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. 2009 Dec;83(23):12631-5.
doi: 10.1128/JVI.01072-09. Epub 2009 Sep 23.

Protonation of individual histidine residues is not required for the pH-dependent entry of west nile virus: evaluation of the "histidine switch" hypothesis

Steevenson Nelson  1 , Subhajit Poddar, Tsai-Yu Lin, Theodore C Pierson
Affiliations

Protonation of individual histidine residues is not required for the pH-dependent entry of west nile virus: evaluation of the "histidine switch" hypothesis

Steevenson Nelson et al. J Virol. 2009 Dec.

Abstract

Histidine residues have been hypothesized to function as sensors of environmental pH that can trigger the activity of viral fusion proteins. We investigated a requirement for histidine residues in the envelope (E) protein of West Nile virus during pH-dependent entry into cells. Each histidine was individually replaced with a nonionizable amino acid and tested functionally. In each instance, mutants capable of orchestrating pH-dependent infection were identified. These results do not support a requirement for any single histidine as a pH-sensing "switch," and they suggest that additional features of the E protein are involved in triggering pH-dependent steps in the flavivirus life cycle.

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Figures

FIG. 1.
FIG. 1.
Impact of mutations at individual histidine residues in the WNV E protein on virus infectivity. (A) The WNV E protein is composed of three structurally distinct domains (DI-DIII). There are 13 histidine residues in the E protein; 3 of these are located in DI (red ribbons), 4 in DII (yellow ribbons), 4 in DIII (blue ribbons), and 2 in the stem-anchor region that anchors the E protein to the viral membrane (not pictured). Five of these histidine residues are conserved among flaviviruses (labeled in red). (B to D) RVPs composed of E proteins incorporating alanine (red) or glutamine (green) histidine substitutions were produced by genetic complementation. The infectious titer of each preparation was determined following infection of Raji-DC-SIGNR cells with serial twofold dilutions of RVPs. Infection was scored as a function of reporter gene expression 48 h postinfection and was measured using flow cytometry. (E) The infectious titer of each glutamine substitution mutant in Raji-DC-SIGNR cells was calculated for multiple independent RVP preparations using data from linear portions of the virus dose/infectivity curves. Hatched bars show results for mutants with substitutions at conserved histidine residues. The means of the results for four or five independent RVP preparations are shown; error bars identify the standard errors. (F) The titers of glutamine substitution mutants were determined following infection of Vero cells with serial twofold dilutions of RVPs. The infectious titer of each mutant was calculated as described above. Hatched bars show results for mutants with substitutions at conserved histidine residues. The means of titers from four independent experiments are shown; error bars represent the standard errors.
FIG. 2.
FIG. 2.
Identification of functional substitutions for H144 and H246. WNV RVPs incorporating E protein histidine substitution mutants were produced by complementation in the presence or absence of a plasmid expressing human furin, followed by incubation at 37°C (A and C) or 28°C (B and D) (1, 7, 23). The infectious titers of RVP populations were determined as described in the Fig. 1 legend. All titer experiments were performed at 37°C, even when lower temperatures were used to produce the RVPs. The titers of RVPs produced in the presence of exogenous furin expression are shown by hatched bars. The means of the results of three or four independent experiments are displayed; error bars identify the standard errors. (E and F) Entry of WNV RVPs incorporating histidine mutants is pH dependent. Raji-DC-SIGNR cells were treated with serial dilutions of the weak base NH4Cl (from 0.2 to 50 mM) for 5 to 10 min at room temperature prior to infection with RVPs. Cells were washed at 12 h postinfection and cultured for an additional 36 h in fresh medium. Infectivity was measured by flow cytometry at 48 h postinfection. The concentration of NH4Cl required to inhibit infection by 50% (IC50) was calculated by nonlinear regression. (F) The means of two or three independent measurements are shown; error bars represent the standard errors. Hatched bars show results for mutants with substitutions at conserved histidine residues.
FIG. 3.
FIG. 3.
Production of infectious RVPs incorporating multiple histidine substitutions at the DI-DII hinge. RVPs incorporating substitutions at one or more histidine positions clustered around the DI-DII hinge were produced by genetic complementation. The infectious titers of multiple independent preparations of RVPs were calculated as described in the Fig. 1 legend. The means of two or three independent measurements are shown; error bars represent the standard errors.
FIG. 4.
FIG. 4.
Mutation of conserved histidine residues in the tick-borne flavivirus LGTV. (A) A panel of mutants at positions H146 and H323 in LGTV was produced by QuikChange mutagenesis employing redundant primers. Mutants were assayed for their capacity to direct virus entry of RVPs produced by complementation. Multiple substitutions for positions H146 and H323 were capable of mediating entry of RVPs (data not shown). The titers of RVPs composed of H146R and H323L were calculated as described in the Fig. 1 legend. The mean titers from three independent RVP preparations are shown; error bars represent the standard errors. (B) The pH dependence of LGTV RVP entry was confirmed as described in the Fig. 2 legend. The means of two or three independent measurements are shown; error bars represent the standard errors.
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