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. 2016 Sep 12;90(19):8855-65.
doi: 10.1128/JVI.00741-16. Print 2016 Oct 1.

Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection

Kyle C Arend  1 , Benjamin Ziehr  1 , Heather A Vincent  1 , Nathaniel J Moorman  2
Affiliations

Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection

Kyle C Arend et al. J Virol. .

Abstract

Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5' untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection.

Importance: The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.

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Figures

FIG 1
FIG 1
Multiple transcription start sites give rise to mRNAs containing MIE exon 2. (A) HFFs were infected with HCMV (MOI of 3) and harvested at 72 h after infection. Cytoplasmic lysates were resolved through a 10 to 50% linear sucrose gradient to separate ribosomal subunits (40S and 60S), monosomes (80S), and polysomes. (B) 5′ RACE was performed on RNA extracted from gradient fractions containing polysomes in the presence (+) or absence (−) of tobacco acid pyrophosphatase (TAP). 5′ RACE PCR products were visualized on agarose gels, cloned, and sequenced. (C) The cartoon shows the structures of the identified transcripts (not to scale). The number to the right of each transcript indicates the length of the 5′ untranslated region (UTR) of each transcript relative to the translation start site (AUG) located in exon 2.
FIG 2
FIG 2
Transcripts originating from each MIE transcription start site give rise to mature IE1 and IE2 mRNAs. Mock-infected (Mock) or HCMV-infected MRC-5 fibroblasts were harvested at 72 h after infection. (A) Polysome-associated RNA was extracted from AD169-infected cells and reverse transcribed using a primer specific for either exon 4 (IE1) or exon 5 (IE2). The resulting cDNA was amplified using a primer specific for either IE1 or IE2 together with a primer specific for each UTR (the primers are indicated by the length [in base pairs] of their their 5′ UTR [UTR70, UTR136, UTR378, and UTR487]), and the PCR products were visualized on agarose gels. Reverse transcriptase was omitted in a set of samples [(-)RT] to ensure the absence of contaminating DNA. (B) Total RNA was extracted and analyzed as in panel A. (C) As in panel B, except cells were infected with the HCMV TB40/E strain (MOI of 3). The PCR products in panels A, B, and C were cloned and sequenced to ensure their specificity.
FIG 3
FIG 3
Temporal analysis of MIE transcript abundance. (A) Primary fibroblasts were infected with HCMV (MOI of 3). The abundance of the indicated MIE transcript at each time point was measured by qRT-PCR using the absolute abundance method as described in Materials and Methods. (B) Cells were infected as described above for panel A in the presence of cycloheximide (gray bars) or absence of cycloheximide (black bars). The relative abundance of the indicated transcript was measured by qRT-PCR at 6 h after infection. Transcript abundance in untreated cells was set at one. (C) Cells were infected as in panel A in the presence (open bars) or absence (black bars) of PAA. Transcript abundance was measured by qRT-PCR using the absolute abundance method. Values that are significantly different (P value < 0.001) are indicated by a bar and asterisk. (D) Cells were infected and treated as in panel C. The reduction of UL99 mRNA expression in the presence of PAA is shown for comparison. NT, not treated.
FIG 4
FIG 4
DNA sequences surrounding the novel MIE transcription start sites have promoter activity. (A) Cartoon showing the location of the 500 nucleotide regions tested for promoter activity. (B) HeLa cells were transfected with the indicated reporter plasmids, and luciferase activity was measured at 24 h after transfection. The graph shows the fold change in luciferase (Luc) activity relative to cells transfected with the empty pGL3 Basic vector, which lacks a promoter for the luciferase gene. (C) The indicated reporters were electroporated into MRC-5 fibroblasts, and luciferase activity was measured at 24 h after electroporation. (D) MRC-5 fibroblasts were electroporated with the indicated reporters and then infected with HCMV (MOI of 3). Luciferase activity was measured at the indicated times after HCMV infection. The graph shows the fold change in luciferase activity relative to the activity of each reporter at 6 h after infection. P-, promoter.
FIG 5
FIG 5
The core promoter region of the MIEP is not necessary for IE1 and IE2 expression outside the context of HCMV infection. (A) Cartoon showing a portion of the MIE locus in pSVH. The numbers show the locations of the MIE transcription start sites for the indicated MIE transcripts shown in Fig. 1. pSVHΔMIEP was created by removing a 158-bp region containing the MIEP core promoter (−94 to +64 relative to the transcription start site). (B) HeLa cells were left untransfected {negative control [(-) control]} or transfected with pSVH or pSVHΔMIEP (ΔMIEP). IE1 and IE2 protein levels were measured by Western blotting at 24 h after transfection. (C) HeLa cells were transfected and harvested as in panel B. The relative abundance of the IE1 and IE2 mRNAs in pSVHΔMIEP-transfected cells compared to pSVH-transfected cells was determined by qRT-PCR using the ΔΔCT method. IE1 and IE2 abundance in pSVH-transfected cells is set at one. (D) HeLa cells were transfected as in panel B. 5′ RACE analysis of polysome-associated RNA was performed using gene-specific primers located in exon 2. 5′ RACE PCR products were visualized on agarose gels and subsequently cloned and sequenced. No PCR products were obtained in cells where tobacco acid pyrophosphatase (TAP) was omitted (−), demonstrating that the PCR products were derived from mRNAs containing a 5′ m7G cap.
FIG 6
FIG 6
Removal of MIE intron A delays HCMV replication. (A) Diagram depicting the genomic regions removed from each recombinant HCMV BAC. (B) MRC-5 fibroblasts were infected with wild-type (WT) HCMV, HCMVΔIntron A (ΔIntron A), or HCMVΔUTR378 (ΔUTR378) (MOI of 0.5). Cell-free virus was quantified at the indicated times after infection by the TCID50 assay. (C) Fibroblasts were infected with HCMV (MOI of 3), and cell-free virus was quantified as in panel B. Values that are significantly different (P < 0.05) from the WT value are indicated by an asterisk. (D) Fibroblasts were infected as in panel B, and the relative abundance of viral DNA was quantified by real-time PCR at 72 h after infection. The amount of viral DNA in cells infected with wild-type virus was set at one. Values that are significantly different (P < 0.001) from the WT value are indicated by an asterisk.
FIG 7
FIG 7
Deletion of MIE intron A results in decreased IE1 and IE2 mRNA and protein expression. (A) MRC-5 fibroblasts were infected with the indicated strains of HCMV (MOI of 0.5). The expression of representative HCMV immediate early (IE1 and IE2), early (UL44), and late (pp28) proteins was measured by Western blotting. (B) Fibroblasts were infected as in panel A. DNA was extracted from infected cells at 2 h after infection, and HCMV genome abundance was measured by qPCR. (C) MRC-5 fibroblasts were infected as in panel A. The relative abundance of IE1, IE2, and UTR136 mRNAs in cells infected with each recombinant virus was measured by qRT-PCR and compared to the levels in cells infected with wild-type HCMV, which was set at one. Values that are significantly different (P < 0.05) from the WT value are indicated by an asterisk.
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