West nile virus structural proteins

A mutation leading to substitution of a key amino acid in the prM protein of West Nile virus WNV occurred during persistent infection of an immunocompetent patient. This case demonstrates active replication of WNV during persistent infection.

West Nile virus WNV is a notable cause of neuroinvasive disease and febrile illness. In humans, WNV generates low viremia levels during infection 1. WNV is endemic in Israel and has been the cause of several disease outbreaks in recent years 2. Several subtypes of WNV lineage 1 have been phylogenetically identified in mosquitoes in Israel 3. However, no WNV sequence or isolation of viruses from humans in Israel had been reported since until the case we report here.

In this study, we isolated and sequenced WNV lineage 1 and identified an amino acid mutation in the prM protein sequence that occurred between day 19 and day 28 of persistent viremia and viruria in a person with confirmed WNV encephalitis. His family recalled that he had received an unusually large mosquito bite 2 weeks before hospital admission.

His medical history was remarkable for a thymoma B2 that was resected 3 years earlier without any evidence of myasthenia gravis. He did not receive immunosuppressive drugs or any other long-term drug therapy. On examination, he was drowsy, disoriented, and noncooperative. Marked neck rigidity was noted. Results of the remainder of the physical and neurologic examination were normal.

Follow-up blood counts, 10 and 50 days after admission, showed 6, and 6, leukocytes, of which 4, and 4, were neutrophils, respectively. Results of a computed tomography scan of his brain, without and with contrast media, were normal. Gram stain results were negative for bacteria. An electroencephalogram showed generalized slowing of brain electrical activity. On the eighth day of hospitalization, pain developed in the left shoulder along with rapidly progressive weakness and atrophy in the left upper limb muscles over several days, mainly in the deltoid, supraspinatus, biceps, and triceps muscles.

Treatment with antiinflammatory drugs and physiotherapy were initiated. Electrophysiologic studies 2 weeks later showed asymmetric denervation in the muscles innervated by C4—C7 nerve roots; the denervation was more prominent on the left side, compatible with an anterior-horn cell lesion. This segmental poliolike syndrome improved slowly and was still present a year later. Because RNA extracted from a urine sample obtained on day 12 was positive 8.

The results demonstrated persistent viremia for 47 days and viruria for 61 days after illness onset Table. In addition, infectious virus was isolated from 2 urine samples taken on days 12 and Sequencing of the premembrane prM , membrane, and envelope protein sequences 2, bp obtained from the first serum sample showed identical sequences to those obtained from all subsequent urine samples taken until day No other nucleotide mutations in the prM or the envelope proteins were identified.

However, the reason the virus persists in some patients but not in others is unknown. In this study, we followed the kinetics of viral clearance and antibody response of a patient with WNV persistence and demonstrated that, despite the development of IgM and IgG, substantial amounts of WNV RNA persisted in serum for 47 days and in urine for 61 days after illness onset. Notably, isolation of infectious virus from urine and appearance of an amino acid mutation in the prM of WNV on day 28 indicate not only persistence of WNV RNA but also active replication.

Although patients with thymoma B2 may, in rare cases, exhibit hypogammaglobulinemia and cellular immune dysfunction 11 , this patient was considered immunocompetent because his medical history and follow-up after this infection did not show any indication of immune deficiency.

The domains are indicated by a colored bar as in Fig. The sequences are truncated at the last residue of the soluble fragment sE of West Nile virus E, which we crystallized.

The conserved glycosylation site in domain I is indicated by a red asterisk and red lettering. Residues lining the hydrophobic pocket in sE are shaded in gray. Residues that are exposed on the viral surface and are conserved in West Nile virus strains but not in other flaviviruses are shaded in magenta.

Residues that are exposed on the viral surface and are conserved in wn, je, d2, and tbe viruses are shaded in orange. The individual domains of West Nile virus sE have a high degree of structural similarity to other flavivirus sE structures 25 , 27 , 31 , 35 , Several loops in West Nile virus sE diverge in conformation.

These include the loops in domain III that are exposed on the viral surface, in particular the DE to and FG to loops, and the glycosylated E 0 F 0 loop to in domain I. Surface-exposed residues determine receptor specificity, vector preference, host range, and tropism of West Nile virus.

A surprisingly small number of residues are responsible for all of these characteristics. Indeed, only 57 residues are conserved in West Nile virus strains but not conserved across flaviviruses including JE virus. Of these residues, only 38 are exposed on the viral surface Fig. Distribution of West Nile virus-specific residues on sE. A and B Two perpendicular views of West Nile virus sE, with residues that are conserved in West Nile virus strains but not in other flaviviruses shown in space-filling representation.

Residues that are exposed on the surface of the mature virus are in magenta; residues that are not exposed are in gray. The I 0 -strand peptide recognized by recombinant antibodies scFv-Fc 11, 71, and 73 residues to is shown in green, with the two essential basic residues Lys and Lys in space-filling representation.

Most West Nile virus-specific neutralizing antibodies bind an epitope that includes Thr 21 , 31 , The view in panel B is perpendicular to the viral surface, such that the outside of the virion is up. The views are the same as in Fig.

C Atomic model of the West Nile virus outer protein shell based on the 9. E assembles into dimers in mature virions. The glycan of West Nile virus E is shown in red, residues lining the putative hydrophobic pocket in dark gray, residues to a partial epitope of scFv-Fcs 11, 71, and 73 in green, and the epitope of therapeutic antibody E16 32 in blue. The fusion loop is in orange. A black triangle connects the icosahedral symmetry axes.

D Close-up of panel C, with the two-, three-, and fivefold icosahedral symmetry axes labeled. A single sE monomer is circled with a semitransparent gray line. The loop bearing the only glycan of the outer protein shell of the mature West Nile virion is the most distinct from other sE structures. The N-linked glycosylation site is conserved in all flaviviruses except yellow fever virus and is located at Asn, in a loop between the fifth and sixth strands of domain I, the so-called E 0 and F 0 strands.

Loss of the glycosylation site at position in certain West Nile virus strains leads to strong attenuation, loss of neuroinvasiveness, and lower stability at mildly acidic pH 3. A similar phenotype is observed when the homologous glycan is lost in dengue virus The loop packs onto strand F 0 and onto the N terminus, which adopts a slightly different conformation than in the dengue and TBE virus sE structures. Despite this shift, the orientation of these glycans in dengue and West Nile virus E is such that they largely overlap and therefore localize to the same positions on the surface Fig.

While the E 0 F 0 loop may be largely protected from immune recognition by its glycan, its structure extends far enough from the glycan in West Nile virus that the E 0 F 0 loop may constitute an attractive target for West Nile virus-specific neutralizing antibodies. We recently used a phage display selection method to generate a panel of human single-chain variable region antibody fragments fused to an IgG1 Fc domain scFv-Fcs.

A similar approach has been used to generate cross-neutralizing recombinant antibodies against dengue virus 12 , Our recombinant antibodies neutralize both West Nile and dengue viruses by binding conserved epitopes in domains I and II Although this method does not allow conformational epitopes to be identified, we found that three of the scFv-Fcs with the best therapeutic properties 13 , scFv-Fcs 11, 71, and 73, recognized a peptide spanning residues to The scFv-Fcs also recognize the homologous E peptide from dengue virus type 2 but not a peptide composed of the same amino acids in a randomized sequence Fig.

The two structures show that the structural assembly of E in the outer protein shell is very similar in the two viruses. The dengue virus cryoEM structure is of sufficient resolution to allow a highly accurate fitting of the atomic coordinates of the dengue virus sE crystal structure into the cryoEM structure By superimposing our West Nile virus sE structure onto the cryoEM-fitted dengue virus sE atomic coordinates and applying icosahedral symmetry, we generated a pseudoatomic model of the entire outer protein shell of the West Nile virion Fig.

Interestingly, it is impossible to achieve a good fit for the subunits of E in the cryoEM structures while avoiding steric clashes, with domain II in the same orientation relative to domain I as in our crystal structure. This suggests that the interface between domains I and II must be flexible in prefusion sE. However, in contrast to West Nile virus sE, these rotations in domain II of dengue virus sE are about an axis that is orthogonal to the axis of rotation of the fusion transition.

A possible explanation for the inability of West Nile virus sE to form dimers in solution is that domain II prefers the postfusion-like orientation observed in the crystal structure over the prefusion-like orientation observed in the mature virion. However, we cannot rule out that the acidic conditions used to elute sE from our immunoaffinity column see Materials and Methods have irreversibly altered the native prefusion structure of sE, causing it to adopt a more postfusion-like monomeric structure.

Since the pH threshold of membrane fusion is approximately 6. It is not known precisely how E senses low pH. Two conserved histidines at the interface between domains I and III have been proposed to form part of the pH sensor of sE in all flaviviruses 4. This could be the initial low-pH trigger that allows domains I and II to rotate out of the viral surface and expose the fusion loop. In West Nile virus sE, the two histidines His and His form similar interactions with domain I as in TBE virus 35 , supporting the notion that the low-pH trigger mechanism for the fusion transition is conserved across flaviviruses.

Our atomic model of the outer glycoprotein layer of the West Nile virus particle reveals the location on the viral surface of potential receptor binding sites and of the epitopes of several previously described neutralizing antibodies. As expected, the epitope of therapeutic antibody E16 maps to a patch on domain III that is fully exposed on the viral surface and also on the postfusion form of E Remarkably, the epitopes of all other strongly neutralizing West Nile virus-specific antibodies localize to the same patch on domain III 32 , 36 , Indeed, most neutralizing antibodies against dengue and JE viruses also map to this region and are also serotype or strain specific.

Within this epitope, the N-terminal loop of domain III residues to and the BC loop to have a dominant role in flavivirus neutralization 21 , 31 , 32 , 36 , 38 , From the data presented above, we conclude that binding of West Nile virus-specific antibodies is likely to involve a subset of the 38 West Nile virus-specific residues that are exposed on the viral surface Fig.

Of these residues, eight are in domain III all in the dominant epitope described above , and five have in fact already been shown to bind neutralizing antibodies directly residues , , , , and 31 , The remaining 27 West Nile virus-specific residues are distributed fairly evenly throughout domains I and II, although there are none in the area around the fusion loop, which is highly conserved across flaviviruses Fig.

While neutralizing antibodies raised against West Nile virus particles invariably bind variable epitopes in domain III, our recombinant scFv-Fc neutralizing antibodies bind conserved epitopes in domains I and II Thus, unlike conventional antibodies, some of our scFv-Fcs cross-neutralize dengue virus type 2 Since the scFv-Fcs were selected for their ability to bind monomeric sE, their epitopes are not necessarily exposed on the viral surface. Three of the scFv-Fcs with the best therapeutic properties, scFv-Fcs 11, 71, and 73 13 , recognize a peptide spanning residues to and the homologous peptide from dengue virus type 2 Fig.

Based on the location and conserved structure of this partial epitope, we conclude that a likely mechanism of neutralization for the cross-neutralizing scFv-Fcs is inhibition of the membrane fusion transition. West Nile virus sE contains an RGE motif in domain III residues to , which is exposed on the viral surface and also forms part of the dominant neutralizing antibody epitope.

Thus, inhibition of receptor binding is the most likely mechanism of neutralization for these antibodies. Since antibody E16 is protective even when administered after cellular attachment has occurred 31 , the virus may achieve initial attachment by binding glycosaminoglycans 18 or, like dengue virus E, by binding a carbohydrate recognition protein through a glycan on the viral surface The latter forms of initial cellular attachment may not be sufficient for infection, and they would probably not interfere with the binding neutralizing antibodies such as E16, because both the glycan and a conserved residue cluster proposed to bind glycosaminoglycans in dengue virus are located in domain I 7.

Indeed, the specificity of carbohydrate recognition of the two lectins depends largely on whether glycans can bind all four carbohydrate recognition domains simultaneously 10 , We find, unexpectedly, that the West Nile virus sE structure shares similarities in the relative orientation of its three domains with both pre- and postfusion structures of dengue and TBE virus sE. It is still unclear whether the West Nile virus sE structure represents a true mechanistic intermediate in the membrane fusion transition, but the incompatibility of the structure with image reconstructions of intact viruses suggests that E may not be in its preferred conformation in the environment of a mature virion.

Does the resulting mechanical energy stored in the outer protein shell, as we propose, serve to drive early steps of membrane fusion in the endosome?

The answer to this question will require careful measurements of kinetic and energetic parameters of flavivirus membrane fusion and assembly. As anticipated, the known epitopes of West Nile virus-specific neutralizing antibodies map to an area of domain III that is exposed on the viral surface. Antibodies against domain III would prevent binding of this type of receptor. In contrast, we show here that our recombinant antibodies recognize an epitope in domain I, which is only briefly exposed during the fusion transition and is at least partly conserved in dengue virus.

We therefore expect that our recombinant antibodies act by inhibiting the fusion transition. Our analysis of the domain organization of West Nile virus sE and of the molecular landscape of the viral surface offers new insight into the membrane fusion mechanism, into likely modes of receptor binding, and into mechanisms of antibody neutralization.

The detailed understanding of specific mechanisms of the viral life cycle gained from our structure provides a framework for the rational design of antiviral vaccines and therapeutics. National Center for Biotechnology Information , U. Journal List J Virol v. J Virol. Published online Aug Marasco , 4 Raymond A. Hannah Gould.

Wayne A. Raymond A. Author information Article notes Copyright and License information Disclaimer. Mailing address: Whitney Ave. Phone: Fax: E-mail: ude. Received Aug 10; Accepted Aug This article has been cited by other articles in PMC. Abstract West Nile virus , a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis.

Crystallization and data collection. TABLE 1. Data collection and refinement statistics. Open in a separate window. Structure determination. Measurement of scFv-Fc binding to sE peptides. Protein structure accession numbers. The hydrophobic pocket. Unique surface features of West Nile virus. Recombinant antibody partial epitope mapping. Implications for membrane fusion.

Implications for immune recognition. Implications for receptor binding. Allison, S. Schalich, K. Stiasny, C. Mandl, C. Kunz, and F. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. Anderson, J. Andreadis, C. Vossbrinck, S. Tirrell, E. Wakem, R. French, A. Garmendia, and H. Van Kruiningen. Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut.

Science : Beasley, D. Whiteman, S. Zhang, C. Huang, B. Schneider, D. Smith, G. Gromowski, S. Higgs, R. Kinney, and A. Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains.

Bressanelli, S. Stiasny, S. Allison, E. Stura, S. Duquerroy, J. Lescar, F. Heinz, and F. Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO J. Adams, G. Clore, W. DeLano, P. Ann N Y Acad Sci. Hayes CG. Bin H, et al. West Nile fever in Israel from geese to humans. Platonov AE, et al. Emerg Infect Dis. Tsai TF, et al. West Nile encephalitis epidemic in southeastern Romania.

Lanciotti RS, et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Mostashari F, et al. Epidemic West Nile encephalitis, New York, results of a household-based seroepidemiological survey. Brilla R, et al. Clinical and neuroradiologic features of 39 consecutive cases of West Nile Virus meningoencephalitis. J Neurol Sci. Virology, pathology, and clinical manifestations of West Nile virus disease.

West Nile virus encephalitis. Tyler KL. West Nile virus infection in the United States. Arch Neurol. Campbell GL, et al. West Nile virus. Lancet Infect Dis.

J Invest Dermatol. Langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovirus. Busch MP, et al. Virus and antibody dynamics in acute west nile virus infection. J Infect Dis. Tonry JH, et al. Persistent shedding of West Nile virus in urine of experimentally infected hamsters. Am J Trop Med Hyg.

Tesh RB, et al. Persistent West Nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections. Ding X, et al. Nucleotide and amino acid changes in West Nile virus strains exhibiting renal tropism in hamsters.

Arjona A, et al. Abrogation of macrophage migration inhibitory factor decreases West Nile virus lethality by limiting viral neuroinvasion. J Clin Invest. Dai J, et al. Icam-1 participates in the entry of west nile virus into the central nervous system. Wang P, et al. Matrix metalloproteinase 9 facilitates West Nile virus entry into the brain. Wang S, et al. Drak2 contributes to West Nile virus entry into the brain and lethal encephalitis. J Immunol. Verma S, et al. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier.

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat Med. West Nile virus: crossing the blood-brain barrier. Garcia-Tapia D, et al. West Nile virus encephalitis: sequential histopathological and immunological events in a murine model of infection. J Neurovirol. Morrey JD, et al.

Increased blood-brain barrier permeability is not a primary determinant for lethality of West Nile virus infection in rodents. Samuel MA, et al. Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis. Tyler KL, et al. CSF findings in patients with serologically confirmed West Nile virus meningitis and encephalitis.

Petropoulou KA, et al. West Nile virus meningoencephalitis: MR imaging findings. Ali M, et al. West Nile virus infection: MR imaging findings in the nervous system. Rossi SL, et al. Adaptation of West Nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action. Anderson J, Rahal JJ. Efficacy of interferon alpha-2b and ribavirin against West Nile virus in vitro. Failure of interferon alpha-2b in a patient with West Nile virus meningoencephalitis and acute flaccid paralysis.

Scand J Infect Dis. Chowers MY, et al. Clinical characteristics of the West Nile fever outbreak, Israel, Kalil AC, et al. Use of interferon-alpha in patients with West Nile encephalitis: report of 2 cases. Clin Infect Dis. Sayao AL, et al. Calgary experience with West Nile virus neurological syndrome during the late summer of Can J Neurol Sci.

Weiss D, et al. Efficacy of orally administered T pyrazine analog on lethal West Nile virus infection in rodents. Antiviral Res. Ben-Nathan D, et al. Using high titer West Nile intravenous immunoglobulin from selected Israeli donors for treatment of West Nile virus infection. BMC Infect Dis. Saquib R, et al.

West Nile virus encephalitis in a renal transplant recipient: the role of intravenous immunoglobulin. Am J Kidney Dis. Sejvar JJ. The long-term outcomes of human West Nile virus infection. Pogodina VV, et al. Study on West Nile virus persistence in monkeys. Siddharthan V, et al. Persistent West Nile virus associated with a neurological sequela in hamsters identified by motor unit number estimation.

Penn RG, et al. Persistent neuroinvasive West Nile virus infection in an immunocompromised patient. Brenner W, et al.

West Nile Virus encephalopathy in an allogeneic stem cell transplant recipient: use of quantitative PCR for diagnosis and assessment of viral clearance. Bone Marrow Transplant. Kapoor H, et al. J Clin Virol. Prince HE, et al. Persistence of West Nile virus-specific antibodies in viremic blood donors. Clin Vaccine Immunol. Roehrig JT, et al. Persistence of virus-reactive serum immunoglobulin m antibody in confirmed west nile virus encephalitis cases. Mutations in West Nile virus nonstructural proteins that facilitate replicon persistence in vitro attenuate virus replication in vitro and in vivo.

Keller BC, et al. Shrestha B, Diamond MS. Shrestha B, et al. Gamma interferon plays a crucial early antiviral role in protection against West Nile virus infection. Wang T, et al. Wang Y, et al. Diamond MS, et al. B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus. Klein RS, et al. Purtha WE, et al. A critical role for induced IgM in the protection against West Nile virus infection. J Exp Med. Ng T, et al.

Equine vaccine for West Nile virus. Dev Biol Basel ; —7. Samina I, et al. An inactivated West Nile virus vaccine for domestic geese-efficacy study and a summary of 4 years of field application. Arroyo J, et al. ChimeriVax-West Nile virus live-attenuated vaccine: preclinical evaluation of safety, immunogenicity, and efficacy.

Minke JM, et al. Arch Virol Suppl. Monath TP, et al. A live, attenuated recombinant West Nile virus vaccine. Pletnev AG, et al. Davis BS, et al. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays.

Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection. Ledizet M, et al. A recombinant envelope protein vaccine against West Nile virus. Lieberman MM, et al. Preparation and immunogenic properties of a recombinant West Nile subunit vaccine. Qiao M, et al.