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Detection of the human cytomegalovirus 2.0-kb immediate early gene 1 transcripts in permissive and nonpermissive infections by RNA in situ hybridization

  • Original Paper
  • Published:
Journal of Biomedical Science

Abstract

The immediate early gene 1 (IE1) is the first gene to be expressed following the entry of the human cytomegalovirus (HCMV) into the cell and it does not require prior protein synthesis for its expression. Therefore, the IE1 gene is a potential candidate for the development of probes to detect HCMV in various states of infection. Using strand-specific32P- or digoxigenin-labeled riboprobes derived from an exon-specific subgenomic fragment of the HCMV Towne IE1 gene, we performed Northern blot analysis and RNA in situ hybridization on HCMV-infected human (permissive cells) and mouse (nonpermissive cells) fibroblasts and on 10 formalin-fixed paraffin-embedded sections of human tissue. By Northern blot analysis and by in situ hybridization, expression of the 2.0-kb IE1 gene was found in permissive as well as in nonpermissive infections. Specific nuclear and cytoplasmic hybridization was found at 5, 10, 24, and 72 h after infection in human fibroblasts. In comparison, hybridization was first detected at 10 h after infection in mouse fibroblasts. Hybridization with the IE1 probe was detected in cells with and without cytopathic changes in the formalin-fixed paraffin-embedded HCMV-infected human tissues. Hybridization patterns of the IE1 riboprobe were compared to those of the HCMV 2.7-kb major early β-riboprobe which we have previously described [Am J Pathol 141:1247–1254;1992]. Although both riboprobes hybridize to their respective target sequences in the consecutive tissue sections, the patterns of hybridization are different. On occasion, sections of HCMV-infected human tissue showing no specific hybridization for the 2.7-kb riboprobe will show specific in situ hybridization when using the IE1 riboprobe. Our results suggest that RNA in situ hybridization with a probe directed at the IE1 transcripts is an effective method of detecting early and late stages of both permissive and nonpermissive HCMV infections.

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Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, The Johns Hopkins Medical Institutions, Baltimore, Md., USA

    Yu-Jiun Chan & Gary S. Hayward

  2. Department of Oncology, The Johns Hopkins Medical Institutions, Baltimore, Md., USA

    T. C. Wu, Wei-Chun Au, Chuang-Jiun Chiou, Ralph H. Hruban & Gary S. Hayward

  3. Department of Obstetrics and Gynecology, The Johns Hopkins Medical Institutions, Baltimore, Md., USA

    Robert J. Kurman

  4. Department of Pathology, The Johns Hopkins Hospital, 600 North Wolfe Street, 21287, Baltimore, MD, USA

    T. C. Wu, Daniel A. Fuentes-Bernardo, William M. Fox III, Ralph H. Hruban & Robert J. Kurman

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  1. T. C. Wu
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  2. Daniel A. Fuentes-Bernardo
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Wu, T.C., Fuentes-Bernardo, D.A., Chan, YJ. et al. Detection of the human cytomegalovirus 2.0-kb immediate early gene 1 transcripts in permissive and nonpermissive infections by RNA in situ hybridization. J Biomed Sci 4, 19–27 (1997). https://doi.org/10.1007/BF02255589

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  • DOI: https://doi.org/10.1007/BF02255589

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