The Sheep Tetherin Paralog oBST2B Blocks Envelope Glycoprotein Incorporation into Nascent Retroviral Virions

Abstract

Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the "tethering" mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A. IMPORTANCEBST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species. BST2/tetherin is an interferon (IFN)-stimulated gene with potent antiviral properties against a variety of enveloped viruses (1-4). Human bone marrow stromal cell antigen 2 (hBST2) restricts viral-particle release of human immunodeficiency virus type 1 (HIV-1) lacking the viral accessory protein Vpu by tethering nascent virions, which are subsequently endocytosed, to the cell membrane (2, 5). Moreover, hBST2 has been shown to be an innate sensor of viral-particle release since, upon HIV-1 virion retention, it activates the NF-B signaling pathway and induces expression of proinflammatory genes (6-8). BST2 is a type II transmembrane protein with a unique topology characterized by a short amino-terminal cytoplasmic tail followed by a transmembrane region, an ectodomain, and a carboxy-terminal glycosylphosphatidylinosotol (GPI) anchor. hBST2 is present in different intracellular compartments, including the trans-Golgi network (TGN) and early and recycling endosomes, and within lipid rafts (9). It is the unique topology of BST2, rather than the primary sequence, that determines the ability of this protein to restrict virus release, since an artificial "art tetherin" with no sequence similarity to BST2, but which maintains its structural features, is still capable of restricting the release of enveloped viral particles (5). At least for HIV, h...

BST2/Tetherin Overexpression Modulates Morbillivirus Glycoprotein Production to Inhibit Cell–Cell Fusion

“…Using RT-PCR, we amplified the ORFs of the BST2A and BST2B genes and generated FLAG-tagged expression constructs in the pcDNA3.1 expression vector. The expression of these proteins was confirmed by immunofluorescence microscopy (Figure 5B) and western blot (Figure 5C), which confirmed previous observations from Murphy et al, i.e., that these two proteins have different subcellular localizations and molecular characteristics [28]. The ability of these proteins to inhibit PPRV cell–cell fusion was then assessed using our standardized HEK293T assay, replacing the MeV glycoproteins and human SLAMF1 with PPRV F and H (pcDNA3.1-based) expression constructs and the ovine SLAMF1 receptor [21].…”

“…To address the specificity of BST2 restriction across the morbillivirus genus, and their respective hosts, we focused on the prevalent morbillivirus peste des petits ruminant virus (PPRV) and its small ruminant host, Ovis aries (sheep). As reported previously [28], the BST2 gene is duplicated in sheep, although the longer isoform (BST2B) is significantly different to the shorter protein (BST2A) which was a feature that was evident following phylogenetic analysis of a range of BST2 proteins from morbillivirus-susceptible mammals (Figure 5A). Using RT-PCR, we amplified the ORFs of the BST2A and BST2B genes and generated FLAG-tagged expression constructs in the pcDNA3.1 expression vector.…”

“Ménage à Trois”: The Evolutionary Interplay between JSRV, enJSRVs and Domestic Sheep

“…Recently, it was proposed that enJSRVs cell tropism within the reproductive tract might have been influenced by host restriction factors, such as bone marrow stromal cell antigen 2 (BST-2) [ 66 , 67 , 68 ]. BST-2, also known as tetherin, is a transmembrane protein that restricts many enveloped viruses, including retroviruses, filoviruses and herpesviruses [ 69 , 70 , 71 , 72 ].…”

“…In vitro experiments reveal that oBST-2A is able to block efficiently enJSRV particles release, whereas oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestrating them within the Golgi apparatus. In turn, oBST2B leads to a reduction in Env incorporation into JSRV viral particles and a significant decrease in the viral infectivity [ 67 , 68 ]. Since the duplication of oBST-2 predated the initial invasion of enJSRVs in the sheep genome, it has been proposed that this gene might have been one of the selective forces that confined enJSRVs within specific areas of the reproductive tract, where these cellular restriction factors were not expressed at all, or at very low levels [ 67 ].…”

Prospects in Innate Immune Responses as Potential Control Strategies against Non-Primate Lentiviruses

“…Ovine tetherin/BST2A impedes viral exit of Jaagsiekte sheep retroviruses (JSRV) while ovine BST2B isoform displays a novel antiviral activity impairing the normal cellular trafficking of JSRV envelope glycoproteins [ 172 ] ( Figure 7 ). Both ovine tetherins show low identity with human (~35%), bovine (~60%) or equine (~30%) tetherins, while BST2A isoform was similar to deer (76%) and fallow deer (72%) tetherins [ 173 ].…”