysaccharide (EPS) produced by Rhizobium sp. strain TAL1145 has been shown to be essential for effective nodulation on Leucaena leucocephala (leucaena). This paper reports the isolation and characterization of an alternative sigma factor gene, rpoH2, involved in the regulation of EPS synthesis in TAL1145. Disruption of this gene in TAL1145 resulted in a Calcofluor-dim mutant RUH102 that produced approximately 18 % of the amount of EPS made by TAL1145. This mutation did not affect the normal growth of RUH102 in free-living state. RUH102 induced few nitrogen-fixing nodules, resulting in a significant reduction in total nitrogen content in leucaena. It was complemented for EPS production and nodulation by a 2.0 kb HindIII fragment of TAL1145. Sequence analysis of this fragment revealed the rpoH2 ORF of 870 bp that encoded a protein of 32 kDa. Expression of the rpoH2 ORF in Escherichia coli also revealed a 32 kDa protein. A PCR-constructed clone of 1263 bp, containing the rpoH2 ORF and its upstream putative regulatory region, complemented RUH102 for EPS defects. Comparison of the RpoH2 sequence to proteins in the databases showed significant similarity to RpoH-like sigma factors of other Gram-negative bacteria. By constructing several exo : : Tn3Hogus fusions and transferring them to the backgrounds of TAL1145 and RUH102, it was demonstrated that RpoH2 positively regulates the transcription of some exo genes.