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. 2004 Aug;78(15):8191-200.
doi: 10.1128/JVI.78.15.8191-8200.2004.

Analysis of splice variants of the immediate-early 1 region of human cytomegalovirus

Sita Awasthi  1 , Jennifer A Isler, James C Alwine
Affiliations

Analysis of splice variants of the immediate-early 1 region of human cytomegalovirus

Sita Awasthi et al. J Virol. 2004 Aug.

Abstract

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) produces multiple mRNAs through differential splicing and polyadenylation. Reverse transcriptase PCR was used to characterize transcripts from exons 1, 2, 3, and 4 (immediate-early 1 [IE1]). The expected IE72 and IE19 mRNAs were detected, as well as two heretofore-uncharacterized transcripts designated IE17.5 and IE9. The IE72, IE19, and IE17.5 transcripts utilized the same 5'-splice site in exon 3. IE9 utilized a cryptic 5'-splice site within exon 3. The IE19, IE17.5, and IE9 transcripts all used different 3'-splice sites within exon 4. These spliced species occur in infected human foreskin fibroblast (HFF) cells, with accumulation kinetics similar to those of IE72 mRNA. IE19 and IE9 RNAs were much more abundant than IE17.5 RNA. Transfection of CV-1 cells with cDNAs resulted in IE19 and IE17.5 proteins detectable by antibodies to either N-terminal or C-terminal epitopes. No IE9 protein product has been detected. We have not been able to detect IE19, IE17.5, or IE9 proteins during infection of HFF, HEL, or U373MG cells. Failure to detect IE19 protein contrasts with a previous report (M. Shirakata, M. Terauchi, M. Ablikin, K. Imadome, K. Hirai, T. Aso, and Y. Yamanashi, J. Virol. 76:3158-3167, 2002) of IE19 protein expression in HCMV-infected HEL cells. Our analysis suggests that an N-terminal breakdown product of IE72 may be mistaken for IE19. Expression of IE19 or IE17.5 from its respective cDNA results in repression of viral gene expression in infected cells. We speculate that expression of these proteins during infection may be restricted to specific conditions or cell types.

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Figures

FIG. 1.
FIG. 1.
The HCMV MIE gene encodes a number of alternatively spliced mRNA species. The schematic illustrates the MIE gene with its five major exons and two alternative polyadenylation (PA) signals. Exons 4 and 5 are traditionally considered the IE1 and IE2 regions, respectively. Previously identified transcripts from both the IE1 and IE2 regions are shown. The locations of primers used in RT-PCR analysis (primers 1, 2, and 3) are indicated with arrows.
FIG. 2.
FIG. 2.
Identification of previously uncharacterized splice variants from the HCMV IE1 region by RT-PCR. RT-PCR was performed using total RNA isolated from HCMV-infected HFFs harvested at the times indicated. Primers 1 and 2 were used (Fig. 1). The RT-PCR products were separated on a 1.2% agarose gel and stained with ethidium bromide. Band sizes (kilobases) are shown on the right. The 0.65-kb band was cloned into the eukaryotic expression vector pCR3.1, and clones were screened by digestion with EcoRI. Three independent clones were identified having inserts of 0.65, 0.60, and 0.55 kb in length (inset).
FIG. 3.
FIG. 3.
Alignment of nucleotide sequences of each alternatively spliced transcript. The start and stop codons within each transcript are marked in bold and underlined. The dashed lines represent deletions relative to the IE19 mRNA.
FIG. 4.
FIG. 4.
Amino acid alignment of protein products predicted for each alternatively spliced transcript. Vertical lines represent exon demarcation. Dashed lines represent deletions relative to IE19. The two amino acids translated in an alternative reading frame in IE9 are underlined.
FIG. 5.
FIG. 5.
IE19 and IE17.5 utilize consensus AU/AG splice sites while IE9 does not. (A) Sequences surrounding the 3′ end of exon 3 and the 5′ end of exon 4 for each transcript are shown, exons in uppercase and introns in lowercase. Sequences of the 5′- and 3′-splice sites are underlined and shaded. The polypyrimidine track (PPT) preceding each 3′-splice site is indicated. (B) Towne strain sequences containing and surrounding the 3′-splice sites (3′-SS) in exon 4 and the 5′-splice sites (5′-SS) in exon 3 were aligned and compared with the sequences of the Ad169 strain and four clinical HCMV isolates (Toledo, fix, phoebe, and tr). Intron-exon junctions are indicated by a slash. Nucleotide differences are shaded.
FIG. 6.
FIG. 6.
Detection of IE19, IE17.5, and IE9 transcripts in HCMV-infected cells. Nuclease protection assays were performed using total RNA isolated from HCMV-infected HFF cells. Antisense RNA probes were generated specifically for IE19, IE17.5, and IE9. A schematic illustrating the design of each probe and the expected protected fragments is shown to the right of each figure. The size of each protected RNA fragment (in nucleotides [nt]) is marked to the right of each gel.
FIG. 7.
FIG. 7.
Expression of IE19 and IE17.5 from cDNAs. (A) CV-1 cells were transfected with the cDNAs indicated. Transfected cells were harvested in RIPA buffer, separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and probed using an antibody specific for exon 2/3. (B) CV-1 cells were transfected with cDNAs encoding IE17.5, IE9, or a plasmid expressing only exons 2 and 3. Lysates were analyzed by Western blotting as described for panel A.
FIG. 8.
FIG. 8.
Repression of IE72 and IE86 by IE19 and IE17.5. (A) CV-1 cells were transfected or cotransfected with the cDNAs indicated. Proteins were separated on an SDS-10% polyacrylamide gel and transferred to nitrocellulose. A polyclonal antibody specific for exons 2 and/or 3 was used to detect immediate-early proteins. (B) U373MG cells were transfected with a Flag-tagged IE19 cDNA and then infected with HCMV after 24 h. Cells were harvested 24 hpi. Lysates were analyzed as described above. The top panel shows a Western blot probed with the exon 2/3 antibody, and the bottom panel shows a Western blot probed with an antibody against the late protein p28. Protein bands are identified at right.
FIG. 9.
FIG. 9.
IE19 and IE17.5 proteins are not detectable during HCMV infection. HFF, HEL, and U373MG cells were infected with HCMV and harvested at the indicated times. Lysates were prepared in RIPA buffer, and IE proteins were immunoprecipitated using MAb 810. CV-1 cells were transfected with cDNAs encoding IE19 and IE17.5 as a positive control for the migration of these two proteins. Immunoprecipitates were separated by SDS-10% polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with an antibody specific for exon 4. Proteins are indicated at the right; sIE72 refers to the sumoylated form of IE72.
FIG. 10.
FIG. 10.
Mutagenesis of the IE19 splice acceptor site in exon 4 eliminates IE19 transcript and protein. (A) IE19(−), a genomic IE1 plasmid (WT IE72) containing a mutation in the IE19 splice acceptor site in exon 4, was generated by site-directed mutagenesis (site of mutation indicated with an asterisk in panel C; see text for details). (B) CV-1 cells were transfected with the plasmids indicated. Total RNA was analyzed by RNase protection assay to confirm that the IE19(−) mutation eliminated production of the IE19 transcript; details of the probe and bands are shown in Fig. 6. Numbers at right are lengths in nucleotides. (C) Western analysis were performed using lysates from transfected and HCMV-infected cells (as indicated) with antibodies specific for exon 4 (top) or exons 2 and 3 (bottom).
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References

    1. Baracchini, E., E. Glezer, K. Fish, R. M. Stenberg, J. A. Nelson, and P. Ghazal. 1992. An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator. Virology 188:518-529. - PubMed
    1. Caswell, R., L. Bryant, and J. Sinclair. 1996. Human cytomegalovirus immediate-early 2 (IE2) protein can transactivate the human hsp70 promoter by alleviation of Dr1-mediated repression. J. Virol. 70:4028-4037. - PMC - PubMed
    1. Cooke, C., and J. C. Alwine. 2002. Characterization of specific protein-RNA complexes associated with the coupling of polyadenylation and last-intron removal. Mol. Cell. Biol. 22:4579-4586. - PMC - PubMed
    1. Cooke, C., H. Hans, and J. C. Alwine. 1999. Utilization of splicing elements and polyadenylation signal elements in the coupling of polyadenylation and last-intron removal. Mol. Cell. Biol. 19:4971-4979. - PMC - PubMed
    1. Ghazal, P., J. Young, E. Giulietti, C. DeMattei, J. Garcia, R. Gaynor, R. M. Stenberg, and J. A. Nelson. 1991. A discrete cis element in the human immunodeficiency virus long terminal repeat mediates synergistic trans activation by cytomegalovirus immediate-early proteins. J. Virol. 65:6735-6742. - PMC - PubMed

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