Virus Isoelectric Point Estimation: Theories and Methods

Abstract

Much of virus fate, both in the environment and in physical/chemical treatment, is dependent on electrostatic interactions. Developing an accurate means of predicting virion isoelectric point (pI) would help to understand and anticipate virus fate and transport, especially for viruses that are not readily propagated in the lab. One simple approach to predicting pI estimates the pH at which the sum of charges from ionizable amino acids in capsid proteins approaches zero. However, predicted pIs based on capsid charges frequently deviate by several pH units from empirically measured pIs. Recently, the discrepancy between empirical and predicted pI was attributed to the electrostatic neutralization of predictable polynucleotide-binding regions (PBRs) of the capsid interior. In this paper, we review models presupposing (i) the influence of the viral polynucleotide on surface charge or (ii) the contribution of only exterior residues to surface charge. We then compare these models to the approach of excluding only PBRs and hypothesize a conceptual electrostatic model that aligns with this approach. The PBR exclusion method outperformed methods based on three-dimensional (3D) structure and accounted for major discrepancies in predicted pIs without adversely affecting pI prediction for a diverse range of viruses. In addition, the PBR exclusion method was determined to be the best available method for predicting virus pI, since (i) PBRs are predicted independently of the impact on pI, (ii) PBR prediction relies on proteome sequences rather than detailed structural models, and (iii) PBR exclusion was successfully demonstrated on a diverse set of viruses. These models apply to nonenveloped viruses only. A similar model for enveloped viruses is complicated by a lack of data on enveloped virus pI, as well as uncertainties regarding the influence of the phospholipid envelope on charge and ion gradients.

Keywords: DNA binding; DNA-binding proteins; RNA binding; RNA-binding proteins; capsid; colloid; electrostatic; modeling; polynucleotide; prediction; predictive model; surface charge; virion; virion structure.

FIG 1

Impact of including only exterior residues in predicted pI calculation using 3D capsid structures. (A to C) The mean empirical pI value for each unique virus is shown in comparison to the virus’ predicted pI calculated from exterior capsid residues. Exterior residues represent increasingly narrow shells determined by fraction of outermost amino acids (A) and distance from the exterior surface (B), as illustrated in panel C. Distances in panel B are displayed on a log color scale to clearly show the impact on all viruses despite large disparities in capsid size and thickness. In both panels A and B, a lighter tint indicates a narrower “slice” of the capsid, while a darker tint indicates that a larger portion of the capsid was considered in the pI calculation. The diagonal line represents equivalent theoretical and empirical pIs; to accept either method of calculating pI based on exterior residues, points of similar tint should be clustered along this line. Two groups that appeared to benefit most from including only exterior residues are labeled in the figure: ssRNA Leviviridae phages with basic, interior beta sheets (bacteriophages fr [EBFR], GA [EBGA], MS2 [EBMS2], and Qβ [EBQB]) and ssRNA viruses with basic, interior N termini (cowpea chlorotic mosaic virus [CCMV], cucumber mosaic virus [CMV], red clover necrotic mosaic virus [RCNM], and southern bean mosaic virus [SBMV]).

FIG 2

Theoretical average charge of virus proteomes before (A) and after (B) modification by removing predicted polynucleotide-binding regions, as reported by Heffron and Mayer (25). Empirical pI values from the literature are shown as purple circles. Good fit between theoretical and empirical pIs is indicated when the purple circles fall within the white space (net-neutral virion surface charge) of the colored bars. Theoretical charge was calculated based on the sum of ionizable amino acids in capsid proteome sequences. Highly represented virus families (>2 representatives) are noted by letters to the right of each graph: L, Leviviridae; P, Picornaviridae; and T, Tymoviridae. A key to the virus abbreviations (y axis) is provided in Table 3.

FIG 3

Hypothesized electrostatic model of the virion including polynucleotide-binding regions. Capsid proteins as a whole contain a balance of acidic and basic residues. At a given pH, these residues range across a broad spectrum of charge from strongly negative (dark red), to neutral (white), to strongly positive (dark blue). However, some viruses have a high concentration of basic residues on the capsid interior which are electrostatically bound to the polynucleotide. The charges of both the polynucleotide-binding regions of the capsid and associated polynucleotide segments are mutually negated. The charge of the polynucleotide core is screened by a hypothesized cloud of counterions retained in the virion core. The overall charge arises from the nonbinding portions of the capsid, which have an acidic pI due to a disproportionately low concentration of basic residues.

FIG 4

Distribution of empirical pI values for enveloped viruses referenced by Michen and Graule (17). Box plots summarizing the pIs for each virus genus are overlaid with individual pI values. Individual pI values are distinguished by method of determination (color) and literature source (shape). Two teams, Douglas et al. (88, 89) and Mouillot and Netter (90), were responsible for all Orthopoxvirus empirical pIs in this plot; all other sources (37, 98–101) are labeled in the figure. Points are horizontally scattered within each group for clarity only.