The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome

466

451

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Coding potential of laboratory and clinical strains of human cytomegalovirus

Murphy

Yü

Grimwood

et al. 2003

466

451

“…Fig. 1 displays the results of the BDGF analysis of the original annotated AD169 ORFs (3), the additional Toledo ORFs (13), and several AD169 ORFs identified by their correspondence to chimpanzee cytomegalovirus orthologues (14). The ORFs in are listed from the highest to the lowest normalized BDGF score.…”

Section: Bdgf Analysis Of Hcmv Orfs Identified In Previous Annotationsmentioning

confidence: 99%

“…Recently, the AD169 genome was compared with that of chimpanzee cytomegalovirus, which has not been extensively passaged in the laboratory (14). It was assumed that proteincoding ORFs would be conserved between the two viruses, but noncoding ORFs would diverge.…”

Section: H Uman Cytomegalovirus (Hcmv) Is a Ubiquitous ␤-Herpes-mentioning

confidence: 99%

Reevaluation of human cytomegalovirus coding potential

Murphy

Rigoutsos

Shibuya

et al. 2003

169

128

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 Ϸ192 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.H uman cytomegalovirus (HCMV) is a ubiquitous ␤-herpesvirus. Infection is usually asymptomatic in healthy individuals, but the virus is not cleared and persists in a latent state. Primary infection or reactivation of latent virus in immunocompromised individuals can lead to severe disease, including pneumonitis (1). The double-stranded DNA genome of HCMV is composed of so-called unique long (UL) and unique short (US) domains, which are flanked on one end by terminal repeated sequences (TRL and TRS) and on the other end by internal repeats (IRL and IRS). In a population of HCMV DNA, four isoforms are attributable to recombination events that result in the US and UL regions being present in both orientations with respect to each other (2).The Ϸ230,000-bp sequence and annotation of the AD169 laboratory strain of HCMV was published in 1990 (3). This first annotation predicted that AD169 has the potential to encode 208 ORFs, of which 14 are duplicated within the TRL͞IRL repeats. The criteria that were used for ORF prediction required that a presumptive ORF encode a polypeptide at least 100 aa in length and that it not overlap a longer ORF by 60% or more. In regions where more than one overlapping ORF was identified, the longer of the two ORFs was generally accepted as the valid ORF. In accordance with the predicted genomic layout of Epstein-Barr virus (4) and herpes simplex virus 1 (5), the AD169 ORFs generally lay in an end-to-end configuration with little noncoding sequence. In a few regions lacking ORFs that fulfilled the above-mentioned criteria, ORFs were predicted by identifying transcriptional regulatory signals upstream of coding sequences, by considering codon usage bias, and by searching for protein motifs. The authors noted that the annotation criteria could miss ORFs that are small or that represent a single exon within a gene that is spliced. Since the original ...

“…The laboratory strain of HCMV, AD169, contains Ϸ150 ORFs likely to encode proteins (2)(3)(4)(5). Most ORFs have not been well studied due to the limited host range and slow growth of HCMV in cultured cells and the lack of efficient tools to generate mutant viruses.…”

mentioning

confidence: 99%

Functional map of human cytomegalovirus AD169 defined by global mutational analysis

Yü

Silva

Shenk

2003

383

468

Human cytomegalovirus has a complex double-stranded DNA genome of Ϸ240,000 bp that contains Ϸ150 ORFs likely to encode proteins, most of whose functions are not well understood. We have used an infectious bacterial artificial chromosome to introduce 413 defined insertion and substitution mutations into the human cytomegalovirus AD169 genome by random and sitedirected transposon mutagenesis. Mutations were produced in all unique ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. The growth of selected mutants was assayed in cultured human fibroblasts, and we now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. Most essential and augmenting genes are located in the central region, and nonessential genes generally cluster near the ends of the viral genome. Human cytomegalovirus (HCMV) is the prototypic ␤-herpes virus and a ubiquitous human pathogen. Although infections in healthy children and adults are generally asymptomatic, HCMV is a leading viral cause of birth defects and a major cause of morbidity and mortality in immunocompromised individuals (1).HCMV contains a complex double-stranded DNA genome of Ϸ240,000 bp, the largest genome for a virus known to infect humans. The laboratory strain of HCMV, AD169, contains Ϸ150 ORFs likely to encode proteins (2-5). Most ORFs have not been well studied due to the limited host range and slow growth of HCMV in cultured cells and the lack of efficient tools to generate mutant viruses. Recently, the HCMV genome has been cloned as an infectious bacterial artificial chromosome (BAC) (6-9), greatly facilitating its genetic manipulation (8, 10).We previously described an infectious BAC clone of HCMV AD169, termed pAD͞Cre (6). The BAC vector is flanked by LoxP sites and contains a Cre-recombinase gene that is modified by the insertion of an intron into its coding sequence. Consequently, Cre is not expressed in bacterial cells, but it is expressed when its transcript is spliced in human cells and the BAC vector is excised from the virus. Now we report the use of both random and site-directed transposon mutagenesis to introduce 413 defined insertion and substitution mutations into the HCMV AD169 genome residing in pAD͞Cre. Mutations were produced in all ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. We have begun to systematically delineate the functions of viral ORFs in HCMVinfected cells by analyzing the growth of HCMV mutants in cultured human fibroblasts. We now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. This work describes a functional map of the complete HCMV genome and provides a foundation for future genetic studies. MethodsCells, Viruses, and Plasmids. Primary human foreskin fibroblasts at passage 8-15 were propagated in medium supplemented with 10% FCS. The HCMV strain AD169 BAC, pAD͞Cre (6), was the wild-type parent of all mutant viruses. A...