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. 2000 Oct;74(19):8867-75.
doi: 10.1128/jvi.74.19.8867-8875.2000.

Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry

E Lee  1 , M Lobigs
Affiliations

Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry

E Lee et al. J Virol. 2000 Oct.

Abstract

The flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (FG loop) in the envelope (E) protein. In some flaviviruses this domain harbors the integrin-binding motif Arg-Gly-Asp (RGD). One of us has shown earlier that host cell adaptation of Murray Valley encephalitis virus (MVE) can result in the selection of attenuated variants altered at E protein residue Asp(390), which is part of an RGD motif. Here, a full-length, infectious cDNA clone of MVE was constructed and employed to systematically investigate the impact of single amino acid changes at Asp(390) on cell tropism, virus entry, and virulence. Each of 10 different E protein 390 mutants was viable. Three mutants (Gly(390), Ala(390), and His(390)) showed pronounced differences from an infectious clone-derived control virus in growth in mammalian and mosquito cells. The altered cell tropism correlated with (i) a difference in entry kinetics, (ii) an increased dependence on glycosaminoglycans (determined by inhibition of virus infectivity by heparin) for attachment of the three mutants to different mammalian cells, and (iii) the loss of virulence in mice. These results confirm a functional role of the FG loop in the flavivirus E protein in virus entry and suggest that encephalitic flaviviruses can enter cells via attachment to glycosaminoglycans. However, it appears that additional cell surface molecules are also used as receptors by natural isolates of MVE and that the increased dependence on glycosaminoglycans for entry results in the loss of neuroinvasiveness.

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Figures

FIG. 1
FIG. 1
Full-length infectious cDNA clone of MVE-1-51. Plasmid pM212 contains the full-length cDNA of MVE-1-51 (11,013 nucleotides), which includes an open reading frame (ORF) encoding a polyprotein of 3,434 amino acids (solid line). Restriction sites used in subcloning and genetic manipulations are shown (numbering is from the 5′-terminal nucleotide in MVE-1-51). Sequences at the 5′ (plus an ApaI site and T7 promoter sequence) and 3′ (plus a NotI site) termini of the viral genome are shown (boxed). The vector pBR322* is a deleted version of pBR322 (see Materials and Methods). The ampicillin resistance gene (Ap), the ROP gene, and the origin of DNA replication (ORI) are shown. NCR, noncoding region.
FIG. 2
FIG. 2
Growth of E protein 390 mutants in cell culture. Vero, SW13, and C6/36 cells were infected with MVE-1-51, the Asp390 control virus, or 1 of 10 E protein 390 mutants at multiplicity of ∼0.1, determined for each cell type. Unbound virus was removed after 1 h of adsorption, and growth medium was added. At 16, 20, 24, and 28 hpi, supernatants were collected from Vero and SW13 cells for titration of virus infectivity, in the respective cell types. For growth assays in C6/36 cells, supernatant samples were taken at 24, 28, and 44 hpi, and virus titers were determined as focus-forming units (FFU) by immunofluorescence in C6/36 cells. Results for Vero and SW13 growth assays are from two separate experiments (shown in different graphs).
FIG. 3
FIG. 3
Uptake of MVE into SW13 cells. The kinetics of uptake of the Asp390 control and His390 mutant viruses into SW13 cells was determined as described in Materials and Methods. Percent uptake for each time point is expressed as the ratio of the number of plaques obtained after acid treatment and the number of plaques obtained on a control monolayer in the absence of acid treatment.
FIG. 4
FIG. 4
Inhibition by heparin of virus infectivity in Vero, SW13, and BHK cells. (A) The Asp390 control and His390, Gly390, and Lys390 mutant viruses (200 PFU) were incubated with heparin (0, 50, 100, and 200 μg/ml) prior to their addition to cells pretreated with HBSS-BSA containing heparin at 0, 50, 100, or 200 μg/ml. Agar overlay was added to allow plaque formation after 1 h of adsorption at 37°C. Inhibition by heparin was calculated using the formula [(plaque number on nontreated cells − plaque number on heparin-treated cells)/(plaque number from non-treated cells)] × 100. (B) Inhibition of infectivity by heparin (200 μg/ml) of 10 E protein 390 mutants was assayed in parallel with the Asp390 control virus as in panel A. The percent inhibition by heparin of the Asp390 virus is subtracted from that for the E protein 390 mutants obtained in the same experiment. Results (mean and standard error) from three independent experiments are shown for each mutant. (C) Inhibition by heparin of virus binding to Vero and SW13 cells. The Asp390 control virus and the His390 mutant were incubated with heparin (0, 20, 50, 100, or 200 μg/ml) for 15 min at 4°C and then added to chilled Vero or SW13 cells treated with heparin (0 to 200 μg/ml). After 1 h of adsorption at 4°C, cell monolayers were washed, and agar overlay was added to allow plaque development. Percent inhibition by heparin is calculated as in panel A. Results (mean and standard error) from two independent experiments are shown.
FIG. 5
FIG. 5
Virulence of infectious clone-derived MVE and E protein 390 mutants. Virus stocks were diluted 10-fold serially in HBSS-BSA and used to inoculate 21-day-old Swiss outbred mice in groups of five by the i.p. or i.c. route. Mortality was recorded for 14 days.
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