In order to further understand the interaction between enJSRVs and their hosts, we investigated the interplay between the ovine BST2
genes and the endogenous betaretroviruses of sheep (enJSRVs). The sheep genome harbors several copies of enJSRV proviruses that are biologically active, abundantly expressed in the reproductive tract of the ewe, and absolutely required for successful pregnancy in this animal species (3
). enJSRVs are highly related to the exogenous and oncogenic JSRV, the causative agent of ovine pulmonary adenocarcinoma, a naturally occurring lung adenocarcinoma of sheep (14
). Interestingly, the sheep (and cow) conceptus secretes IFNT, a type I interferon, which functions as a pregnancy recognition signal in this animal species, although it maintains potent antiviral properties. Thus, the sheep represents a unique model system to investigate the coevolution of endogenous and exogenous retroviruses with host antiviral defenses.
The present study found that the BST2
gene is duplicated in sheep as in cattle. Phylogenetic analysis supports the idea that this duplication occurred approximately 25 million years ago (MYA) before speciation in the Bovinae
subfamily within the ruminants (19
). Thus, duplication of BST2
predates the initial integration of enJSRVs in the host genome that we estimated occurred between 5 and 7 MYA (1
). Both oBST2A and oBST2B blocked viral particle release of enJSRV-18 and the exogenous JSRV. As expected, the antiretroviral activity of these proteins is not specifically targeted to the sheep betaretroviruses, but they have a broader spectrum, considering that HIV-1 ΔVpu can also be blocked by oBST2A/B. These data support the idea that oBST2A has more antiretroviral activity than oBST2B. We speculate that the difference in antiretroviral activities between oBST2A and -2B is due to the proteins per se
and not to JSRV/enJSRV-18 (viral) factors. Interestingly oBST2B does not possess any predicted N-glycosylation site. We were also unable to identify any GPI anchor signals in the C terminus of this protein. The use of oBST2B mutants and more mechanistic studies will be necessary to determine the molecular basis at the origin of the poor antiviral activity of this protein. Indeed, it is possible that oBST2B may have evolved to serve other functions besides viral restriction.
The two oBST2 proteins displayed stronger restriction activity in sheep cells than in human 293T cells. The difference in these two cell types could be due to ovine-specific cofactors important for oBST2 restriction. As mentioned above, oBST2A inhibits equally well HIV-1 ΔVpu, JSRV, and enJSRV-18 viral particle release. These data support the argument that JSRV/enJSRV-18 do not possess a viral protein counteracting oBST2 restriction.
In the present study, we found that oBST2A
genes were induced by IFNT both in vitro
and, importantly, in vivo
. Pregnant sheep showed much higher levels of oBST2A/B
expression in their endometria than cyclic (i.e., nonpregnant) sheep. The levels of oBST2A/B were correlated to the amount of IFNT produced by the mononuclear trophoblast cells of the elongating conceptus. By using an in vivo
hormone replacement model (4
), we showed that IFNT induces expression of oBST2A/B in the endometrium of the sheep uterus. Indeed, oBST2A is expressed at higher levels than oBST2B. Using a probe that would detect both BST2 mRNAs, in situ
hybridization analyses revealed that expression of BST2 was restricted to the stromal cells of the endometrium of the ewe. Thus, enJSRVs and BST2 are expressed in different cellular compartments of the same organ. enJSRVs are expressed in the luminal and glandular epithelium of the uterus, while oBST2A and oBST2B are expressed in the stroma. As mentioned above, duplication of the BST2
gene predates the initial integration of enJSRVs of several million years. Therefore, oBST2A/B could have been one of the driving forces during evolution that shaped the tropism of enJSRV-related exogenous viruses within specific areas of the reproductive tract (i.e., where the BST2 proteins are not expressed). Subsequently, germ line infections by those exogenous viruses led to the selection of enJSRVs present today within the sheep genome. Alternatively, it is possible that sheep have evolved mechanisms to repress BST2 expression in the luminal epithelial cells in order to create a favorable environment for the function of these endogenous retroviruses in host reproductive biology (10
). However, we believe that the latter scenario is unlikely because cows, which do not harbor enJSRVs in their genome (1
), have a pattern of BST2 expression identical to that of sheep.
In the ovine uterus, the luminal and superficial glandular epithelial cells express a potent transcriptional repressor, IFN regulatory factor 2 (IRF2), that inhibits the activation of many genes induced by IFNT during pregnancy, including many antiviral proteins and classical IFN-stimulated genes (5
). Thus, the luminal epithelial cells could have represented a window of opportunity for the replication of enJSRVs. Curiously, we detected oBST2A expression also in the uterine epithelium in sheep treated with progesterone, IFNT, and the progesterone antagonist RU486. It is possible that in the presence of RU486, IRF2 is not expressed or is not active in the luminal epithelium cells of the uterus. Indeed, progesterone normally acts on the stroma cells (which are progesterone receptor positive) and stimulates growth factors, such as fibroblast growth factor 10 (FGF10). In turn, FGF10 acts on the luminal epithelium via its receptor in order to activate signaling pathways that are known to modify transcription factors like IRFs (5
). Thus, treatment with RU486 likely deregulates the normal homeostasis of the sheep endometrium and could explain the upregulation of BST2 in the luminal epithelium of ewes infused with IFNT receiving the RU486 antiprogestin.
As mentioned above, the exogenous and pathogenic JSRV is also blocked as efficiently as enJSRV-18 by both oBST2A and -2B. One could have expected that the exogenous JSRV was able to overcome the block induced by BST2. Thus, replication in cellular compartments devoided of BST2 expression may be one of the strategies used by retroviruses to avoid the antiviral response of the host.