Comparative sequence analysis of the largest E1A proteins of human and simian adenoviruses

Abstract

The early region 1A (E1A) gene is the first gene expressed after infection with adenovirus and has been most extensively characterized in human adenovirus type 5 (hAd5). The E1A proteins interact with numerous cellular regulatory proteins, influencing a variety of transcriptional and cell cycle events. For this reason, these multifunctional proteins have been useful as tools for dissecting pathways regulating cell growth and gene expression. Despite the large number of studies using hAd5 E1A, relatively little is known about the function of the E1A proteins of other adenoviruses. In 1985, a comparison of E1A sequences from three human and one simian adenovirus identified three regions with higher overall levels of sequence conservation designated conserved regions (CR) 1, 2, and 3. As expected, these regions are critical for a variety of E1A functions. Since that time, the sequences of several other human and simian adenovirus E1A proteins have been determined. Using these, and two additional sequences that we determined, we report here a detailed comparison of the sequences of 15 E1A proteins representing each of the six hAd subgroups and several simian adenoviruses. These analyses refine the positioning of CR1, 2, and 3; define a fourth CR located near the carboxyl terminus of E1A; and suggest several new functions for E1A.

FIG. 1.

Phylogenetic tree for the adenovirus E1A proteins. An unrooted tree was generated for the E1A proteins (Table 1) with the program TreeView. Each species of E1A is labeled at the tip of its representative branch. The adenoviral subgroups are labeled A to F according to convention.

FIG. 2.

Sequence alignment of the adenovirus E1A proteins. The sequences of the indicated adenovirus E1A proteins were aligned and shaded for conservation. Darker shading corresponds to higher levels of conservation. Gaps are indicated as dots. The positions of the CR are indicated as solid bars. The binding sites for Rb and CtBP in CR2 and CR4, respectively, are indicated with asterisks.

FIG. 3.

Variations in the level of sequence identity along E1A. Protein sequences were aligned (Fig. 2), and the plurality of the consensus sequence was calculated as described in Materials and Methods and plotted as a function of the amino acid position. Note that gaps introduced during the alignment are reflected in the numbering of the consensus sequence, which therefore does not correspond to any of the individual sequences. The axis on the left indicates the number of sequences out of 15 that were identical. A nonintegral value indicates that two or more different amino acids were equally prevalent at a given position.

FIG. 4.

Graph of average sequence identities along E1A. Four CR within the E1A sequences were identified as described in Materials and Methods. The percent identity of each adenoviral E1A sequence to that of hAd5 was calculated for each of the indicated regions and averaged. Note that hAd2 E1A was excluded from these calculations as it is virtually identical to that of hAd5.

FIG. 5.

Sequence alignment of the region unique to hAd2 and hAd5 E1As with precursor IL-16. The sequences of hAd2 and -5 E1As (residues 202 to 234) and precursor IL-16 proteins of bovine (b), mouse (m), and human (h) were aligned and shaded for conservation. Darker shading corresponds to higher levels of sequence conservation. The percentages of identical and similar amino acid residues are shown to the right. Cleavage of precursor IL-16 by caspase-3 occurs following the aspartic acid and is indicated with an arrow.

FIG. 6.

Secondary-structure predictions for the E1A proteins. Prediction of secondary structure for each of the indicated E1A proteins was performed using the PSIPRED program as described in Materials and Methods. Predicted α-helices and β-strands four or more residues in length are shown as blocks or arrows, respectively. The scale at the top indicates the amino acid positions within each E1A protein.