Reevaluation of human cytomegalovirus coding potential

Abstract

The Bio-Dictionary-based Gene Finder was used to reassess the coding potential of the AD169 laboratory strain of human cytomegalovirus and sequences in the Toledo strain that are missing in the laboratory strain of the virus. The gene-finder algorithm assesses the potential of an ORF to encode a protein based on matches to a database of amino acid patterns derived from a large collection of proteins. The algorithm was used to score all human cytomegalovirus ORFs with the potential to encode polypeptides >/=50 aa in length. As a further test for functionality, the genomes of the chimpanzee, rhesus, and murine cytomegaloviruses were searched for orthologues of the predicted human cytomegalovirus ORFs. The analysis indicates that 37 previously annotated ORFs ought to be discarded, and at least nine previously unrecognized ORFs with relatively strong coding potential should be added. Thus, the human cytomegalovirus genome appears to contain approximately 192 unique ORFs with the potential to encode a protein. Support for several of the predictions of our in silico analysis was obtained by sequencing several domains within a clinical isolate of human cytomegalovirus.

Fig. 1.

BDGF analysis of HCMV ORFs. All previously annotated ORFs of HCMV are listed from high (upper left) to low (lower right) normalized BDGF (N-BDGF) scores. Expression of the ORF was scored as positive if a report exists in the published literature directly demonstrating the expression of a protein or showing that a mutation within the ORF generates a viral growth phenotype. NR indicates that no report was found. The presence of orthologues corresponding to HCMV ORFs within the genomes of chimpanzee cytomegalovirus (CCMV), rhesus cytomegalovirus (RhCMV), and murine cytomegalovirus (MCMV) are indicated. Green boxes designate characteristics favoring the conclusion that an ORF encodes a protein, and red boxes mark characteristics arguing that an ORF does not encode a protein. The length of the polypeptide potentially encoded by each ORF and the presence of a Kozak translational initiation motif (Kozak AUG) are provided for informational purposes. The UL65 ORF was reported to be expressed (26), but the reported polypeptide does not match the UL65 sequence (3).

Fig. 2.

Map of the HCMV genome. The green arrows represent previously annotated ORFs of HCMV likely to encode a bona fide polypeptide, red arrows designate ORFs unlikely to encode a polypeptide, and blue arrows indicate previously unrecognized ORFs that the present analysis predicts have high potential to encode proteins. The gray box marks the additional sequence found in the HCMV Toledo strain, locating it with respect to the AD169 genome. Rectangles superimposed on the line represent the sequence-identify terminal repeats. Each mark on the sequence line represents 1,000 bp.