The Interferon-Inducible Human PLSCR1 Protein Is a Restriction Factor of Human Cytomegalovirus

Abstract

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and it has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the levels of human cytomegalovirus (HCMV) plaque formation in OUMS-36T-3 (36T-3) cells with high basal expression of PLSCR1 were significantly lower than those in human embryonic lung (HEL) cells with low basal expression of PLSCR1. In addition, the levels of HCMV plaque formation and replication in PLSCR1-knockout (KO) 36T-3 cells were significantly higher than those in parental 36T-3 cells and were comparable to those in HEL cells. Furthermore, compared to that in PLSCR1-KO cells, the expression of HCMV major immediate early (MIE) proteins was repressed and/or delayed in parental 36T-3 cells after HCMV infection. We also showed that PLSCR1 expression decreased the levels of the cAMP-responsive element (CRE)-binding protein (CREB)•HCMV immediate early protein 2 (IE2) and CREB-binding protein (CBP)•IE2 complexes, which have been suggested to play important roles in the IE2-mediated transactivation of the viral early promoter through interactions with CREB, CBP, and IE2. Interestingly, PLSCR1 expression repressed CRE- and HCMV MIE promoter-regulated reporter gene activities. These observations reveal, for the first time, that PLSCR1 negatively regulates HCMV replication by repressing the transcription from viral MIE and early promoters, and that PLSCR1 expression may contribute to the IFN-mediated suppression of HCMV infection. IMPORTANCE Because several IFN-stimulated genes (ISGs) have been reported to suppress HCMV replication, HCMV replication is thought to be regulated by an IFN-mediated host defense mechanism, but the mechanism remains unclear. PLSCR1 expression is induced in response to viral infection and IFN treatment, and PLSCR1 has been reported to play an important role in IFN-dependent antiviral responses. Here, we demonstrate that HCMV plaque formation and major immediate early (MIE) gene expression are significantly increased in PLSCR1-KO human fibroblast cells. PLSCR1 reduces levels of the CREB•IE2 and CBP•IE2 complexes, which have been suggested to play important roles in HCMV replication through its interactions with CREB, CBP, and IE2. In addition, PLSCR1 expression represses transcription from the HCMV MIE promoter. Our results indicate that PLSCR1 plays important roles in the suppression of HCMV replication in the IFN-mediated host defense system.

Keywords: genome editing; human cytomegalovirus; interferon-stimulated genes; phospholipid scramblase 1; protein-protein interactions; restriction factors.

FIG 1

The expression level and pattern of PLSCR1 induced by HCMV infection differs in HEL and OUMS-36T-3 (36T-3) cells. (A) Total cell lysates were prepared using RIPA buffer, and a total of 5 μg of total cell lysates were subjected to SDS-PAGE. Immunoblotting (IB) was performed using an anti-PLSCR1 antibody to detect endogenous PLSCR1 or an anti-actin antibody for endogenous β-actin. (B) HEL cells were infected with the HCMV AD169 strain at a multiplicity of infection (MOI) of 3 PFU per cell, and cell lysates were prepared at the indicated time points (hours postinfection). Equivalent amounts of lysates were subjected to SDS-PAGE. IB was performed using an anti-PLSCR1 antibody to detect endogenous PLSCR1 or an anti-MxA antibody to detect endogenous MxA. Then, endogenous β-actin, as a loading control, was detected by reprobing the same membrane with an anti-actin antibody (upper panel). Band intensity was quantified using NIH ImageJ software, and levels were normalized to those of the internal β-actin control (lower panel). (C) 36T-3 cells were infected with HCMV AD169, and cell lysates preparation, IB, and quantification of band intensity were performed as described in panel B.

FIG 2

HCMV plaque formation is increased in PLSCR1-KO cells compared to parental cells. HEL, 36T-3, PLS1KO-A, and PLS1KO-B cells were infected with a 10-fold series of diluted HCMV and incubated for 6 to 10 days. The cell monolayer was fixed and then stained with methylene blue. Plaques were counted microscopically under low power, and the PFU were measured. The Data represent the average relative values from four experiments with duplicate cultures per experiment, and the error bars indicate the standard deviations. **, P < 0.01 by Student's t test.

FIG 3

Generation of PLSCR1-KO 36T-3 cells. 36T-3 and CRISPR/Cas9-generated PLSCR1-KO 36T-3 (PLS1KO-A and PLS1KO-B) cells were treated with or without 3,000 units/mL IFN-α-2b for 16 h. Total cell lysates were prepared using RIPA buffer (Sigma-Aldrich), and a total of 4 μg of total cell lysates were subjected to SDS-PAGE. IB was performed using an anti-PLSCR1 antibody to detect endogenous PLSCR1, an anti-ISG15 antibody to detect endogenous ISG15, or an anti-actin antibody to detect endogenous β-actin.

FIG 4

HCMV replicates to higher titers in PLSCR1-KO cells than in parental cells. HEL, 36T-3, PLS1KO-A and PLS1KO-B cells were infected with HCMV at an MOI of 3 PFU per cell. At 5 and 7 days p.i., the supernatants were collected, and viral titers were determined by plaque formation assays in HEL cells from three experiments with duplicate cultures per experiment. Data represent average relative values, and error bars indicate standard deviations. **, P < 0.01 by Student's t test.

FIG 5

Expression levels of HCMV MIE genes in PLS1KO cells were higher than those in the parental cell line. HEL, 36T-3, PLS1KO-A, and PLS1KO-B cells were infected with HCMV at an MOI of 0.1 PFU per cell. At 6 h (A) or 48 h (B) (for MIE) and 72 h (C) (for gB) p.i., the cells were fixed and then subjected to immunofluorescence staining with an anti-HCMV MIE antibody (A, B) and an anti-HCMV gB antibody (C), respectively. Nuclear and cytoplasmic fluorescence-positive cells were counted as MIE and gB antigen-positive cells, respectively, under a fluorescence microscope. Data represent means and standard deviations of three independent experiments. Data represent average relative values, and error bars indicate standard deviations. **, P < 0.01 by Student's t test. (D) HEL, 36T-3, and PLS1KO-A cells were infected with the HCMV AD169 strain at an MOI of 3 PFU per cell, and cell lysates were prepared at the indicated time points (hours). Equivalent amounts of lysates were subjected to SDS-PAGE. IB was performed using an anti-HCMV MIE antibody to detect endogenous IE1 and IE2. Detection of endogenous β-actin was performed as described in Fig. 1B and C. (E) Band intensity was quantified as described in Fig. 1B and C.

FIG 6

PLSCR1 specifically interacts with HCMV IE1 and IE2. HEK-293 cells (1 × 10⁶) were plated in 60-mm cell culture plates and transfected with 2 μg of pcDNA3 or S-PLSCR1 and 0.5 μg of pUCIE1 or 3 μg of pUCIE2. A total of 300 μg of total cell lysate prepared in NP-40 lysis buffer was incubated with S-protein beads to precipitate PLSCR1. Following pulldown assays, 1 μg (IE1) or 5 μg (IE2) of total cell lysate and the precipitated complexes were divided into two parts and subjected to SDS-PAGE. IB was performed using an anti-S tag antibody to detect PLSCR1 or an anti-HCMV MIE antibody to detect IE1 and IE2.

FIG 7

PLSCR1 decreases the levels of the CREB•IE2 and CBP•IE2 complexes. (A) HEK-293 cells (3 × 10⁶) were plated in 90-mm cell culture plates and transfected with 2 μg of pcDNA3 or 3FG-CREB, 1 μg of pcDNA3 or pUCIE2, and 5 μg of pcDNA3 or 3M-PLSCR1. A total of 160 μg of total cell lysate prepared in NP-40 lysis buffer was incubated with FLAG M2 beads to precipitate CREB. Following immunoprecipitation, 15 μg of total cell lysate and the precipitated complexes were divided into three parts and subjected to SDS-PAGE. IB was performed using a FLAG M2 antibody to detect CREB, an anti-myc-tag antibody to detect PLSCR1, or an anti-HCMV MIE antibody to detect IE2. To detect mouse-derived primary antibodies, Mouse TrueBlot ULTRA: Anti-mouse Ig HRP was used as a secondary antibody. (B) HEK-293 cells (4.5 × 10⁶) were plated into 90-mm cell culture plates and transfected with 4 μg of pcDNA3 or 3FG-CBP, 0.75 μg of pcDNA3 or pUCIE2, and 3 μg of pcDNA3 or 3M-PLSCR1. A total of 1,300 μg of total cell lysate prepared in NP-40 lysis buffer was incubated with FLAG M2 beads to precipitate CBP. Following immunoprecipitation, 25 μg of total cell lysate and the precipitated complexes were divided into three parts and subjected to SDS-PAGE. IB was performed as in panel A, except that a FLAG M2 antibody was used to detect CBP.

FIG 8

PLSCR1 represses transcription from the CRE- and HCMV MIEP-regulated reporter constructs. (A) HEK-293 cells (1 × 10⁵) were plated in 24-well cell culture plates and transfected with 40 ng of CRE-responsive reporter plasmid (pCRE-Luc), 40 ng of TAL-pGL4.70 (53), and 300 ng of pcDNA3 or S-PLSCR1 using jetPEI. After 24 h of transfection, the cells were treated with or without 5 μM Forskolin (Cayman Chemical) for 16 h. Then the cells were lysed, and their luciferase activity levels were determined. The firefly luciferase/Renilla luciferase activity ratio of cells transfected with the vector only was defined as 100%. Data represent the average relative values from triplicates, and error bars indicate standard deviations. **, P < 0.01 by Student's t test. (B) HeLa-PLSKO cells (5 × 10⁴) were plated in 24-well cell culture plates and transfected with 2 ng of pMIEP-Luc, 40 ng of pGL4.74 (Promega), and 350 ng of pcDNA3 or S-PLSCR1 using TransIT-X2. After 24 h of transfection, the cells were treated with or without 5 μM Forskolin for 16 h. Then the cells were lysed, and their luciferase activity levels were determined. The firefly luciferase/Renilla luciferase activity ratio of cells transfected with the vector only was defined as 100%. Data represent average relative values from triplicates, and error bars indicate standard deviations. **, P < 0.01 by Student's t test.