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Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region.

ABSTRACT: The immediate-early region exon 4 sequences of six clinical cytomegalovirus strains were determined and compared with those of laboratory strains AD169 and Towne. Of 407 codons in exon 4, 33 (8.1%) showed interstrain variation at the peptide level and 74 (18%) showed interstrain variation at the nucleotide level. Variation occurred sporadically throughout the exon, and no grouping of strains was apparent. Published oligonucleotide primers proposed for diagnostic detection of cytomegalovirus by polymerase chain reaction have often been based on exon 4 sequences. Some of these primers show sequence mismatches with strains sequenced here. Amplification sensitivity for mismatched strains was reduced up to 100-fold. More-uniform detection sensitivity was achieved with primers of conserved sequence.

SUBMITTER: Chou S

PROVIDER: S-EPMC265497 | biostudies-other | 1992 Sep

REPOSITORIES: biostudies-other

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Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region.

Chou S S

Journal of clinical microbiology 19920901 9

The immediate-early region exon 4 sequences of six clinical cytomegalovirus strains were determined and compared with those of laboratory strains AD169 and Towne. Of 407 codons in exon 4, 33 (8.1%) showed interstrain variation at the peptide level and 74 (18%) showed interstrain variation at the nucleotide level. Variation occurred sporadically throughout the exon, and no grouping of strains was apparent. Published oligonucleotide primers proposed for diagnostic detection of cytomegalovirus by p ...[more]

PMID: 1328286

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Project description:The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18-25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

Project description:The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

Dataset Information

Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region.

Publications

Effect of interstrain variation on diagnostic DNA amplification of the cytomegalovirus major immediate-early gene region.

Similar Datasets

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