Single amino acid changes in the virus capsid permit coxsackievirus B3 to bind decay-accelerating factor

Abstract

Many coxsackievirus B isolates bind to human decay-accelerating factor (DAF) as well as to the coxsackievirus and adenovirus receptor (CAR). The first-described DAF-binding isolate, coxsackievirus B3 (CB3)-RD, was obtained during passage of the prototype strain CB3-Nancy on RD cells, which express DAF but very little CAR. CB3-RD binds to human DAF, whereas CB3-Nancy does not. To determine the molecular basis for the specific interaction of CB3-RD with DAF, we produced cDNA clones encoding both CB3-RD and CB3-Nancy and mutated each of the sites at which the RD and Nancy sequences diverged. We found that a single amino acid change, the replacement of a glutamate within VP3 (VP3-234E) with a glutamine residue (Q), conferred upon CB3-Nancy the capacity to bind DAF and to infect RD cells. Readaptation of molecularly cloned CB3-Nancy to RD cells selected for a new virus with the same VP3-234Q residue. In experiments with CB3-H3, another virus isolate that does not bind measurably to DAF, adaptation to RD cells resulted in a DAF-binding isolate with a single amino acid change within VP2 (VP2-138 N to D). Both VP3-234Q and VP2-138D were required for binding of CB3-RD to DAF. In the structure of the CB3-RD-DAF complex determined by cryo-electron microscopy, both VP3-234Q and VP2-138D are located at the contact site between the virus and DAF.

Fig. 1.

Amino acid sequence differences between CB3-Nancy and CB3-RD. Regions where the sequences of CB3-Nancy and CB3-RD diverge are shown. Conserved amino acids are marked with hyphens. Sequence differences are in bold.

Fig. 2.

Mapping of amino acid residues required for CB3-RD interaction with DAF. (A) Mutant constructs were made by overlap extension PCR from full-length cDNA clones encoding CB3-RD (in gray) or CB3-Nancy (in white). Mutations are indicated with stars: black stars represent amino acid residues from CB3-RD, and open stars represent amino acid residues from CB3-Nancy. An arrow indicates VP3-234. (B) CHO cells stably transfected with DAF (CHO-hDAF) and CAR (CHO-hCAR) were incubated with radiolabeled viruses at room temperature for 1 h to measure binding to specific receptors. Data are presented as percentages of input virus bound to cells ± the standard deviation (SD) for triplicate samples. Mutants of CB3-RD in which VP3-234Q was replaced by 234E (constructs 2, 3, 7, 9, and 10) lost the capacity to bind DAF. A mutant of CB3-Nancy in which VP3-234E was replaced by 234Q (construct 11) acquired the capacity to bind DAF. N, Nancy.

Fig. 3.

Mapping of amino acid residues required for interaction with RD cells. (A) Percentages of bound radiolabeled virus, determined as for preceding figures. (B) Virus infection. RD cell monolayers were incubated with viruses (10 PFU/cell) at 37°C for 72 h and then stained with anti-enterovirus VP1 MAb to measure virus infection. All DAF-binding virus strains infected RD cells, whereas non-DAF-binding strains did not.

Fig. 4.

Adaptation of cloned CB3-Nancy to growth in RD cells selects for DAF binding. (A) Infection of RD cells by the readapted virus, Nancy-RD. (B) The readapted virus binds to DAF.

Fig. 5.

VP2-138D is also important for virus interaction with DAF. (A) Adaptation of CB3-H3 to growth in RD cells. RD cells were exposed to CH3-H3 or the adapted variant CB3-H3-RD and then stained at 72 h with anti-VP1 antibody to detect infection. (B) RD-adapted CB3-H3 binds to DAF. CHO cells expressing DAF or CAR, control CHO-pcDNA cells, and HeLa cells were incubated with radiolabeled viruses, and virus binding was measured as described in Materials and Methods. (C) In H3-VP2-138D, VP2-138D was introduced into cloned H3; this virus gained the capacity to bind DAF. In RD-VP2-138N, VP2-138D in the RD clone was replaced with VP2-138N; this virus lost the capacity to bind DAF. (D) Replacement of VP2-151S with T does not affect binding of CB3-RD to DAF.

Fig. 6.

Critical capsid residues are in contact with DAF. (A) Cryo-EM reconstruction of DAF bound to CVB3-RD at 9-Å resolution displayed at 1 sigma. Density further than 160 Å from the center of the virus is shown in blue. (B) Surface representation of CVB3-RD protomers surrounding one 5-fold icosahedral axis of the virus with DAF shown in yellow ribbon. The DAF molecule attaches at the 3-fold vertex by way of the C-terminal His tag; SCRs 4 and 3 stretch across the virus surface of the red protomer (in standard orientation), crossing the canyon, and SCR2 interacts with the puff region of the blue (neighboring) protomer. SCR1 makes no contacts, rising above the virus surface. Residues VP2-138D and VP3-234Q are highlighted in green. (C) Close-up view of residue VP2-138D (in green, stick rendering) with DAF colored according to electrostatic potential. The light blue coloring of DAF indicates an overall slightly positive charge in the region nearest to VP2-138D. (D) Close-up view of residue VP3-234Q (in green, stick rendering with oxygen and nitrogen colored red and blue, respectively) with DAF surface rendered and colored according to electrostatic potential; the side chain configuration shown is derived from the crystal structure of CB3-RD and may differ in virus-DAF complex. The light-red coloring of DAF indicates a negative charge in the region nearest to VP2-234Q.