The HIV-1 accessory protein Vpu counteracts a host factor that restricts virion release from infected cells. Here we show that the interferon-induced cellular protein BST-2/HM1.24/CD317 is such a factor. BST-2 is downregulated from the cell surface by Vpu, and BST-2 is specifically expressed in cells that support the vpu phenotype. Exogenous expression of BST-2 inhibits HIV-1 virion release, while suppression of BST-2 relieves the requirement for Vpu. Downregulation of BST-2 requires both the transmembrane/ion channel domain and conserved serines in the cytoplasmic domain of Vpu. Endogenous BST-2 colocalizes with the HIV-1 structural protein Gag in endosomes and at the plasma membrane, suggesting that BST-2 traps virions within and on infected cells. The unusual structure of BST-2, which includes a transmembrane domain and a lumenal GPI anchor, may allow it to retain nascent enveloped virions on cellular membranes, providing a mechanism of viral restriction counteracted by a specific viral accessory protein.

RESULTS-TLR1-6 and 9 mRNA species were detected in both whole endometrium and separated endometrial epithelial cells. Ishikawa cells expressed TLR2 and TLR5, while RL95-2 cells expressed TLR3, 5, and 9. Response of RL95-2, Ishikawa, and U-937 cells to TLR2-5 ligands was consistent with RT-PCR findings except response to flagellin by Ishikawa cells.CONCLUSION-These studies provide the first evidence of TLR expression in the endometrium of any species and suggest the usefulness of endometrial cell lines to study TLR function.

Retroviruses like human immunodeficiency virus type 1 (HIV-1), as well as many other enveloped viruses, can efficiently produce infectious virus in the absence of their own surface glycoprotein if a suitable glycoprotein from a foreign virus is expressed in the same cell. This process of complementation, known as pseudotyping, often can occur even when the glycoprotein is from an unrelated virus. Although pseudotyping is widely used for engineering chimeric viruses, it has remained unknown whether a virus can actively recruit foreign glycoproteins to budding sites or, alternatively, if a virus obtains the glycoproteins through a passive mechanism. We have studied the specificity of glycoprotein recruitment by immunogold labeling viral glycoproteins and imaging their distribution on the host plasma membrane using scanning electron microscopy. Expressed alone, all tested viral glycoproteins were relatively randomly distributed on the plasma membrane. However, in the presence of budding HIV-1 or Rous sarcoma virus (RSV) particles, some glycoproteins, such as those encoded by murine leukemia virus and vesicular stomatitis virus, were dramatically redistributed to viral budding sites. In contrast, the RSV Env glycoprotein was robustly recruited only to the homologous RSV budding sites. These data demonstrate that viral glycoproteins are not in preformed membrane patches prior to viral assembly but rather that glycoproteins are actively recruited to certain viral assembly sites.

Toll-like receptor 3 (TLR3) responds to dsRNA, a product of most viral life cycles, and initiates production of proinflammatory and antiviral cytokines. The role of TLR3 in human mucosal immunity of the endometrium has not been examined. The effects of TLR3 ligation in endometrial epithelium could be significant as the endometrium is a significant site for viral entry and infection. Additionally, the cytokine milieu plays an essential role in normal functions of the endometrium such as uterine cycle progression, epithelial proliferation and shedding, and embryo implantation. In this study, we demonstrated cycle dependent expression of functional TLR3 in primary endometrial epithelial tissue and expression of intracellular TLR3 in human endometrial epithelial cell lines. We established that stimulation of TLR3-positive cell lines and primary human endometrial epithelial cells with dsRNA leads to TLR3-dependent expression of interleukin (IL)-6, IL-8, interferon (IFN)-inducible protein 10, RANTES, and IFN-β. These results indicate that the cytokine profile of human endometrial epithelial cells can be modified through TLR3 stimulation. Our findings suggest that TLR3 is involved in the immune responses of endometrial epithelial cells after exposure to dsRNA and has the potential to alter the cytokine milieu and influence the outcome and consequences of infection.

Objective. The U1-70-kd RNP is a prominent target of autoimmunity in connective tissue diseases. In this study, we explored whether its endogenous ligand, U1 RNA, mediates a proimmune signal and may be immunogenic.Methods. We assayed the proliferation of control and MyD88-knockout splenocytes in response to in vitro-synthesized U1 RNA, and measured interleukin-6 (IL-6) and IL-8 secretion induced by U1 RNA in a human cell line competent for signaling through Tolllike receptor 3 (TLR-3) and TLR-5. Conclusion. U1 RNA is capable of inducing manifestations consistent with TLR-3 activation. The ability of U1 RNA (which has a substantial double-stranded secondary structure) to activate TLR-3 may contribute to the immunogenicity of the U1-70-kd autoantigen.Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins.