A New Human Cytomegalovirus Isolate Has an Invertible Subsegment within Its L Component Producing Eight Genome Isomers

Takekoshi, Masataka; Ihara, Shinji; Tanaka, Shigeaki; Maeda-Takekoshi, Fumiko; Watanabe, Y.

doi:10.1099/0022-1317-68-3-765

Cited by 15 publications

(6 citation statements)

References 29 publications

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“…Intriguingly, one of the breakpoints in both Towne and Toledo DNA occurs precisely at the nucleotide identified as the U L /bЈ boundary of the AD169 strain. For HCMV, examples of differences near the unique b-sequence boundaries have been described (20,42,48). Specifically, the Tanaka strain has a sequence difference of ϳ7.8 kbp relative to Towne near the b/U L boundary (42).…”

Section: Discussionmentioning

confidence: 99%

“…Additional nucleotide sequence information is available for only a few loci or genes of other strains (for example, see references 8, 9, 13, 22, 25, 26, 40, 41, and 43). Although these lines of evidence suggest a high degree of similarity among HCMV strains, there have been reports of substantial variability near the U L and b-repeat boundaries (20,42,48). We performed nucleotide sequence analysis of the U L /bЈ region, comparing the genomes of the low-passage strain Toledo and the two high-passage strains Towne and AD169.…”

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confidence: 99%

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Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains

Cha¹,

Tom²,

Kemble³

et al. 1996

J Virol

578

193

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Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (U L)/b (IR L) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the U L /b region during laboratory passage.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains

Cha¹,

Tom²,

Kemble³

et al. 1996

J Virol

578

193

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“…The observed Mr of 48K is accounted for by N-glycan modification and predicted O-glycan modification (Chang et al, 1989a). Deletion mutants lacking UL4 have demonstrated the non-essential nature of this gene in cell culture (Ripalti & Mocarski, 1991;Takekoshi et al, 1987Takekoshi et al, , 1991. The function of this structural glycoprotein is currently unknown.…”

Section: (Io Gpul75 (Gh)mentioning

confidence: 99%

Human cytomegalovirus structural proteins

Spaete¹,

Gehrz²,

Landini³

1994

Journal of General Virology

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“…Although engineered mutations have provided important information on gene function in other viruses such as herpes simplex virus type 1 (HSV-1; Roizman & Sears, 1993), human CMV has not yet been subjected to such systematic analysis. Comparisons of different strains (Takekoshi et al, 1987), adventitious deletions (Kollert-Jons et al, 1991), and deletion mutagenesis using indicator gene insertions (Spaete & Mocarski, 1987;Ripalti & Mocarski, 1991;Takekoshi et al, 1991;Jones et al, 1991;Jones & Muzithras, 1992;Browne et al, 1992;Kaye et aL, 1992 that 29 open reading frames (ORFs; UL1-UL10, UL16, UL18, UL20, UL33, UL144, UL128, US1-US13, US27 and US28), and one copy of repeated ORFs (TRL/ IRL4-14, IRS1), are dispensable for growth in human fibroblast cells. Most mutagenesis of human CMV has employed indicator genes encoding #-galactosidase (Spaete & Mocarski, 1987;Takekoshi et al, 1991;Browne et al, 1992;Kaye et al, 1992) and flglucuronidase (Jones et al, 1991;Jones & Muzithras, 1992) in conjunction with laborious co-transfection methods.…”

Section: Introductionmentioning

confidence: 99%

Selectable insertion and deletion mutagenesis of the human cytomegalovirus genome using the Escherichia coli guanosine phosphoribosyl transferase (gpt) gene

Greaves

Brown

Vieira

et al. 1995

Journal of General Virology

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We describe the mutagenesis of the IRS1-US5 region of the human cytomegalovirus genome, demonstrating the potential of the E. coli guanosine phosphoribosyl transferase (gpt) gene as a selectable marker for insertion and deletion mutagenesis of high passage (AD169, Towne) as well as low passage (Toledo) strains of virus.Despite evidence suggesting that the US3 gene product may play a regulatory role, disruption of this gene with a gpt insert had no effect on growth of any of these strains of virus in resting or dividing human fibroblasts, or in human thymus plus liver implants in SCID-hu mice. Transcripts of the gpt gene, under control of the herpes simplex virus thymidine kinase promoter adjacent to the US3 enhancer in the viral genome, accumulated with delayed early (fl) kinetics. Mutants with'deletions in the IRS1 and US3-US5 regions were isolated by backselection against gpt with the drug 6-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthineguanine phosphoribosyl transferase deficient) skin fibroblasts immortalized with human papillomavirus oncogenes. Thus, we demonstrate a dependable method for insertion and deletion mutagenesis that can be applied to any region of the viral genome.

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A New Human Cytomegalovirus Isolate Has an Invertible Subsegment within Its L Component Producing Eight Genome Isomers

Cited by 15 publications

References 29 publications

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains

Human cytomegalovirus structural proteins

Selectable insertion and deletion mutagenesis of the human cytomegalovirus genome using the Escherichia coli guanosine phosphoribosyl transferase (gpt) gene

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