Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases

The 72 known members of the flavivirus genus include lethal human pathogens such as Yellow Fever, West Nile, and Dengue viruses. There is at present no known chemotherapy for any flavivirus and no effective vaccines for most. A common genomic organization and molecular mechanisms of replication in hosts are shared by flaviviruses with a viral serine protease playing a pivotal role in processing the viral polyprotein into component polypeptides, an obligatory step in viral replication. Using the structure of the dengue serine protease complexed with a protein inhibitor as a template, we have identified five compounds, which inhibit the enzyme. We also describe parallel inhibitory activity of these compounds against the West Nile virus Protease. A few of the compounds appear to provide a template for design of more potent and specific inhibitors of the dengue and West Nile virus proteases. Sequence similarities among flaviviral proteases suggests that such compounds might also possibly inhibit other flaviviral proteases.     

Infection of mouse neurones by West Nile virus is modulated by the interferon-inducible 2'-5' oligoadenylate synthetase 1b protein

Over the past 7 years, West Nile zoonosis has been an emerging concern for public health in Europe, Middle East and more recently in North America. West Nile virus causes epidemic outbreaks in humans and infected patients may exhibit severe neurological symptoms. Because susceptibility and sensitivity to West Nile virus infections may depend on host genetic factors, a mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile virus. A nonsense mutation in gene encoding the 1b isoform of the 2'-5'oligoadenylate synthetase (OAS1b) was constantly associated with the susceptibility of mouse strains to experimental West Nile virus infection. Oligoadenylate synthetase are interferon-inducible proteins playing a role in the endogeneous antiviral pathway. It was of interest to establish whether interferon-alpha and OAS 1B were sufficient to mediate resistance to West Nile virus infection. In the present study, we showed that interferon-alpha had the ability to modulate West Nile virus infection in mouse. In vitro, interferon-alpha protected mouse neuroblastoma cells against West Nile virus infection if cells have been pretreated with the cytokine for several hours. As a consequence of the presence of a stop codon, the Oas1b gene of the susceptible mice encodes a truncated and presumably inactive form, while resistant mice have a normal copy of the gene. Stable mouse neuroblastoma cell clones overexpressing mutant or wild-type OAS 1B were established. Replication of West Nile virus was less efficient in cells that produce the normal copy of OAS 1B as compared to those expressing the truncated form. Our data illustrate the notion that interferon-alpha and Oas genes may be critical for West Nile virus pathogenesis.     

Species composition of mosquitoes (Diptera, Culicidae) and possibility of the West Nile virus natural foci formation in the South of Western Siberia

In 2004 June-July collections of mosquito adults and small mammals were carried out in two areas of Novosibirsk Region (forest-steppe and steppe zones), where the West Nile virus (WNV) was for the first time recorded in birds with different migration status in 2002-2004. Seventeen species of mosquitoes were found; significant changes in their species composition and abundance, as compared with latest faunistic studies made in the sixties-seventies of the last century, are revealed. WNV markers (antigen, RNA) are found in small mammals; highly sensitive to the WNV replication mosquito species are also found. These facts allow supposing a possibility of the formation of stable West Nile virus natural foci in the South of Western Siberia, under conditions of forest-steppe and steppe zones.     

Nuclear localization of flavivirus RNA synthesis in infected cells

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.     

West Nile fever/encephalitis as one of the arboviral infections

West Nile virus maintains natural infection cycle between birds and mosquitoes. It has been known that about 200 species of birds are infected with West Nile virus and the virus is isolated from more than 40 species of mosquitoes. This suggests that West Nile virus has an ability to be transmitted by many species of mosquitoes and infect many kinds of animals. Approximately 20% of infected humans develop symptoms. West Nile fever, an acute febrile illness, is the main disease, and meningitis and encephalitis (meningoencephalitis) occasionally occur. Cases with flaccid paralysis or polyneuritis have been recently reported. Thus, West Nile virus causes multiple types of symptoms in humans. The endemic area has expanded in North America and Siberia. West Nile virus may enter Japan in the near future; therefore, we should keep paying attention to the endemic and epidemic situations in the world.     

Silencing early viral replication in macrophages and dendritic cells effectively suppresses flavivirus encephalitis

West Nile (WN) and St. Louis encephalitis (SLE) viruses can cause fatal neurological infection and currently there is neither a specific treatment nor an approved vaccine for these infections. In our earlier studies, we have reported that siRNAs can be developed as broad-spectrum antivirals for the treatment of infection caused by related viruses and that a small peptide called RVG-9R can deliver siRNA to neuronal cells as well as macrophages. To increase the repertoire of broad-spectrum antiflaviviral siRNAs, we screened 25 siRNAs targeting conserved regions in the viral genome. Five siRNAs were found to inhibit both WNV and SLE replication in vitro reflecting broad-spectrum antiviral activity and one of these was also validated in vivo. In addition, we also show that RVG-9R delivers siRNA to macrophages and dendritic cells, resulting in effective suppression of virus replication. Mice were challenged intraperitoneally (i.p.) with West Nile virus (WNV) and treated i.v. with siRNA/peptide complex. The peritoneal macrophages isolated on day 3 post infection were isolated and transferred to new hosts. Mice receiving macrophages from the anti-viral siRNA treated mice failed to develop any disease while the control mice transferred with irrelevant siRNA treated mice all died of encephalitis. These studies suggest that early suppression of viral replication in macrophages and dendritic cells by RVG-9R-mediated siRNA delivery is key to preventing the development of a fatal neurological disease.     

我国西尼罗病毒和Tahyna病毒的发现与流行

已发现100余种蚊传虫媒病毒在世界各地流行,其引发的人兽共患病是全世界关注的公共卫生问题。长期以来我国仅发现乙型脑炎和登革热两种蚊传虫媒病毒病,但近年来新发现西尼罗病毒和Tahyna病毒及其感染疾病流行。从我国新疆维吾尔自治区采集的蚊虫标本中分离到西尼罗病毒,大量血清学研究证明当地不仅存在西尼罗病毒感染所致疾病,还发生过西尼罗病毒感染引发的病毒性脑炎流行。目前已从新疆维吾尔自治区、青海省和内蒙古自治区采集的蚊虫标本中分离到Tahyna病毒,并发现其在自然界动物中的循环和导致的人类感染流行。西尼罗病毒和Tahyna病毒及其相关感染性疾病的发现为我国虫媒病毒及虫媒病毒病的预防与控制提出了新的挑战。     

Flaviviruses

Family Flaviviridae genus flavivirus contains numerous pathogenic viruses such as Japanese encephalitis virus, dengue virus, West Nile virus, etc, which cause public health problems in the world. Since many mammals and birds can act as amplifying hosts and reservoir hosts in nature and those viruses are transmitted by haematophagous mosquitoes or ticks, those viruses could not be eradicated from the nature. In the recent few decades, the viral replication mechanism and the ultrastructure of viral proteins as well as the viral immune evasion mechanism have been elucidated extensively, leading to develop novel types of antivirals and vaccines. In this review, the flavivirus nature and epidemiology, replication mechanism, immune response and immune evasion, and antivirals and vaccines against flaviviruses were described.     

NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease

Background

The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation) in the absence of inhibitor and lining the substrate binding site (closed conformation) in the presence of an inhibitor.

Methods

In this work, nuclear magnetic resonance (NMR) spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution.

Findings

In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation) occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors.

Conclusion

Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.     

Protective and therapeutic capacity of human single-chain Fv-Fc fusion proteins against West Nile virus

West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.     

Synthesis and biological evaluation of α-ketoamides as inhibitors of the Dengue virus protease with antiviral activity in cell-culture

The development of small molecule inhibitors of the viral protease is of considerable interest for the treatment of emergent flaviviral diseases such as Dengue or West Nile fever. Until today little progress has been made in finding drug-like compounds that inhibit the protease and provide a starting point for lead optimization. We describe here the initial steps of a drug discovery effort that focused on the styryl pharmacophore, combined with a ketoamide function to serve as electrophilic trap for the catalytic serine. This resulted in a fragment-like lead compound with reasonable target affinity and good ligand efficiency, which was extensively modified to explore structure-activity relationships. Selected compounds were cross-tested against the West Nile virus protease and thrombin, indicating that selectivity for one or more flaviviral proteases can be achieved. Finally, the antiviral activity of several protease inhibitors was confirmed in a cell-culture model of Dengue virus replication. The SAR presented here may serve as starting point for further drug discovery efforts with the aim of targeting flaviviral proteases.     

Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.     

Differential induction of antiviral effects against West Nile virus in primary mouse macrophages derived from flavivirus-susceptible and congenic resistant mice by alpha/beta interferon and poly(I-C)

A cell model of primary macrophages isolated from the peritoneal cavity of flavivirus-susceptible and congenic resistant mice has been used to study the extent and kinetics of antiviral effects against West Nile virus upon priming with alpha/beta interferon (IFN-alpha/beta) or poly(I-C) (pIC). The pattern of flavivirus resistance expressed after priming of cells in this model was in good agreement with the pattern of flavivirus resistance described in the brains of the corresponding mouse strains. While priming with either IFN-alpha/beta or pIC completely blocked flavivirus replication in macrophages from resistant mice, it only transiently reduced flavivirus replication in macrophages from susceptible mice. It was only the combined pretreatment with IFN-alpha/beta and pIC that elicited strong antiviral responses that completely prevented flavivirus replication in macrophages from susceptible mice. Primary macrophages isolated from the blood of healthy human donors expressed a similar need for double-stranded RNA (dsRNA) cofactor in developing efficient antiviral responses against West Nile virus. These findings reveal that the inefficient IFN-alpha/beta-induced antiviral effects against flaviviruses in cells from susceptible hosts could be successfully complemented by an external dsRNA factor leading to the complete eradication of the virus. This treatment appears to compensate for the lack of an inborn resistance mechanism in cells from the susceptible host. Furthermore, it may also provide useful clues for the prevention and treatment of flavivirus infections.     

Ecological correlates of risk and incidence of West Nile virus in the United States

West Nile virus, which was recently introduced to North America, is a mosquito-borne pathogen that infects a wide range of vertebrate hosts, including humans. Several species of birds appear to be the primary reservoir hosts, whereas other bird species, as well as other vertebrate species, can be infected but are less competent reservoirs. One hypothesis regarding the transmission dynamics of West Nile virus suggests that high bird diversity reduces West Nile virus transmission because mosquito blood-meals are distributed across a wide range of bird species, many of which have low reservoir competence. One mechanism by which this hypothesis can operate is that high-diversity bird communities might have lower community-competence, defined as the sum of the product of each species’ abundance and its reservoir competence index value. Additional hypotheses posit that West Nile virus transmission will be reduced when either: (1) abundance of mosquito vectors is low; or (2) human population density is low. We assessed these hypotheses at two spatial scales: a regional scale near Saint Louis, MO, and a national scale (continental USA). We found that prevalence of West Nile virus infection in mosquito vectors and in humans increased with decreasing bird diversity and with increasing reservoir competence of the bird community. Our results suggest that conservation of avian diversity might help ameliorate the current West Nile virus epidemic in the USA     

West Nile Complement Fixing antibodies in Nigerian domestic animals and humans

A survey for West Nile Complement Fixing (CF) antibody was carried out in humans and domestic animals in Nigeria. Human sera were obtained from two communities namely Ibadan and Ogbomoso but animal sera were collected from Ibadan and Maiduguri. The overall CF antibody to West Nile virus in the two localities surveyed was 65%. Of 170 persons tested, 53% and 75% were positive in Ibadan and Ogbomoso respectively. Antibody prevalence increased with age in both communities. Tests for antibody against other flaviviruses revealed that monotypic complement fixation reactions were found frequently in young people, but broadly reacting sera were common among the older age groups. Sex distribution of West Nile CF antibody showed that 49/82 (60%) of females and 62/88 (75%) of males had West Nile CF antibody. Tests on animal sera showed that 33% contained CF antibody to West Nile virus. Prevalence of CF antibody in different animal species was 62% in camels, 4% in cattle and 0% in goats.     

The West Nile virus: its recent emergence in North America

West Nile fever emerged in New York in the summer of 1999 when seven people, several horses and thousands of wild birds died. It was soon established that the human disease and the mortality of birds were related. Continued surveillance detected West Nile virus in mosquitoes, birds, horses, small mammals, bats and humans, and has shown its spread to several northeastern states. These events confirm the establishment of West Nile virus endemically in the United States.     

Cholesterol manipulation by West Nile virus perturbs the cellular immune response

Complex membrane structures induced by West Nile virus (WNV), an enveloped RNA virus, are required for efficient viral replication. How these membranes are induced and how they facilitate the viral life cycle are unknown. We show that WNV modulates host cell cholesterol homeostasis by upregulating cholesterol biosynthesis and redistributing cholesterol to viral replication membranes. Manipulating cholesterol levels and altering concentrations of cellular geranylgeranylated proteins had a deleterious effect on virus replication. Depletion of the key cholesterol-synthesizing enzyme 3-hydroxy-methyglutaryl-CoA reductase drastically hampered virus replication. Significantly, virus-induced redistribution of cellular cholesterol downregulated the interferon-stimulated Jak-STAT antiviral signaling response to infection. This defect could be partially restored by exogenous addition of cholesterol, which increased the ability of infected cells to respond to interferon. We propose that, by manipulating cellular cholesterol, WNV utilizes the cellular response to cholesterol deficiency and dependence of antiviral signaling pathways on cholesterol-rich microdomains to facilitate viral replication and survival.     

虫媒黄病毒亚基因组RNA

黄病毒是一大科人类致病性的单股正链RNA病毒。黄病毒包括登革病毒、西尼罗脑炎病毒及日本脑炎病毒等成员,主要径通过节肢动物的叮咬进行传播,即为虫媒黄病毒。研究发现,在虫媒黄病毒复制过程中,除病毒基因组正链RNA、互补的负链RNA及两者的杂合RNA分子外,在病毒感染细胞后还能产生一种病毒亚基因组RNA(subgenomic RNA,sgRNA)。近年对这种sgRNA展开了比较多的研究,结果表明,其产生机制与已知的其他病毒sgRNA产生机制并不相同。该sgRNA的产生与虫媒黄病毒基因组3’非编码区所形成的保守二级结构有关,同时宿主核酸酶对其的不完全降解亦有重要作用。虫媒黄病毒基因组3’非编码区中带有多个与病毒复制相关的RNA元件,而sgRNA的发现有助于全面地认识病毒RNA与宿主RNA代谢途径间的相互作用,为最终阐明病毒的致病机制奠定基础。     

Structure of hepatitis C virus polymerase in complex with primer-template RNA

The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory β-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory β-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.