In this study, we have analyzed the determinants of the NP that determine its aberrant migration. We show that the region responsible for this effect also mediates incorporation of the nucleocapsid into the viral particle, and this property can be conferred to a homologous NP from the measles virus that is not normally incorporated into filoviruses. Specifically, two domains within this COOH terminal domain, one between aa 439 to 492 and another at aa 589 to 739, are responsible for its unusual biochemical properties. We suggest that its unexpected migration on SDS-PAGE gels is likely due to the high acidic content of the two domains. Informatic analysis of the protein suggests such behavior. In addition, extensive analysis of the COOH-terminal domain shows no evidence of posttranslational modification, including evaluation of all peptides by MALDI-TOF MS.
Previous studies have shown that NP migrates aberrantly by SDS-PAGE (3
), but the biochemical basis of this effect is not well understood. A previous report of phosphorylation of Marburg virus NP may suggest Ebola virus NP is also phosphorylated and account for this size change (3
). However, this protein is not substantially phosphorylated, and such phosphorylation is unlikely to cause substantial changes in the migration of this protein. The possibility of posttranslational carbohydrate modification has been raised in two independent studies (13
). The finding that [3
H]glucosamine could be incorporated into the protein further supported this possibility. However, lectin binding has been observed in the absence of carbohydrate modification, and in vitro labeling may be nonspecific.
Glycomic and proteomic analysis failed to identify distinct posttranslational modification sites. Instead, we find that there are two discontinuous subdomains that give rise to this aberrant migration, which are notable for their highly charged acidic character. Point mutations of a variety of amino acids in these regions suggest that neither serines nor threonines nor prolines within these affected regions were responsible for aberrant migration. In addition to posttranslational modifications, acidic domains have also been shown to cause aberrant migration of proteins. For example, in an early observation of this phenomenon, a 52-kDa component of the U1 small nuclear ribonucleoprotein was found to migrate on PAGE at 70 kDa (22
). Its aberrant electrophoretic migration was attributed to a carboxy-terminal charged domain. Similarly, the anomalous electrophoretic behaviors of the human papillomavirus type 16 E7 protein and the DNA repair protein XPA were found to be due to the high content of acidic amino acid residues (1
). Within the two subdomains of NP that cause aberrant migration, such acidic regions comprise ca. 30% of the sequence, well beyond the remainder of this protein, ca. 10%, or of other proteins in general. In addition, this region of the protein is predicted to show significant disorder (Fig. ). Many proteins are now known to require regions of disorder for function, particularly when molecular recognition is used (27
). Indeed, recent bioinformatics studies have revealed a small but significant correlation between intrinsic structural disorder and participation in multiple protein interactions (12
). Such disordered regions generally have a higher percentage of polar and charged residues, as well as Gly, Ala, and Pro. Despite their lack of rigidly defined structure, disordered regions of proteins involved in macromolecular interactions can have numerous advantages over structured regions such as more rapid association rates, lower entropy losses upon binding, greater topological flexibility, the potential to use shorter amino acid sequences for molecular recognition, the flexibility to recognize multiple partners depending on the microenvironment, and the ability to provide entropic exclusion of other macromolecules in spacer regions. Furthermore, many, although not all, disordered regions are induced to fold into distinct structures upon interaction with target molecules (6
). The prediction of a large intrinsically disordered region in the COOH-terminal end of an NP with high acidic content is thus consistent with NP's role as a critical component of a large macromolecular assembly involving VP35 and VP24. These results suggest that the COOH-terminal region of NP may play a direct role in the assembly of the nucleocapsid.
The filoviruses are characterized by their filamentous nucleocapsids, a feature not observed with uniformity in their most closely related phylogenetic class of viruses, the paramyxoviruses. Interestingly, the region responsible for this aberrant migration is lacking in the paramyxoviruses. When the COOH-terminal region is fused to either measles or RSV NP, it associated in nucleocapsids with VP35 and was incorporated into viruslike particles by biochemical analysis. This finding suggests that these Ebola virus subdomains are required for incorporation of NP into the virion. Taken together, these data indicate that the subdomains of the Ebola virus NP responsible for its aberrant biochemistry contribute to NP localization in the virus and may help to determine the unique characteristics and morphology of this class of viruses.