Biochemical and Biophysical Research Communications
The role of the structural domains of human BST-2 in inhibiting the release of xenotropic murine leukemia virus-related virus
Highlights
► We show that BST-2 tethers nascent XMRV virions to cellular membranes. ► We determine the structural domains that are essential for BST-2 to restrict XMRV. ► Protease treatment releases the tethered XMRV particles from the cell surface. ► Illustrating the role of BST-2 structural domain in BST-2 cellular localization.
Introduction
Host restriction factors often determine the tissue tropism of a virus and limit virus cross-species transmission [1], [2]. One such restriction factor is BST-2 (also named tetherin, CD317 or HM1.24), which was discovered through its ability to inhibit the release of human immunodeficiency virus type 1 (HIV-1) [3], [4]. BST-2 is a homodimeric, lipid raft-associated type II integral membrane glycoprotein. BST-2 contains an N-terminal cytoplasmic tail (CT), a transmembrane domain (TM), a coiled-coiled ectodomain, two potential N-linked glycosylation sites, and a C-terminal glycosylphosphatidylinositol (GPI) anchor [5], [6]. The antiviral function of BST-2 is attributed to its unique two-membrane-anchor topology. Since the discovery of its anti-HIV-1 activity, BST-2 has been shown to restrict the release of many enveloped viruses, including HIV-2, simian immunodeficiency virus (SIV), Kaposi’s sarcoma herpes virus (KSHV), Lassa virus, Marburg virus, and Ebola virus [7], [8], [9], [10]. The importance of BST-2 in host antiviral defense is demonstrated by the study showing that BST-2 knockout mice exhibited severe pathology upon infection by murine leukemia virus (MLV) [11].
Xenotropic murine leukemia virus-related virus (XMRV) was originally reported in prostate cancer tissues from patients with a low-activity variant of RNase L [12], [13]. This finding has been questioned by subsequent studies [14]. XMRV has also been linked to chronic fatigue syndrome (CFS) [15], but this observation could not be reproduced in other studies [16], [17]. Results of several groups argued that the XMRV DNA detected in human clinical samples resulted from the contamination by mouse DNA containing an MLV-like sequence [18], [19], [20], [21], [22]. XMRV was traced back to the recombination of two endogenous MLVs, PreXMRV-1 and PreXMRV-2, during in vivo tumor passaging in nude mice [23], [24]. These data led to the retraction of the papers by Lombardi et al. and Lo et al. [25], [26], [27]. It is now well received that XMRV does not circulate in the general human population [28].
Although the association of XMRV with human diseases now appears unlikely, XMRV can still replicate in human cell lines in vitro [29]. This provides an opportunity to study how XMRV responds to host restriction in human cells. Indeed, studies have shown that XMRV infection is inhibited by human APOBEC3G and human BST-2 [30]. Here, we confirmed and extended the observation that XMRV is sensitive to the inhibition by BST-2 [30]. We further showed that human BST-2 was sufficient to tether nascent XMRV particles to the cell surface. We also used mutagenesis analysis to characterize the features of BST-2 that are required for its anti-XMRV activity.
Section snippets
Plasmid DNA and antibodies
The human BST-2 (hBST-2) cDNA was a gift from Chen Liang (Lady Davis Institute for Medical Research, McGill University). The pcDNA-BST2-HA construct expresses internally HA-tagged BST-2 proteins (with the HA tag inserted immediately 3′ to BST-2 codon 154). Single amino acid mutations at BST-2 cysteine codons and glycosylation sites were generated by PCR-based mutagenesis. The BST-2-HA mutants delCC, delGPI and delTM were generated according to methods that were described previously [31]. The
BST-2 significantly decreases XMRV release from mammalian cells
We first established Vero cell lines that stably express human BST-2. These cells, when transfected with XMRV DNA, produced significantly less viruses than the control cells (Fig. 1A). We then investigated whether XMRV is sensitive to inhibition by endogenous BST-2. HeLa cells, which express endogenous BST-2, were transduced with lentiviral vectors expressing control shRNA or BST-2 shRNA. As shown in Fig. 1C, the knockdown of endogenous BST-2 led to a significant increase in XMRV particle
Discussion
In this study, we made several novel observations. First, through the results of our imaging studies, we confirmed that BST-2 tethers nascent XMRV particles to the cell surface. Second, we performed systematic mutagenesis studies to assess the contribution of BST-2 structural and modification features to virus inhibition. We concluded that the anti-XMRV activity of BST-2 depends on its unique membrane topology, cysteine-mediated dimerization and N-linked glycosylation. Third, we provided a
Acknowledgments
We especially thank Chen Liang and Stephen Goff for providing various reagents. We also thank Chen Liang and Lawrence Kleiman for helpful discussion and comments on the manuscript. This research was funded by grants from the Ministry of Science and Technology of China (2012CB911100, 2011CB504800, 2008ZX10001-002, 2009ZX1004-303 and 2010DFB30870) and from the Nature Science Foundation of China (30970155). The funding agencies had no role in the study design, data collection and analysis, the
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These authors contributed equally to this work.