Interplay between ovine bone marrow stromal cell antigen 2/tetherin and endogenous retroviruses

Abstract

Endogenous betaretroviruses (enJSRVs) of sheep are expressed abundantly in the female reproductive tract and play a crucial role in conceptus development and placental morphogenesis. Interestingly, the colonization of the sheep genome by enJSRVs is likely still ongoing. During early pregnancy, enJSRV expression correlates with the production of tau interferon (IFNT), a type I IFN, by the developing conceptus. IFNT is the pregnancy recognition signal in ruminants and possesses potent antiviral activity. In this study, we show that IFNT induces the expression of bone marrow stromal cell antigen 2 (BST2) (also termed CD317/tetherin) both in vitro and in vivo. The BST2 gene is duplicated in ruminants. Transfection assays found that ovine BST2 proteins (oBST2A and oBST2B) block release of viral particles produced by intact enJSRV loci and of related exogenous and pathogenic jaagsiekte sheep retrovirus (JSRV). Ovine BST2A appears to restrict enJSRVs more efficiently than oBST2B. In vivo, the expression of BST2A/B and enJSRVs in the endometrium increases after day 12 and remains high between days 14 and 20 of pregnancy. In situ hybridization analyses found that oBST2A is expressed mainly in the endometrial stromal cells but not in the luminal and glandular epithelial cells, in which enJSRVs are highly expressed. In conclusion, enJSRVs may have coevolved in the presence of oBST2A/B by being expressed in different cellular compartments of the same organ. Viral expression in cells unable to express BST2 may be one of the mechanisms used by retroviruses to escape restriction.

FIG. 1.

The BST2 gene is duplicated in sheep and is induced by IFNT in vitro. (A) PCR amplification of two distinct ovine BST2 cDNAs by RT-PCR from total mRNA of sheep choroid plexus (CPT-Tert) cells treated with IFNT. The succinate dehydrogenase (SDHA) cDNA was used as a control of the RNA quality of each sample. Mk indicates the DNA molecular marker, while M is the mock DNA negative control. (B) Alignment of the amino acid sequences of the ovine BST2 proteins (oBST2A and -B), gorilla BST2 (gorBST2), mouse BST2 (mBST2), and human BST2 (hBST2). The arrow indicates a predicted N-glycosylation site that is present in all the above-described sequences, with the exception of oBST2B. (C) Unrooted phylogenetic tree of the BST2 orthologs constructed by using the neighbor-joining method. Clade support was evaluated based on 1,000 bootstrap replicates using the same algorithm. Bootstrap values are indicated. Note that the BST2 paralogs in both bBST2 and oBST2 cluster in different branches of the tree, suggesting that they are conserved and were duplicated before speciation. Each ortholog is differentiated using a letter in front of “BST2” as follows: bBST2, bovine (Bos taurus); oBST2, ovine (Ovis aries); cpzBST2, chimpanzee (Pan troglodytes troglodytes); hBST2, human (Homo sapiens); macBST2, macaque (Macaca mulatta); chlBST2, vervet monkey (Chlorocebus aethiops); gorBST2 (Gorilla gorilla); rBST2, rat (Rattus norvegicus); mBST2, mouse (Mus musculus); canBST2, dog (Canis familiaris); swBST2, pig (Sus scrofa domesticus).

FIG. 2.

oBST2A and -2B block viral particle release by JSRV, enJSRV-18, and HIV-1 ΔVpu. Western blotting of concentrated viral particles from supernatants (Virus) and cellular extracts (lysates) of 293T or CPT-Tert cells cotransfected with expression plasmids for viruses and BST2 proteins was as indicated in each panel. Cells were transfected in a 10-cm-diameter petri dish with a fixed amount of expression plasmids for either JSRV (D and I), enJSRV-18 (A to C and F to H), or HIV-1 ΔVpu (E, J, and K), and increasing amounts of expression plasmids for oBST2A, oBST2B (either the untagged or HA-tagged versions), or hBST2 (the HA-tagged version). Blots were incubated with the appropriate antisera, as indicated in each panel. Viral particle release was quantified, and values represent arbitrary units relative to the values of each virus transfected in the absence of BST2 (which was assigned a value of 100). Representative experiments are shown and were repeated at least three times independently.

FIG. 3.

Schematic diagram of the sheep endometrium and early ovine embryo development. (A) Cross-section of the ovine uterus. The main cellular compartments important for this study are the luminal and glandular epithelia of the endometrium and the stroma. (B) Early embryo development. The fertilized oocyte develops into a multicellular morula at day 4 of pregnancy and then into a hatched spherical blastocyst by days 7 to 8 that consists of an inner cellular mass surrounded by a layer of mononuclear trophoblast cells. Between days 12 and 16, the spherical blastocyst elongates to form a filamentous conceptus that secretes the pregnancy recognition signal, IFNT, and begins the process of implantation.

FIG. 4.

Expression of oBST2 in the endometrium of cyclic and pregnant ewes. In situ hybridization analyses of BST2 mRNA in uteri of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic (10C, 12C, 14C; A, B, E, F, I, and J) and pregnant (12P, 14P, 16P, 18P, and 20P; G, H, K, L, O, P, Q, R, S, and T) ewes were hybridized with radiolabeled antisense ovine BST2 cRNA probe. Numbers (10C, 12C, 12P, etc.) indicate the day of the cycle (C) or pregnancy (P) of the ewes used in these experiments. (C and D) A sense ovine BST2 cRNA probe was used as a negative control. Images of representative fields are shown both in bright-field (panels A, C, E, G, I, K, M, O, Q, and S) and dark-field illumination (panels B, D, F, H, J, L, N, P, R, and T). BST2 mRNA was observed with endometrial stroma of sheep beginning at day 14 of pregnancy (panels L, N, P, R, and T) but not in the luminal epithelia (LE), superficial glandular epithelia (sGE), middle or deep glandular epithelia (GE) of the endometrium, or in the conceptus trophectoderm (Tr). A superimposed red line is shown in panels K to T in order to facilitate the visualization of the LE and GE of the uterus in the sections shown under dark-field illumination. Car, caruncle; S, stroma; Tr, trophectoderm.

FIG. 5.

Expression of oBST2 in the endometrium of pregnant cows. In situ hybridization analyses of BST2 mRNA in uteri of pregnant cows. Cross-sections of the uterine wall from pregnant cows (13P, 16P, 19P; A to F) were hybridized with a radiolabeled antisense ovine BST2 cRNA probe. A sense ovine BST2 cRNA probe was used as a negative control (G and H). Numbers (13P, 16P, etc.) indicate the day of the pregnancy of the cows used in these experiments. Images of representative fields are shown both in bright-field (A, C, E, and G) and dark-field illumination (B, D, F, and H). BST2 mRNA was observed in endometrial stroma (S) of cows beginning at day 16 of pregnancy but not in the luminal epithelia (LE) or glandular epithelia (GE) of the endometrium.

FIG. 6.

Relative quantification of BST2A and -2B RNA in cyclic and pregnant ewes. (A) Steady-state levels of BST2A/B mRNAs in endometria from cyclic and early pregnant ewes were determined by slot blot hybridization analysis. In cyclic ewes, BST2 mRNA was low between days 3 and 16. In contrast, BST2 mRNA increased (P < 0.01) in pregnant ewes between days 12 and 14 and remained maximal between days 14 and 20. Data are expressed as LSM relative units (RU) with standard errors (SE). (B) Relative levels of BST2A and BST2B mRNA in the endometria of day 16 cyclic and pregnant ewes. The relative mRNA abundance was calculated from the quantitative RT-PCR analysis as described in Materials and Methods.

FIG. 7.

IFNT induces expression of oBST2 in vivo. Effects of progesterone and IFNT on BST2 mRNA in the ovine uterus. (A) Experimental design. See Materials and Methods for complete description. CX, control serum proteins; P4, progesterone; IFNT, recombinant ovine tau interferon; RU, RU486 antiprogestin. (B) Steady-state levels of oBST2 mRNA in endometria as determined by slot blot hybridization analysis. Intrauterine infusion of IFNT increased oBST2 mRNA in the endometrium of P4-treated ewes (P4+CX versus P4+IFNT, P < 0.001) as well as in ewes receiving the RU486 antiprogestin (P4+IFNT versus P4+RU+IFNT, P < 0.001). (C to L) In situ hybridization analysis of oBST2 mRNA expression similar to that described in the legends for Fig. 4 and 5. IFNT increased BST2 mRNA in a cell-type-specific manner in P4-treated ewes, consistent with increased expression in uteri from day 14, 16, and 18 pregnant ewes. Intrauterine injections of IFNT increased BST2 mRNA in endometrial stroma but not in LE, GE, or myometrium. A red line has been superimposed to facilitate the visualization of the luminal and glandular epithelia of the uterine stroma. (M to V) In situ hybridization analysis of enJSRV env RNA expression. The enJSRV env RNA was specifically and abundantly present in the endometrial LE and GE but not in the stroma or myometrium.