The Pentamer glycoprotein complex inhibits viral Immediate Early transcription during Human Cytomegalovirus infections

Abstract

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

Keywords: entry; glycoprotein; transcription; virus.

Fig. 1.

TB40/E-Pentamer decreases HCMV productive replication in fibroblasts. (A) NHDF (Left) or ARPE-19 (Right) cells were infected with parental TB40/E or TBm131 at an MOI of 1 for four days before being fixed and imaged for GFP+ cells. Nuclei were counterstained with Hoechst. (B) Quantitation of GFP+ cells from experiments in panel A. (C) Growth curve of respective viruses, TB40/E, TBm131, or a Revertant (TBr131) in fibroblasts. NHDF were infected with the indicated virus at an MOI of 0.05 and virus was harvested at the indicated day postinfection and analyzed by the plaque assay on naive NHDFs. Data represent the mean ± SEM from three biological replicates. *P < 0.05 by two-tail Student’s t test. ns: not significant (P > 0.05).

Fig. 2.

The Pentamer represses IE transcript accumulation during infection of fibroblast and myeloid cells. (A) Equal numbers of PFU from viral stocks were used to isolate viral DNA, and relative genomes per PFU were determined using qPCR for the UL123 locus and plotted relative to the Pentamer-null virus for each matched pair. (B) RNA and DNA isolated from NHDFs infected for 4 h with AD169 or AD169 with a repaired Pentamer (ADrUL131) at the indicated MOI. Viral DNA (Left panel) was quantitated by qPCR relative to cellular GAPDH. Viral IE transcripts (Right panel) were quantitated by RT-qPCR relative to cellular GAPDH, normalized to viral DNA, and plotted relative to Pentamer-negative virus. (C) Viral DNA and IE transcripts from NHDF infected with TB40/E or TBm131 as in panel B. (D–F) Viral IE transcripts from THP1 monocytes infected with AD169 (panel D), Towne (panel E), or TB40/E (panel F)-based viruses at an MOI of 1 in the absence (−) or presence (+) of 1 mM VPA for 18 h were quantitated by RT-qPCR relative to cellular GAPDH. Fold induction was then quantitated relative to respective no VPA condition. (G and H) RNA was isolated from CD34⁺ HPCs infected with AD169 (panel G) or TB40/E (panel H)-based viruses at an MOI of 1 in the absence (−) or presence (+) of VPA for 24 h and IE transcripts were analyzed as above. Towne-UL130rep (TN-rUL130). “r131”: ADrUL131; “m131”: TBm131. Data represent the mean ± SEM from a minimum of three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tail Student’s t test. ns: not significant (P > 0.05).

Fig. 3.

IE1 expressed in trans complements the growth defect of Pentamer-positive virus in fibroblasts. (A) Western blots of lysates from NHDFs stably transduced with an empty vector (NHDF-EV) or Flag-tagged IE1 (NHDF-IE1). Tubulin serves as a loading control. (B) Relative titers from plaque assays of CR208 grown in NHDF-IE1 cells compared to NHDF-EV cells at 11 d postinfection at an MOI of 0.01. (C and D) Cells were infected with the indicated virus at an MOI of 0.01 and collected at the indicated days postinfection. Titers were determined by plaque assay on the same cells in which the virus was produced. (E) Relative titers of AD169 and ADrUL131 (r131) at 11 d postinfection for either NHDF-EV or NHDF-IE1. Data represent the mean ± SEM from three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tail Student’s t test. ns: not significant (P > 0.05).

Fig. 4.

Virion-incorporated Pentamer suppresses IE transcription. (A) Western blot for the indicated proteins in lysates from NHDFs infected with live or UV-inactivated AD169 or ADrUL131 at an MOI of 0.1 for 4 h. (B) RNA and DNA were isolated from NHDFs infected with the indicated viruses, each at an MOI of 0.1, and analyzed by qPCR. Viral IE transcripts were normalized to viral genomes and are shown relative to WT AD169-only infected controls. (C and D) ADrUL131 was incubated with the indicated antibody for 1 h before infecting either ARPE-19 (panel C) or NHDF (panel D) at an MOI of 0.5. GFP-positive cells were quantified 4 d postinfection. (E) ADrUL131 was incubated with the indicated antibody for 1 h before infecting NHDF cells at an MOI of 0.1. RNA and DNA were isolated and analyzed by qPCR. Viral IE transcripts were normalized to viral genomes and are shown relative to WT AD169 infected controls. “RSV”: anti-RSV-F antibody. “128”: anti-UL128 antibody. “130”: anti-UL130 antibody. “”131”: anti-UL131 antibody. Data represent the mean ± SEM from a minimum of three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tail Student’s t test. ns: not significant (P > 0.05).

Fig. 5.

CRE/ATF or NFκB sites are required for the Pentamer to repress IE transcription. (A) RNA and DNA were isolated from NHDFs infected with the indicated virus and analyzed by qPCR. Viral IE transcripts were normalized to viral genomes and shown relative to WT AD169 infected controls. (B) NHDF cells were pretreated with either a PBS vehicle control or 25 µg/mL of recombinant Pentamer before being infected and analyzed as in panel A. AD-CRE-: AD169 with all MIEP CRE/ATF sites mutated; AD-κB-: AD169 with all MIEP NFκB sites mutated; AD-κB-/CRE-: AD169 with all MIEP CRE/ATF and NFκB sites mutated. “Pent”: recombinant Pentamer. Data represent the mean ± SEM from a minimum of three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tail Student’s t test. ns: not significant (P > 0.05).

Fig. 6.

The UL130 subunit of the Pentamer represses the MIEP. (A) Western blots for the indicated proteins in lysates from 293T cells transfected with plasmids encoding the indicated Pentamer subunit or empty vector (EV) control for 24 h. Tub: α-Tubulin. Tag: anti-Flag, gH and UL130; anti-HA, gL and UL128; anti-Myc, rUL131. (B) Dual-Luciferase Reporter assay in which plasmids for the indicated individual proteins or all five Pentamer subunits (All Subunits, open bar) were cotransfected into THP1 monocytes along with an MIEP-driven Firefly-luciferase and a control Renilla-luciferase for 48 h. MIEP-driven Firefly-luciferase activity for each condition was normalized to the internal Renilla-luciferase control and plotted relative to the EV condition. UL128 (128), UL130 (130), and rUL131 (131). (C–F) Dual-luciferase reporter assays were carried out the same as in panel B except the MIEP driving Firefly luciferase was either wild-type (wt) (panel C), mutated for all five CRE/ATF sites (CRE-) (panel D), mutated for all four NFkB sites (κB-) (panel E), or mutated for all CRE/ATF and all NFκB sites (κB-/CRE-) (panel F), and cotransfected with either EV or UL130. Data represent the mean ± SEM from a minimum of three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tail Student’s t test. ns: not significant (P > 0.05).