Herpes Simplex Virus 1 Small Noncoding RNAs 1 and 2 Activate the Herpesvirus Entry Mediator Promoter

Abstract

Herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) plays a significant role in efficient establishment of latency and reactivation. LAT has antiapoptotic activity and downregulates expression of components of the type I interferon pathway. LAT also specifically activates expression of the herpesvirus entry mediator (HVEM), one of seven known receptors used by HSV-1 for cell entry that is crucial for latency and reactivation. However, the mechanism by which LAT regulates HVEM expression is not known. LAT has two small noncoding RNAs (sncRNAs) that are not microRNAs (miRNAs), within its 1.5-kb stable transcript, which also have antiapoptotic activity. These sncRNAs may encode short peptides, but experimental evidence is lacking. Here, we demonstrate that these two sncRNAs control HVEM expression by activating its promoter. Both sncRNAs are required for wild-type (WT) levels of activation of HVEM, and sncRNA1 is more important in HVEM activation than sncRNA2. Disruption of a putative start codon in sncRNA1 and sncRNA2 sequences reduced HVEM promoter activity, suggesting that sncRNAs encode a protein. However, we did not detect peptide binding using two chromatin immunoprecipitation (ChIP) approaches, and a web-based algorithm predicts low probability that the putative peptides bind to DNA. In addition, computational modeling predicts that sncRNA molecules bind with high affinity to the HVEM promoter, and deletion of these binding sites to sncRNA1, sncRNA2, or both reduced HVEM promoter activity. Together, our data suggest that sncRNAs exert their function as RNA molecules, not as proteins, and we provide a model for the predicted binding affinities and binding sites of sncRNA1 and sncRNA2 in the HVEM promoter. IMPORTANCE HSV-1 causes recurrent ocular infections, which is the leading cause of corneal scarring and blindness. Corneal scarring is caused by the host immune response to repeated reactivation events. LAT functions by regulating latency and reactivation, in part by inhibiting apoptosis and activating HVEM expression. However, the mechanism used by LAT to control HVEM expression is unclear. Here, we demonstrate that two sncRNAs within the 1.5-kb LAT transcript activate HVEM expression by binding to two regions of its promoter. Interfering with these interactions may reduce latency and thereby eye disease associated with reactivation.

Keywords: HSV-1; HVEM; cornea; infection; luciferase; promoters; transfection; transfection systems; virus replication.

FIG 1

Chromatin immunoprecipitation (ChIP) and PCR amplification of HVEM promoter. 293T cells were transfected with WT sncRNA1-FLAG or WT sncRNA2-FLAG as described in Materials and Methods. Chromatin from transfected cells was sheared and immunoprecipitated with anti-FLAG beads. Immunoprecipitated chromatin and input chromatin were amplified with primers corresponding to HVEM promoter sequences. Experiments were repeated twice.

FIG 2

sncRNA1 and sncRNA2 plasmids used in these studies. (A) Plasmid containing the 1.5-kb LAT (pGEM5317) and its promoter has been described previously (17, 18). Shaded boxes indicate sncRNA1 and sncRNA2 sequences within LAT. sncRNA1 (62 nt) (B) and sncRNA2 (36 nt) (C) were generated and inserted into the pSilencer and pcDNA3.1-FLAG plasmids. LAT sequences lacking ΔsncRNA1 sequence (D), ΔsncRNA2 sequence (E), or both sequences (E) and under the LAT promoter were generated and inserted into the pUC57 plasmid. sncRNA1 TTG (F) and sncRNA2 TTG (G) mutant constructs were established by inserting a 3,007-bp region of LAT corresponding to LAT 118641–121660 and containing a single base pair mutation of ATG to TTG in either sncRNA1 or sncRNA2 and inserted into pUC57. All constructs except those in panels B and C are under the LAT promoter.

FIG 3

Expression of sncRNA1 or -2 does not affect expression of viral transcripts. RS cells were transfected with WT sncRNA1, sncRNA2, or LAT and infected with 1 PFU/cell of HSV-1 strain McKrae 48 h after transfection. Cells were collected at 2, 4, 8, and 12 h postinfection in TRIzol. RNA was extracted and amplified by qRT-PCR with gB (A), ICP0 (B), or ICP4 (C) primers. The copy number of any of the three sequences did not differ at any time point. Each point represents the means ± SEM for 3 experiments.

FIG 4

Expression of sncRNA1 and 2 increases HVEM promoter activity. 293T cells were transfected with pGL4-pHVEM and plasmids containing LAT, WT sncRNA1, WT sncRNA2, or empty vector (A), WT sncRNA1, sncRNA1 deletion plasmid (ΔsncRNA1), or empty vector (B), or WT sncRNA2, sncRNA2 deletion plasmid (ΔsncRNA2), or empty vector (C). HVEM promoter activity was measured 8, 24, and 48 h posttransfection. Expression relative to the empty vector is shown as fold change. Each point represents the mean ± SEM (n = 10 for A, n = 6 for B, C) from three separate experiments. *, P < 0.002.

FIG 5

Deletion of both sncRNA1 and sncRNA2 sequences results in loss of HVEM promoter activity. 293T cells were transfected with pGL4-pHVEM and LAT or sncRNA1 and sncRNA2 deletion plasmid (ΔsncRNA1&2). The effects on HVEM promoter activity were determined 8, 24, and 48 h posttransfection. *, P < 0.0001.

FIG 6

sncRNA ATG is required to enhance HVEM promoter activity. 293T cells were transfected with pGL4-pHVEM and WT sncRNA1 or sncRNA1 TTG (A) or WT sncRNA2 or sncRNA2 TTG (B). The effects on HVEM promoter activity were determined 8, 24, and 48 h posttransfection. Each point represents the means ± SEM (n = 9) from three separate experiments. *, P < 0.0001.

FIG 7

HVEM promoter activation levels by TTG sncRNA and sncRNA deletion mutants are similar. 293T cells were transfected with pGL4-pHVEM and sncRNA1 TTG or sncRNA1 deletion plasmid (A) or sncRNA2 TTG or sncRNA2 deletion plasmid (B). Effect on HVEM promoter activity was determined as described for Fig. 3. Each point represents the mean ± SEM (n = 5) from two separate experiments. *, P < 0.01.

FIG 8

Computational modeling predicts binding of sncRNA1 and sncRNA2 to mouse HVEM promoter. (A) Mouse HVEM promoter sequence corresponding to nucleotides 539/−478 upstream of the HVEM ATG start codon is predicted to bind to sncRNA1 with a free energy of binding of −50.6 kcal/mol. (B) mHVEM promoter sequence corresponding to −484/−421 upstream of the HVEM start codon is predicted to bind to sncRNA2 with a free energy of binding of −19.9 kcal/mol. Mfe refers to free energy of binding. Red boxes show the position of the ATGs in sncRNA sequences. Schematic diagram of (C) the wild-type mouse HVEM promoter construct, (D) the mouse HVEM promoter construct lacking the predicted sncRNA1 binding site, (E) the mouse HVEM promoter construct lacking the predicted sncRNA2 binding site, and (F) the mouse HVEM promoter construct lacking both the predicted sncRNA1 and sncRNA2 binding sites.

FIG 9

sncRNA1 and -2 sequences are necessary for HVEM promoter activation. 293T cells were transfected with either LAT or empty vector and pGL4-pHVEM constructs lacking sncRNA1, sncRNA2, or both sncRNA1 and sncRNA2 binding sites. HVEM promoter activity was measured 8 h posttransfection. Expression relative to empty vector is shown as fold change. Each point represents the mean ± SEM (n = 10) from two separate experiments. *, P < 0.0001.