Interaction of Yellow Fever Virus French ... - Journal of Virology

JOURNAL OF VIROLOGY, Mar. 2000, p. 2903–2906 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 6

Interaction of Yellow Fever Virus French Neurotropic Vaccine Strain with Monkey Brain: Characterization of Monkey Brain Membrane Receptor Escape Variants HAOLIN NI,1,2 KATE D. RYMAN,1,2† HEIMAN WANG,1,2 MOHAMMAD F. SAEED,2,3 ROBIN HULL,4 DAVID WOOD,4 PHILIP D. MINOR,4 STANLEY J. WATOWICH,5 AND ALAN D. T. BARRETT1,2,3* Department of Pathology,1 Center for Tropical Diseases,2 Department of Microbiology and Immunology,3 and Sealy Center for Structural Biology,5 University of Texas Medical Branch, Galveston, Texas 77555-0609, and Division of Virology, National Institute of Biological Standards and Control, Potters Bar EN6 3QG, United Kingdom4 Received 14 June 1999/Accepted 17 December 1999

Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRPR) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRPR variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRPR variant selected at pH 6.0 and two mouse brain MRPR variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458). Wild-type yellow fever (YF) virus, the prototype member of the Flavivirus genus (family Flaviviridae), characteristically causes hepatitis and hemorrhagic fever (i.e., viscerotropic disease) and not neurotropic disease in humans and monkeys. YF is controlled by the use of live attenuated vaccines, known as 17D and FNV (for French neurotropic vaccine) (see reference 1 for a review), which demonstrate various degrees of neurotropism, but not viscerotropism, in humans and monkeys. The attenuated FNV strain was derived by passage of wildtype strain French viscerotropic virus (FVV) in mouse brain (6). Although the FNV strain was attenuated for viscerotropism, it was found to have enhanced neurotropic properties, including lethality for monkeys following intracerebral inoculation and a high incidence of postvaccination complications in children under the age of 11 years (3). In comparison, the 17D vaccine strain (derived by passage of wild-type strain Asibi in chicken tissue) was found to be attenuated for both viscerotropism and neurotropism to the extent that 17D virus rarely kills monkeys following intracerebral inoculation, and the incidence of postvaccination complications is so low that the 17D vaccine can safely be given to 1-year-old children. The more attenuated phenotype of 17D relative to FNV resulted in 17D virus replacing FNV virus as the vaccine of choice to control YF. The molecular basis of the enhanced neurotropism and the cell receptor(s) for FNV are still unknown and are the subject of this communication. The first step of a virus infection is attachment to the host cell (5). Binding of the virion to specific cellular receptor-

binding site molecules is mediated by the viral spike protein(s), which in the case of the flaviviruses is the envelope (E) protein. Recent studies have elucidated the three-dimensional structure of a 50-kDa fragment of the ectodomain of the E protein of Central European tick-borne encephalitis (TBE) virus and assigned three domains (termed, I, II, and III) to the protein structure (8). We have previously reported a methodology for investigation of the interaction of the flavivirus Japanese encephalitis (JE) virus with mouse brain cells by selection of mouse brain membrane receptor preparation (MRP) binding escape variants (MRPR) and used this technique to identify potential E protein amino acids of JE virus that interact with mouse brain cell binding sites (7). This study proposed that E-306 in domain III was involved in the interaction of the E protein with the binding site in mouse brain cells. This methodology (described in detail in reference 7) has now been used to investigate the interaction of FNV with monkey brain cells. Briefly, the brain and liver of a cynomolgus monkey and the brains of NIH Swiss mice were dissected, weighed, and then homogenized in 50 mM Tris buffer, pH 7.6. The homogenates were centrifuged at 35,600 ⫻ g for 10 min to obtain MRPs. The pellets were resuspended in Tris buffer, and the process was repeated twice. The final pellets were resuspended in Tris buffer at a final protein concentration of 20 to 40 mg (wet weight) of brain or liver tissue/ml and stored at ⫺70°C. MRPs from monkey brain, monkey liver, and mouse brain will be referred to as MKB, MKL, and MS MRPs, respectively. The specificity of binding of FNV to MKB MRPs was determined by comparison of wild-type and vaccine strains of YF virus with other flaviviruses, namely, JE virus strain P3 and dengue virus serotype 2 (DEN-2) strain New Guinea C (NGC). Neurotropic JE virus strain P3 bound to MKB MRP but bound less well to MKL MRP (Table 1), while nonneurotropic, non-

* Corresponding author. Mailing address: Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. Phone: (409) 772-6662. Fax: (409) 747-2415. E-mail: abarrett@utmb .edu. † Present address: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514. 2903

2904

NOTES

J. VIROL.

TABLE 1. Comparison of binding of YF, JE, and DEN-2 to MKB and MKL MRPs Virus

Strain

Infectivitya (log10 PFU) Virus ⫹ buffer

Virus ⫹ MRP

Binding indexb

3.6 (MKB) 5.0 (MKL) 5.5 (MKB) 5.6 (MKL) 5.0 (MKB) 3.8 (MKL) 2.3 (MKB) 5.0 (MKL) 5.8 (MKB) 5.0 (MKL) 6.3 (MKB) 6.0 (MKL)

2.1 (MKB) 0.7 (MKL) 0.3 (MKB) 0.2 (MKL) 0.6 (MKB) 1.8 (MKL) 4.2 (MKB) 1.5 (MKL) 0.5 (MKB) 1.3 (MKL) 0.5 (MKB) 0.8 (MKL)

JE

P3

5.7

DEN-2

NGC

5.8

YF

FVV

5.6

FNV-Yale

6.5

Asibi

6.3

17D-204

6.8

a Infectious virus remaining in the supernatant following incubation of virus with MRP or buffer and pelleting of the virus-MRP complex as described previously (7). b Log10 reduction in infectivity titer (log10 infectivity in buffer/infectivity in MRP supernatant).

viscerotropic DEN-2 NGC strain bound poorly to both MKB and MKL MRPs (i.e., there was a correlation with virus tissue specificity in vivo). Wild-type YF virus strains Asibi and FVV bound well to MKL MRP but bound poorly to MKB MRP, while, interestingly, vaccine strain 17D-204 bound poorly to both MKB and MKL MRPs. This may be indirectly involved in the attenuated phenotype of 17D virus. FNV was the only strain of YF virus to bind well to MKB MRPs. Four MKB MRP binding-resistant (MRPR) variants were generated for FNV “strain” Yale (10) as described previously (7), except that MRPs were derived from the brain of a cynomolgus monkey. Briefly, MRPR variants were generated by incubating virus with MRPs for 30 min at 37°C in 50 mM Tris, pH 7.6 (or pH 6.0 in some experiments) and then centrifuging at 13,000 ⫻ g for 10 min to remove MRP and bound virus. Residual infectious virus in the supernatant was titrated in Vero cell monolayers, and individual MRPR variant plaques were picked and amplified in Vero cells. Putative MRPR variants were examined for binding to MRPs, and lack of reduction in infectivity following incubation of the virus with fresh MRPs confirmed that they were true MRPR variants. Using this procedure, three MKB MRPR variants were selected in

buffer at pH 7.6 and designated MKB MRPR I, MKB MKRPR II, and MKB MRPR IV. A fourth variant was selected in 50 mM Tris buffer, pH 6.0, to investigate the potential effect of pH-induced conformational changes in the E protein; it was termed MKB MRPR (pH 6.0). Two MRPR variants, selected from FNV-Yale after incubation with mouse brain MRPs at pH 7.6, were designated MS MRPR I and MS MRPR II. The plaque morphology of the six MRPR variants was indistinguishable from that of parental FNV. Similarly, there were no differences in growth characteristics or infectivity titers in Vero cell cultures. FNV-Yale, which is known to be highly neurovirulent for mice after intracerebral inoculation (10), was found to have a Vero cell PFU/50% lethal dose (LD50) ratio of 0.08 in 4-weekold female NIH-Swiss mice, while MKB MRPR variant viruses selected at pH 7.6 were attenuated at least 400-fold (e.g., 32 PFU/LD50 for MKB MRPR II) (Table 2). MKB MRPR (pH 6.0) was attenuated 87-fold compared to parent FNV-Yale virus and was less attenuated than MKB MRPR variant viruses selected at pH 7.6. The two MS MRPR variants were attenuated approximately 50- to 100-fold compared with parental FNV-Yale virus (Table 2). Two of the MKB MRPR variant viruses selected at pH 7.6 were indistinguishable from the parental FNV-Yale when tested in monkeys by the World Health Organization neurovirulence test inoculation, dose, and scoring procedure (Table 2) (12), but just one monkey per preparation was tested. Sequencing of premembrane and E protein genes of parent and MRPR variant viruses revealed that each variant had only a single nucleotide change, resulting in one amino acid substitution in the E protein, compared with the parental FNV-Yale virus. No changes in the M protein gene were identified. The MKB MRPR (pH 6.0) variant had one amino acid substitution at E-458 (G3R), and the three MKB MRPR variants selected at pH 7.6 each had a single amino acid substitution at E-260 (G3A), E-274 (Y3H), or E-237 (P3Y) for MKB MRPR I, MKB MRPR II, and MKB MRPR IV, respectively. These substitutions were found to be unique; they have not been found in the E protein of any other YF virus strain sequenced to date (2, 11). The two MS MRPR variants had identical amino acid substitutions at E-457 (M3I). Note that unlike the JE MS MRPR variants reported previously (7), none of the YF MRPR variants had amino acid substitutions in domain III of the E protein. This suggests that the interaction of YF and JE virus E proteins with MS MRPs may be different.

TABLE 2. Comparison of mouse and monkey neurovirulence following intracerebral inoculation, and amino acid differences between FNVYale and MRPR variants Monkey results Virus

FNV-YALE MKB MRPR I (pH 7.6) MKB MRPR II (pH 7.6) MKB MRPR IV (pH 7.6) MKB MRPR (pH 6.0) MS MRPR I (pH 7.6) MS MRPR II (pH 7.6)

PFU/ mouse LD50

Mouse avg survival timeb (days ⫾ SEM)

Amino acid substitution (parent3variant)

0.08 ⬎100 32 80 7 5 8

5.4 ⫾ 0.4 NAc 6.5 ⫾ 0.3 6.3 ⫾ 0.3 7.2 ⫾ 0.2d 7.6 ⫾ 0.4d 7.2 ⫾ 0.2d

E-260 (G3A) E-274 (Y3H) E-237 (H3Y) E-458 (G3R) E-457 (M3I) E-457 (M3I)

Actual survival time (days)

Clinical scorea

5 6 5

Histology scorea Discriminator area

Discriminator plus target area

3.5

1.75

2.72

3.4 3.5

1.52 1.31

2.49 2.12

a The variants were indistinguishable from the parental virus on the basis of clinical score and histology score in either the discriminator areas or target plus discriminator areas, which are the usual criteria for evaluation of the World Health Organization neurovirulence test (12). b ASTs were calculated based on an inoculum of 100 PFU given by the intracerebral route to 4-week-old female NIH Swiss mice. c NA, not applicable. d AST statistically longer than that of parental FNV-Yale virus, P ⬍ 0.2.

VOL. 74, 2000

NOTES

2905

FIG. 1. Diagrammatic representation of the structure of the ectodomain of the flavivirus E protein (modified from reference 8). (A) Top view; (B) side view. White-highlighted amino acids are different in wild-type FVV and FNV; gray-shaded amino acids are substitutions found in MKB MRPR variants.

Previous studies have shown that the E protein of wild-type FVV differed from four strains of FNV in having three common amino acid substitutions at E-54, E-227, and E-249 (10). E-227 is not considered important to the phenotype of FNV, since FNV has an E residue at this position, like all YF virus strains sequenced except for FVV. Figure 1 shows a diagrammatic representation of the three-dimensional structure of the soluble portion of YF virus E protein based on the structure of the TBE virus E protein reported by Rey et al. (8), with the amino acid substitutions found in the FNV viruses (E-54, E-227, and E-249) highlighted in white. The single amino acid substitutions in the E proteins of MKB MRPR I, MKB MRPR II, and MKB MRPR IV give rise to mutations that are clustered in domain II at E-237, E-260, and E-274, respectively, and are shown as gray-shaded amino acids in Fig. 1. This clustering is adjacent to the E-54 substitution found in the FNV viruses, suggesting that this region of the E protein is involved in binding of virus to monkey brain cells and may be in part responsible for the enhanced neurotropism of FNV viruses in monkeys. The substitutions at E-260, E-274, or E-237 in the MKB MRPR variants selected at pH 7.6 were distinct from those of the MKB MRPR variant selected at pH 6.0, which had an amino acid substitution at E-458 in the proposed transmembrane region. However, selection of an MRPR variant at pH 6.0 did not affect the viability of the virus, since both MKB MRPR (pH 7.6) and MKB MRPR (pH 6.0) variants and pa-

rental FNV were not sensitive to pH, as evidenced by their infectivity at pHs from 7.6 to 5.0. Infectivity titers varied by twofold or less. We speculate that the E-458 substitution is due to the conformational change in the E protein that takes place because of the decrease in pH and is consistent with the proposal for TBE virus (9). Also, the substitution found in the two MS MRPR variants of YF FNV-Yale strain selected at pH 7.6 was at E-457 in the transmembrane region of the E protein. It is considered unlikely that the proposed transmembrane region of the E protein interacts directly with a cell receptor. Rather, the amino acid substitutions at E-457 and E-458 may induce a conformational change in the E protein that affects the interaction of the E protein with the cell receptor. Support for this proposal comes from comparison of levels of virus binding to MRPs. MS MRPR and MKB MRPR (pH 6.0) variants did not bind to either MKB or MS MRPs, while MKB MRPR (pH 7.6) variants bound to MS MRPs (albeit poorly compared to parental FNV) but not to MKB MRPs (Table 3). Presumably, mutations at E-237 to E-274 in domain II affect the conformation of the E protein less than mutations in the transmembrane region, and MKB MRPR variants can still bind to MS MRPs. Interestingly, the three MRPR variants with amino acid substitutions at E-457 or E-458 had statistically longer average survival times (ASTs) in mice than did parental FNV-Yale and MKB MRPR variants I, II and IV, even though these last three MKB MRPR variants had higher PFU/LD50 ratios than the other variants (Table 2). Thus, mutations in

2906

NOTES

J. VIROL.

TABLE 3. Comparison of binding indices of FNV-Yale and its MO and MKB MRPR variants to MO or MKB MRPs Virus strain

Parent FNV-Yale (pH 7.6) FNV-Yale (pH 6.0) MKB variants MRPR I MRPR II MRPR IV MRPR (pH 6.0) MS variants MRPR I MRPR II

Binding indexa Virus ⫹ MS MRP

Virus ⫹ MKB MRP

2.9 2.9

4.6 5.1

1.3 0.9 0.9 0.1

0.1 0.2 0.1 0.1

0.2 0.5

⫺0.2 0.3

a Log10 infectivity in buffer/infectivity in MRP supernatant (see legend to Table 1 for more details).

domain II appear to be associated with attenuation in terms in PFU/LD50 ratios, while mutations in the transmembrane region appear to alter both the PFU/LD50 ratios and the ASTs. The differences in neurotropism among the MRPR variants may be associated with the binding of these variants to MS MRPs; namely, variants with amino acid substitutions in domain II still bind to MS MRPs, while the variants with substitutions in the transmembrane region do not (Table 3). Also, mutations in the transmembrane region may affect the uptake of virus into cells in the mouse brain and may be in part responsible for the increased ASTs of mice infected with these MRPR variants. The above proposals remain to be directly tested using infectious clones and site-directed mutagenesis. Overall, these results suggest that, at the very least, the interaction of the E protein of FNV with mouse brain cell binding sites differs from that with monkey brain cell binding sites, and this may indicate that FNV recognizes different cell receptors on mouse brain and monkey brain cells. This sug-

gestion is consistent with biological studies showing that all strains of YF virus are lethal for mice while only FNV kills monkeys following intracerebral inoculation (for a review, see reference 4). This work was supported, in part, by the Clayton Foundation for Research. We thank Steve Harrison and Felix Rey for helpful discussions. REFERENCES 1. Barrett, A. D. T. 1997. Yellow fever vaccines. Biologicals 25:17–25. 2. Chang, G.-J. J., B. C. Cropp, R. M. Kinney, D. W. Trent, and D. J. Gubler. 1995. Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus. J. Virol. 69:5773–5780. 3. Durieux, C. 1956. Mass yellow fever vaccination in French Africa south of the Sahara, p. 31–66. In Yellow fever vaccination. World Health Organization, Geneva, Switzerland. 4. Freestone, D. S. 1995. Yellow fever vaccines, p. 741–779. In S. A. Plotkin and E. A. Mortimer, Jr. (ed.), Vaccines, 2nd ed. W. B. Saunders, Philadelphia, Pa. 5. Lentz, T. L. 1990. The recognition event between virus and host cell receptor: a target for antiviral agents. J. Gen. Virol. 71:751–766. 6. Mathis, C., A. W. Sellards, and J. Laigret. 1928. Sensibilite´ du Macac rhesus au virus de la fievre´ jaune. C. R. Hebd. Seances Acad. Sci. 186:604–606. (In French.) 7. Ni, H., and A. D. T. Barrett. 1998. Attenuation of Japanese encephalitis virus by selection of it mouse brain membrane receptor preparation escape variants. Virology 241:30–36. 8. Rey, F. A., F. X. Heinz, C. Mandl, C. Kunz, and S. C. Harrison. 1995. The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375:291–298. 9. Stiasny, K., S. L. Allison, A. Marchler-Bauer, C. Kunz, and F. X. Heinz. 1996. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus. J. Virol. 70:8142– 8147. 10. Wang, E., K. D. Ryman, A. D. Jennings, D. J. Wood, E. Taffs, P. D. Minor, P. D. Sanders, and A. D. T. Barrett. 1995. Comparison of the genomes of the wild-type French viscerotropic strain of yellow fever virus with its vaccine derivative French neurotropic vaccine. J. Gen. Virol. 76:2749–2755. 11. Wang, E., S. C. Weaver, R. E. Shope, R. B. Tesh, D. M. Watts, and A. D. T. Barrett. 1996. Genetic variation in yellow fever virus: duplication in the 3⬘ noncoding region of strains from Africa. Virology 225:274–281. 12. World Health Organization. 1976. Requirements for yellow fever vaccine (revised, 1975). WHO Tech. Rep. Ser. 594:23–49.