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Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus
Abstract:
Human cytomegalovirus particles contain a phosphoprotein of 150,000 (pp150) apparent molecular weight in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera. The gene for pp150 was mapped by screening a bacteriophage lambda gt11 cDNA expression library with monospecific rabbit antisera. Subsequent hybridization of cDNA with cosmid and plasmid clones containing the human cytomegalovirus strain AD169 genome mapped the gene to HindIII fragments J and N. The gene is transcribed into a late 6.2-kilobase RNA. The nucleotide sequence of this region was determined, and a transcription initiation site and two polyadenylation sites of an abundant transcript were located by primer extension and nuclease protection experiments. The reading frame for pp150, deduced from computer analyses, gives rise to a polypeptide of 1,048 amino acids in length; protein secondary structure analysis revealed multiple beta-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide.
Insights
Researchers identified the gene for human cytomegalovirus phosphoprotein 150 (pp150), a matrix protein. This discovery aids in understanding viral structure and potential immunogenic properties.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Human cytomegalovirus (HCMV) contains a matrix phosphoprotein, pp150, with apparent molecular weight of 150,000.
- This pp150 protein shows high reactivity in Western blot analyses using human antisera.
Purpose of the Study:
- To map the gene encoding the HCMV pp150 protein.
- To determine the transcriptional and translational characteristics of the pp150 gene.
- To investigate the structural basis for pp150's immunogenicity.
Main Methods:
- Screening a lambda gt11 cDNA expression library with rabbit antisera.
- Hybridization of cDNA with HCMV genomic clones (cosmids, plasmids).
- Nucleotide sequencing, primer extension, and nuclease protection assays.
Main Results:
- The pp150 gene was mapped to HindIII fragments J and N of the HCMV AD169 genome.
- The gene is transcribed into a late 6.2-kilobase RNA with identified initiation and polyadenylation sites.
- Computer analysis predicted a 1,048-amino acid polypeptide with potential beta-pleated sheet structures.
Conclusions:
- The study successfully localized and characterized the gene for HCMV pp150.
- Understanding the pp150 gene and protein structure provides insights into HCMV replication and immune response.
- The identified structural features may explain the immunogenic nature of pp150.