The major open reading frame of the beta2.7 transcript of human cytomegalovirus: in vitro expression of a protein posttranscriptionally regulated by the 5' region

Abstract

beta2.7 is the major early transcript produced during human cytomegalovirus infection. This abundantly expressed RNA is polysome associated, but no protein product has ever been detected. In this study, a stable peptide of 24 kDa was produced in vitro from the major open reading frame (ORF), TRL4. Following transient transfection, the intracellular localization was nucleolar and the expression was posttranscriptionally inhibited by the 5' sequence of the transcript, which harbors two short upstream ORFs.

FIG. 1

Schematic representations of the constructed plasmids. (a) Localization of TRL4 and IRL4 within the inverted repeats (TR_L and IR_L) flanking the long unique segment (U_L) of the HCMV genome. (b) Eukaryotic expression plasmids pAD/ORF3∗ and pTo/ORF3∗, containing ORF3 from AD169 and Towne attached to the FLAG sequence (∗), and constructs pAD/ORF1-2-3∗ and pTo/ORF1-2-3∗, in which the corresponding inserts are extended by the respective 5′ regions including uORF1 and uORF2. pTo/ORF3 harbors ORF3 from Towne without the FLAG sequence. Differences between Towne and AD169 regarding the coding information of the 5′ terminal parts are illustrated by proportional depictions of the transcribed products (black lines) of the predicted ORFs (black boxes). MIEP, major intermediate early promoter; T7, T7 promoter.

FIG. 2

(a) In vitro transcription and translation assay with RRL. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography allowed detection of a band corresponding to a product of 24 kDa in the reaction carried out with the construct pTo/ORF3 (lane 2), while no product was detected in the control reaction with the plasmid pcDNA3 (lane 1). With the plasmid pTo/ORF3∗, a product of 25 kDa (lane 3) was obtained. Lanes 4 and 5 correspond to the in vitro assays performed with the plasmids pTo/ORF1-2-3∗ and pAD/ORF1-2-3∗, respectively. It is clear that production of the 25-kDa protein is greatly reduced compared to that when the reaction was carried out on the construct lacking the 5′ region. (b) Northern blot analysis of RNA from in vitro transcription and translation assays. Total RNAs extracted from a reaction mixture containing the two sets of plasmids pTo/ORF3∗ and pTo/ORF1-2-3∗ (lane 1) and pAD/ORF3∗ and pAD/ORF1-2-3∗ (lane 2) were analyzed by using a ³²P-labeled fragment as a probe corresponding to ORF3. Equivalent amounts of transcripts were detected for ORF1-2-3∗ (0.8 kb) and ORF3∗ (0.5 kb).

FIG. 3

Intracellular localization of pTRL4∗ in U373-MG cells. Following transfection with pAD/ORF3∗, cells were fixed and probed with the anti-FLAG MAb M2. The fluorescence signal is always localized to the nucleoli (a and b), as verified by comparison with the phase-contrast images (c and d). In approximately 50% of the positive cells, some positivity was also detectable throughout the cytoplasm (b).

FIG. 4

Northern blot analysis of total RNA extracted from COS7 cells transfected with the control vector (lane 1) and with plasmids pTo/ORF3∗ (lane 2), pTo/ORF1-2-3∗ (lane 3), and pAD/ORF1-2-3∗ (lane 4). Detection of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) transcript confirmed the presence of equal quantities of cellular RNA. Autoradiography showed that transcripts produced from all of the three plasmids were present in similar amounts in transfected cells (lanes 1, 2, and 3).