ABSTRACT
Interplay between the host and human cytomegalovirus (HCMV) has a pivotal role in the outcome of infection. A region (referred to as UL/b’) present in the Toledo strain of HCMV and low passage clinical isolates contains 19 additional genes, which are absent in the highly passaged laboratory strain AD169. Products of the UL/b’ genes may determine the manifestations of HCMV infection in vivo. However, little is known about the host factors, which interact with UL/b’ proteins. This study was conducted to investigate the function of the HCMV UL136 protein. By yeast two-hybrid screening, the β1 subunit of the host Na+/K+-ATPase (ATP1B1) was identified to be a candidate protein, which interacts with the HCMV UL136 protein. The interaction was further evaluated both in vitro by pull-down assay and in vivo by immunofluorescent co-localization. The results showed that the UL136 protein can interact with ATP1B1 in vitro. Co-localization of UL136-EGFP and ATP1B1-DsRed in cell membranes suggests that ATP1B1 was a partner of the UL136 protein. It can be proposed that the HCMV UL136 protein may have important roles in processes such as cell-to-cell spread, and in maintaining cell osmotic pressure and intracellular ion homeostasis during HCMV infection.
Subject(s)
Humans , Cytomegalovirus/chemistry , Protein Interaction Mapping , Sodium-Potassium-Exchanging ATPase/metabolism , Two-Hybrid System Techniques , Viral Envelope Proteins/metabolism , Sequence Analysis, ProteinABSTRACT
Interplay between the host and human cytomegalovirus (HCMV) has a pivotal role in the outcome of infection. A region (referred to as UL/b') present in the Toledo strain of HCMV and low passage clinical isolates contains 19 additional genes, which are absent in the highly passaged laboratory strain AD169. Products of the UL/b' genes may determine the manifestations of HCMV infection in vivo. However, little is known about the host factors, which interact with UL/b' proteins. This study was conducted to investigate the function of the HCMV UL136 protein. By yeast two-hybrid screening, the ß1 subunit of the host Na+/K+-ATPase (ATP1B1) was identified to be a candidate protein, which interacts with the HCMV UL136 protein. The interaction was further evaluated both in vitro by pull-down assay and in vivo by immunofluorescent co-localization. The results showed that the UL136 protein can interact with ATP1B1 in vitro. Co-localization of UL136-EGFP and ATP1B1-DsRed in cell membranes suggests that ATP1B1 was a partner of the UL136 protein. It can be proposed that the HCMV UL136 protein may have important roles in processes such as cell-to-cell spread, and in maintaining cell osmotic pressure and intracellular ion homeostasis during HCMV infection.
Subject(s)
Cytomegalovirus/chemistry , Protein Interaction Mapping , Sodium-Potassium-Exchanging ATPase/metabolism , Two-Hybrid System Techniques , Viral Envelope Proteins/metabolism , Humans , Sequence Analysis, ProteinABSTRACT
OBJECTIVE: To study and research the transcription pattern of UL131A-128 mRNA in human cytomegalovirus (HCMV) clinical low passage strains. METHODS: The UL131A-128 mRNAs of from different clinical strains and kinetic periods were amplified using 3' RACE and analyzed by sequencing. Meanwhile, clones containing UL131A-128 transcripts in a HCMV cDNA library of clinical strain were selected and sequenced. RESULTS: It is successful to obtain the transcription pattern of UL131A, UL130 and UL128 gene in HCMV clinical low passage strains, the UL131A gene consisted of two exons and the coding region of UL130 gene was not interrupted by any intron in the region as reported before. However, the transcript of UL128 gene showed two patterns, one pattern consisted of the three exons, the length is 519bp, and the other one contained the three exons and the sequence of the first intron further, the length is 642bp. The quantities of UL128 transcript containing the sequence of the first intron were higher than that of transcript only containing the three exons in the studied clinical strains at all kinetic classes. It was demonstrated that the UL131A-128 mRNA were expressed with immediately early, early and late kinetics. The result of 3'RACE and HCMV cDNA library of clinical strain is conformity. CONCLUSIONS: Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with 3'-coterminal, although the initiation points of their mRNA may be different. The variation of the transcripts found in our study indicated complex nature of transcription of UL131A-128 genes in HCMV clinical strains.