Phospholipid scramblase 1 potentiates the antiviral activity of interferon

Abstract

Phospholipid scramblase 1 (PLSCR1) is an interferon (IFN)- and growth factor-inducible, calcium-binding protein that either inserts into the plasma membrane or binds DNA in the nucleus depending on its state of palmyitoylation. In certain hematopoietic cells, PLSCR1 is required for normal maturation and terminal differentiation from progenitor cells as regulated by select growth factors, where it promotes recruitment and activation of Src kinases. PLSCR1 is a substrate of Src (and Abl) kinases, and transcription of the PLSCR1 gene is regulated by the same growth factor receptor pathways in which PLSCR1 potentiates afferent signaling. The marked transcriptional upregulation of PLSCR1 by IFNs led us to explore whether PLSCR1 plays an analogous role in cellular responses to IFN, with specific focus on antiviral activities. Accordingly, human cells in which PLSCR1 expression was decreased with short interfering RNA were rendered relatively insensitive to the antiviral activity of IFNs, resulting in higher titers of vesicular stomatitis virus (VSV) and encephalomyocarditis virus. Similarly, VSV replicated to higher titers in mouse PLSCR1(-/-) embryonic fibroblasts than in identical cells transduced to express PLSCR1. PLSCR1 inhibited accumulation of primary VSV transcripts, similar to the effects of IFN against VSV. The antiviral effect of PLSCR1 correlated with increased expression of a subset of IFN-stimulated genes (ISGs), including ISG15, ISG54, p56, and guanylate binding proteins. Our results suggest that PLSCR1, which is itself an ISG-encoded protein, provides a mechanism for amplifying and enhancing the IFN response through increased expression of a select subset of potent antiviral genes.

FIG. 1.

Decreasing levels of PLSCR1 with siRNA suppresses the anti-VSV activity of IFN in human Hey1B cells. Hey1B cells stably transfected with pSUPER lacking insert (vector control) or pSUPER expressing the siRNA mismatch control or siRNA to PLSCR1 were incubated with or without human IFN-β (1,000 U per ml) for 8 h and were then infected with purified VSV at an MOI of 0.1 for 16 and 24 h. (A) Levels of hPLSCR1, VSV N protein, p56, and β-actin were determined at 24 h postinfection from cell extracts in Western blots probed with antibodies. (B) VSV yields were determined by plaque assays after combining media from triplicate cultures of infected cells preincubated in the presence (+) or absence (−) of IFN-β (as indicated).

FIG. 2.

PLSCR1 suppresses EMCV replication in Hey1B cells. Hey1B cells stably transfected with empty vector (vector control, white bars) or with vector expressing mismatched siRNA (hatched bars) or PLSCR1 siRNA (black bars) were treated with IFN-β (1,000 U per ml) for 8 h and infected with EMCV (MOI of 0.01) for 24 and 40 h. Viral titers from combining media of triplicate infected cultures of cells were determined by plaque assays. +, present; −, absent.

FIG. 3.

PLSCR1 enhances expression of a set of ISGs as determined in DNA microarrays. Hey1B cells expressing siRNA mismatch or siRNA to PLSCR1 were incubated with or without IFN-β (1,000 U/ml) for 8 h. Gene array results are from RNA samples isolated from triplicate cultures of IFN-treated or control cells. Numbers represent increases (n-fold) in RNA levels after IFN treatment.

FIG. 4.

PLSCR1 enhances the expression of a subset of ISGs as determined by Western immunoblots. Hey1B cells containing empty vector (vector) or expressing siRNA mismatch or siRNA to PLSCR1 were incubated with (+) or without (−) IFN-β (1,000 U/ml) for 16 h. Levels of proteins (indicated) were determined by probing Western blots of cell extracts with specific antibodies (see Materials and Methods).

FIG. 5.

VSV replicates to higher titers in MEFs lacking PLSCR1. (A) Wild-type (black bars) and PLSCR1^−/−KO1 (white bars) MEFs were infected with VSV at an MOI of 0.1. (B) PLSCR1^−/− KO2 (white bars) MEFs and reconstituted, PLSCR1-expressing knock-in KI cells (black bars) were infected with VSV at an MOI of 0.1. At different times postinfection (x axes), virus was harvested. Viral yields, determined by plaque assays on indicator L929 cells, were from combined triplicate cultures of infected cells.

FIG. 6.

Adsorption and penetration of ³⁵S-labeled VSV is unaffected by PLSCR1. KO2 (PLSCR1^−/−) and KI (PLSCR1 reconstituted) cells were infected with purified ³⁵S-VSV (MOI of 4) (see Materials and Methods). Cell-associated proteins were separated by SDS-polyacrylamide gel electrophoresis, and an autoradiogram of the dried gel was prepared. The positions of the VSV G, N, and M proteins are indicated (arrows).

FIG. 7.

PLSCR1 and IFN-α inhibit accumulation of primary VSV N transcripts. Cells were incubated with or without IFN-α A/D (1,000 U per ml) for 16 h followed by treatment with cycloheximide (3 μg/ml) for 2.5 h. Infections were with purified VSV (MOI of 0.5) for 0, 3, 5, and 8 h in the continuous presence of cycloheximide to prevent replication. The Northern blot was probed with ³²P-cDNA to the N gene of VSV and was normalized with a radiolabeled cDNA to β-actin.

FIG. 8.

VSV protein accumulation is reduced in cells expressing PLSCR1. KO2 and KI cells were infected with purified VSV at an MOI of 0.1 for 5, 8, and 11 h (as indicated). Levels of VSV proteins from released virus (Media) and associated with intact cells (Cells) were determined on Western blots probed with antibodies to the VSV L, G, N, and M proteins.

FIG. 9.

Replication of VSV with a late-budding domain mutation (AAPA) in the M protein and wild-type VSV were similarly inhibited by PLSCR1. KO2 (white bars) and KI (black bars) cells were infected with wild-type VSV and VSV-AAPA mutant virus (MOI of 0.1) for 16 h. The viral yields in the media combined from three separate infections of cells were determined by plaque assays on indicator cells.

FIG. 10.

IFN-induced and basal levels of PLSCR1, GBP-2, PKR, and Stat1 in IFN-treated and control MEFs that contain or lack PLSCR1. Cells were incubated for 8 or 24 h in the presence or absence of different concentrations of IFN-α A/D (as indicated). Cells harvested at 8 and 24 h were subconfluent and confluent, respectively. Western blots probed with antibodies to PLSCR1, GBP-2, PKR, and Stat1 are shown.