西尼罗病毒与西尼罗热

从西尼罗热的症状、传播、流行规律、疾病发生史、潜在爆发的危险性和西尼罗病毒的分类地位、病毒粒子形态结构、病毒核酸和蛋白质结构特征、病毒感染及增殖周期、病毒的病理学特性诸方面对西尼罗热和西尼罗病毒的研究进展作了综合评述     

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.     

N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.     

Domain-based homology modeling and mapping of the conformational epitopes of envelope glycoprotein of west nile virus

Knowledge-based modeling has proved significantly accurate for generating the quality models for proteins whose sequence identity with the structurally known targets is greater than or equal to 40%. On the other hand, models obtained for low sequence identities are not reliable. Hence, a reliable and alternative strategy that uses knowledge of domains in the protein can be used to improve the quality of the model generated by the homology method. Here, we report a method for developing a 3D-model for the envelope glycoprotein (Egp) of west nile virus (WNV), using knowledge of structurally conserved functional domains amongst the target sequence (Egp of WNV) and its homologous templates belonging to the same protein family, flaviviridae. This strategy is found to be highly effective in reducing the root mean square deviation (RMSD) value at the C positions of the target and its experimental homologues. The 3D structure of a protein is a prerequisite for structure-based drug design as well as for identifying the conformational epitopes that are essential for the designing vaccines. The conformational epitopes are mapped from the 3D structure of Egp of WNV modeled using the concept of an antigenic domain. A total of five such epitope regions/sites have been identified. They have been found distributed in the loop regions (surface) of the whole protein model composed of dimerization, central and immunological domains. These sites are proposed as the binding sites for HLA proteins/B-cell receptors. Binding is required to activate the immune response against WNV.Figure The conformational epitopes that are distributed in all the domains. They are found out by the algorithm by Kolaskar et al.     

Recent advances in the molecular biology of west nile virus

Since the mid-1990s, West Nile virus (WNV) has emerged as a significant agent of arboviral encephalitis in several regions of the world. In 1999, WNV was introduced into the northeastern United States and was associated with an outbreak of encephalitis affecting humans, birds and horses. Subsequently, the virus has spread across the country, and across southern Canada, and in 2002 and 2003 was associated with the largest outbreaks of arboviral encephalitis recorded in the Western hemisphere. Interestingly, the more recent spread of WNV into Mexico, Central America and the Caribbean has not been associated with the high levels of clinical disease observed in North America. This review addresses the most recent results from studies investigating the molecular biology and evolution of WNV, as well as progress in the development of diagnostic and therapeutic reagents.     

Antiviral activity of human immunodeficiency virus type 1 Gag-specific cytotoxic T lymphocyte targeting is not necessarily intrinsically superior to envelope targeting

Across several cohorts, human immunodeficiency virus type 1 (HIV-1) Gag- and Env-specific CD8(+) T lymphocyte (CTL) responses have demonstrated inverse and positive correlations, respectively, to viremia. The mechanism has been proposed to be superior antiviral activity of Gag-specific CTLs in general. Addressing this hypothesis, we created two HIV-1 constructs with an epitope translocated from Gag (SLYNTVATL, SL9) to Env, thereby switching the protein source of the epitope. A virus expressing SL9 in Env was similar to the original virus in susceptibility to SL9-specific CTLS. This finding suggests that Env targeting is not intrinsically inferior to Gag targeting for CTL antiviral activity.     

Icam-1 participates in the entry of west nile virus into the central nervous system

Determining how West Nile virus crosses the blood-brain barrier is critical to understanding the pathogenesis of encephalitis. Here, we show that ICAM-1(-/-) mice are more resistant than control animals to lethal West Nile encephalitis. ICAM-1(-/-) mice have a lower viral load, reduced leukocyte infiltration, and diminished neuronal damage in the brain compared to control animals. This is associated with decreased blood-brain barrier leakage after viral infection. These data suggest that ICAM-1 plays an important role in West Nile virus neuroinvasion and that targeting ICAM-1 signaling may help control viral encephalitis.     

Amino acid sequence of various tryptic peptides of the envelope protein of the tick-borne encephalitis virus

Protein E of the tick-borne encephalitis virus (TBEV) (strain Sofin) was treated with purified trypsin in 1% Triton X-100, and the peptides thus obtained were separated by micro-column reversed-phase chromatography. Four of the purified peptides were sequenced, their structures being in accordance with the nucleotide sequence of the viral protein E gene. Amino acid sequences of peptides deduced from the cDNA primary structure are: Ser-Val-Leu-Ile-Pro-Ser-His-Ala-Gln-Gly-Asp-Leu-Thr-Gly-Arg (N-terminal peptide of protein E); Thr-Glu-Gly-Ala-Gln-Asn-Trp-Asn-Ala-Glu-Arg: Trp-Leu-Glu-Gly-Asp-Ser-Leu-Arg; Leu-Val-Glu-Phe-Gly-Ala-Pro-His-Ala-Val-Lys.     

Factors affecting the direct targeting of murine leukemia virus vectors containing peptide ligands in the envelope protein

To develop a reliable strategy for cell-specific delivery of retroviral vectors, we genetically modified the envelope (Env) protein of the ecotropic Moloney murine leukemia virus. We found a site in the variable region A, where the insertion of ligands, epidermal growth factor (EGF) and stromal-derived factor-1α (SDF-1α), was possible without abolishing virion incorporation of the Env protein and its ecotropic entry function. The vector containing the EGF–Env did not show the EGF receptor-dependent transduction. The vector containing the SDF-1α–Env, however, specifically transduced human cells expressing CXCR4, the receptor for SDF-1α, at titers of 10³–10⁴ c.f.u./ml. Further experiments showed that the CXCR4-dependent transduction was based on the specific interaction between the SDF-1α moiety of the SDF-1α–Env and CXCR4 and was independent of the ecotropic entry function. The direct targeting of the retroviral vector may be possible if the proper chimeric Env structure and the appropriate ligand–receptor system are employed.     

Membrane-perturbing properties of three peptides corresponding to the ectodomain of hepatitis C virus E2 envelope protein

Based on the predicted capacity to interact with membranes at the interface, we have found three regions in the ectodomain of the hepatitis C virus envelope glycoprotein E2 (430-449, 543-560 and 603-624) with the ability to destabilize membranes. Three peptides corresponding to the sequence of these regions have been synthesized and their interaction with liposomes have been characterized. The three peptides were able to insert deeply into the hydrophobic core of negatively charged phospholipids as stated by fluorescence depolarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Peptides E2(430-449) and E2(603-624) were able to induce aggregation of phosphatidylglycerol vesicles in a concentration-dependent manner both at neutral and acidic pH while peptide E2(543-560) did not induce any increase of optical density at 360 nm in the concentration range studied. The three peptides induced lipid mixing and the release of the internal contents in a dose-dependent manner when acidic phospholipids were used. Fourier transformed infrared spectroscopy indicated that the peptides adopted mainly a beta-sheet conformation which is not modified by the presence of acidic phospholipids. Taken together, our results point out to the involvement of these three regions in the fusion mechanism of HCV at the plasma membrane level.     

Membrane-perturbing properties of three peptides corresponding to the ectodomain of hepatitis C virus E2 envelope protein

Based on the predicted capacity to interact with membranes at the interface, we have found three regions in the ectodomain of the hepatitis C virus envelope glycoprotein E2 (430-449, 543-560 and 603-624) with the ability to destabilize membranes. Three peptides corresponding to the sequence of these regions have been synthesized and their interaction with liposomes have been characterized. The three peptides were able to insert deeply into the hydrophobic core of negatively charged phospholipids as stated by fluorescence depolarization of the probe 1,6-diphenyl-1,3,5-hexatriene. Peptides E2_430-449 and E2_603-624 were able to induce aggregation of phosphatidylglycerol vesicles in a concentration-dependent manner both at neutral and acidic pH while peptide E2_543-560 did not induce any increase of optical density at 360 nm in the concentration range studied. The three peptides induced lipid mixing and the release of the internal contents in a dose-dependent manner when acidic phospholipids were used. Fourier transformed infrared spectroscopy indicated that the peptides adopted mainly a β-sheet conformation which is not modified by the presence of acidic phospholipids. Taken together, our results point out to the involvement of these three regions in the fusion mechanism of HCV at the plasma membrane level.     

Golgi network targeting and plasma membrane internalization signals in vaccinia virus B5R envelope protein

The vaccinia virus B5R type I integral membrane protein accumulates in the Golgi network, from where it becomes incorporated into the envelope of extracellular virions. Our objective was to determine the domains of B5R responsible for Golgi membrane targeting in the absence of other viral components. Fusion of an enhanced green fluorescent protein to the C terminus of B5R allowed imaging of the chimeric protein without altering intracellular trafficking and Golgi network localization in transfected cells. Deletion or swapping of B5R domains with corresponding regions of the vesicular stomatitis virus G protein, which is targeted to the plasma membrane, indicated that (i) the N-terminal extracellular domain of B5R had no specific role in Golgi apparatus localization, (ii) the transmembrane domain of B5R was sufficient for exiting the endoplasmic reticulum, and (iii) removal of the cytoplasmic tail impaired Golgi network localization and increased the accumulation of B5R in the plasma membrane. Further experiments demonstrated that the cytoplasmic tail mediated internalization of B5R from the plasma membrane, suggesting a retrieval mechanism. Mutagenesis revealed residues required for Golgi membrane localization and efficient plasma membrane retrieval of the B5R protein: a tyrosine at residue 310 and two adjacent leucines at residues 315 and 316.     

A novel approach for the rapid mutagenesis and directed evolution of the structural genes of west nile virus

Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (～10(7) infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the "pr" portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.     

Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3'-terminal 6 nucleotides (nt) (5'-GGAUCU(OH)-3') of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3'-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i). The flavivirus-conserved terminal 3' U is optimal for WN virus replication. Replacement of the wild-type 3' U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3' A or G mutants, with only partial reversion. (ii). The flavivirus-conserved 3' penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3'-terminal CU(OH), are absolutely essential for viral replication. (iii). The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv). The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v). Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3' flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.     

Reconstitution of protein targeting to the inner envelope membrane of chloroplasts

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), we investigated the targeting of the nucleus-encoded integral IM protein, atTic40. We found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. We also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. Collectively, our experiments are consistent with a "postimport" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.     

Impaired virus clearance, compromised immune response and increased mortality in type 2 diabetic mice infected with west nile virus

Clinicoepidemiological data suggest that type 2 diabetes is associated with increased risk of West Nile virus encephalitis (WNVE). However, no experimental studies have elucidated the role of diabetes in WNV neuropathogenesis. Herein, we employed the db/db mouse model to understand WNV immunopathogenesis in diabetics. Nine-week old C57BL/6 WT and db/db mice were inoculated with WNV and mortality, virus burden in the periphery and brain, and antiviral defense responses were analyzed. db/db mice were highly susceptible to WNV disease, exhibited increased tissue tropism and mortality than the wild-type mice, and were unable to clear the infection. Increased and sustained WNV replication was observed in the serum, peripheral tissues and brain of db/db mice, and heightened virus replication in the periphery was correlated with enhanced neuroinvasion and replication of WNV in the brain. WNV infection in db/db mice was associated with enhanced inflammatory response and compromised antiviral immune response characterized by delayed induction of IFN-α, and significantly reduced concentrations of WNV-specific IgM and IgG antibodies. The compromised immune response in db/db mice correlated with increased viremia. These data suggest that delayed immune response coupled with failure to clear the virus leads to increased mortality in db/db mice. In conclusion, this study provides unique mechanistic insight into the immunopathogenesis of WNVE observed in diabetics and can be used to develop therapeutics for the management of WNVE among diabetic patients.     

HCV envelope protein function is dependent on the peptides preceding the glycoproteins

Although significant advances have been made on the studies of HCV glycoproteins (E1 and E2) recently, the role of the peptides preceding each glycoprotein remains unclear. We expressed E1 and E2 using two individual plasmids to form HCV pseudoparticles (HCVpp) in order to characterize the peptides preceding E1 and E2.Our data show that 14 amino acids from the HCV core and 12 amino acids from the E1 C-terminus are required for E1 and E2 function, respectively. The lack of a long enough peptide preceding E1 or E2 will abolish HCVpp infectivity, and the presence of fewer than 14 amino acids ahead of E1 and 12 amino acids ahead of E2 may alter their glycosylation. Furthermore, the peptides preceding E1 and E2 may be interchanged or may be replaced by those from genotype 2a. Our findings may contribute to the future development of new anti-HCV drugs.     

Crystal structure of the Japanese encephalitis virus envelope protein

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.