Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon

Beihua Dong

Quansheng Zhou

Ji Zhao

Aimin Zhou

Jeanna Guenther+4 authors

The Departments of Cancer Biology,^{Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation,}^{The Department of Chemistry, Cleveland State University, Cleveland, Ohio,}^{The Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California,}^{The Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania}⁴

^{Corresponding author. Mailing address: Department of Cancer Biology, NB40, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Phone: (216) 445-9650. Fax: (216) 445-6269. E-mail:}gro.fcc@rrevlis.

^{These authors were equal contributors to this study.}

Received 2004 Mar 14; Accepted 2004 Apr 14.

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.}

Abstract

Interferons (IFNs) are the principal cytokines responsible for mediating innate immunity against viral infections (7). How IFNs establish an antiviral state in cells has been a subject of investigation since their discovery (21). Nevertheless, mechanisms of IFN action against viral infections remain incompletely understood. IFN antiviral studies have largely focused on several types of IFN-stimulated genes (ISGs), including the double-stranded RNA (dsRNA)-activated protein kinase (PKR), human myxovirus resistance proteins (Mx), 2′,5′-oligoadenylate synthetase (OAS) and its effector protein RNase L, ISG56 (p56), dsRNA-specific adenosine deaminase, and guanylate binding proteins (GBP) (35). Given the critical role of innate immunity in survival from infections, it is not surprising that the antiviral action of IFNs is complex and involves multiple overlapping or related pathways. For instance, mice that are triply deficient for RNase L, PKR, and Mx1 are nevertheless able to mount a substantial, residual IFN antiviral response (48). Therefore, identification of all of the antiviral ISGs is an important step toward a more complete appreciation and understanding of innate immunity. In this regard, within the past several years, global gene expression profiles from IFN-treated cells, obtained by DNA microarrays, have expanded the number of known ISGs from about 33 to >200 (12, 13).

Phospholipid scramblase 1 (PLSCR1) is a novel ISG identified as such by way of DNA microarray analysis and confirmed by detailed analysis of the PLSCR1 promoter (12, 49, 50). In fact, PLSCR1 is highly induced by IFN-α, -β, and -γ and also by various growth factors, including epidermal growth factor (EGF), stem cell factor, and granulocyte colony-stimulating factor (30, 51). PLSCR1 is a multiply palmitoylated, lipid-raft-associated endofacial plasma membrane protein, with a proline-rich cytoplasmic domain containing several SH3 and WW domain binding motifs (38). PLSCR1 is proposed to accelerate bidirectional movement of plasma membrane phospholipids during conditions of elevated calcium (50). Transmembrane movement of phospholipids in response to calcium, however, is unaffected by either IFN treatment or PLSCR1 deletion (14, 49, 51).

Although the precise biologic function(s) of PLSCR1 and its related isoforms PLSCR2 to 4 remain to be determined (38), recent studies provide intriguing evidence of a role in cell signaling, maturation, and apoptosis. For instance, proliferation and terminal differentiation of certain hematopoietic stem cells (granulocyte precursor) populations is impaired in PLSCR1-null mice (51), and in both monocytic and granulocytic lineages, expression of this protein markedly increases with terminal differentiation into polymorphonuclear leukocytes or macrophages. Conversely, mutations affecting murine PLSCR1 have been associated with a leukemogenic phenotype, which is reversed upon expression of the wild-type (full-length) protein (24, 25). PLSCR1 suppressed ovarian carcinoma in an animal model (37), and elevated expression of PLSCR1 has been shown to be required for normal myeloid differentiation (51). Finally, there is recent evidence that the level of expression of this protein correlates with overall survival in acute myelogenous leukemia (46). PLSCR1 is phosphorylated by select protein kinases, including Abl and Src, tyrosine kinases that participate in multiple growth factor receptor signaling pathways (30, 32, 41). Tyrosine phosphorylation of PLSCR1 by c-Src occurs in response to growth factors such as EGF, resulting in association of phosphorylated PLSCR1 with Shc and the activated EGF receptor complex (30). In the absence of PLSCR1, the activation of c-Src kinase through EGF receptor (and related receptors) is markedly attenuated, suggesting that PLSCR1 plays a role in growth factor-dependent recruitment or activation of c-Src kinase, potentially through its interaction in membrane lipid rafts (30, 40). Palmitoylation of PLSCR1 is required for insertion into the plasma membrane (44). However, when palmitoylation does not occur, the importin α/β nucleopore transport system has recently been shown to import PLSCR1 into the nucleus where it binds DNA (6, 44). Accordingly, newly synthesized PLSCR1 appeared in nuclei after IFN induction of PLSCR1 in the human ovarian carcinoma cell line, Hey1B (44). PLSCR1 is the only member of the PLSCR family thus far shown to be inducible by IFNs. These findings raise the possibility that PLSCR1 may contribute to the antiviral effects of IFNs by affecting viral penetration, IFN-stimulated cell signaling pathways at the plasma membrane, the transcription of antiviral genes in the nucleus, and/or by directly blocking specific stages in the viral replication cycle. To determine the involvement of PLSCR1 in the IFN-induced antiviral state, we have compared viral replication in wild-type and PLSCR1^{mouse cells as well as in human cells in which PLSCR1 levels were decreased with short interfering RNA (siRNA). Our results demonstrate a marked suppression of viral replication by PLSCR1 which is accompanied by the enhanced expression of a specific subset of antiviral ISGs.}

Acknowledgments

We thank Ganes Sen, Ernest Borden (Cleveland, Ohio), and Deborah Vestal for gifts of antibodies and Jonathan Leis (Chicago, Ill.) and Xiaoxia Li (Cleveland, Ohio) for discussions.

This investigation was supported by grant CA89132 (to R.H.S. and P.J.S.) and grant P01 CA62220 (to B.R.G.W. and R.H.S.) from the National Cancer Institute, National Institutes of Health, by grant HL63819 (to P.J.S.) from the National Heart, Lung, and Blood Institute, National Institutes of Health, and by U.S. Army grant DAMD17-01-C-0065 (to B.R.G.W. and R.H.S.).

Acknowledgments

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