Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l ( ERVWE1 )/syncytin‐1 and endogenous retrovirus group FRD, member 1 ( ERVFRDE1 )/ syncytin‐2, encoded by human endogenous retrovirus (HERV) envelope ( env ) genes, are present at the surface of exosomes produced by placenta‐derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum‐derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead‐associated flow cytometry approach, and a syncytin‐2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome–cell interactions were compared between cells incubated in the presence of control exosomes, syncytin‐1 or syncytin‐2‐deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin‐1‐ and syncytin‐2‐dependent manner and that syncytin‐2 is reduced in serum‐derived exosomes from women with PE when compared to exosomes from normal pregnant women.—Vargas, A., Zhou, S., Éthier‐Chiasson, M., Flipo, D., Lafond, J., Gilbert, C., Barbeau, B. Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia. FASEB J. 28, 3703–3719 (2014). www.fasebj.org

Background: Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. Results: This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. Conclusion: These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.

Background: We and others have recently demonstrated that the human retrovirus HTLV-I was producing a spliced antisense transcript, which led to the synthesis of the HBZ protein. The objective of the present study was to demonstrate the existence of antisense transcription in HIV-1 and to provide a better characterization of the transcript and its regulation.Results: Initial experiments conducted by standard RT-PCR analysis in latently infected J1.1 cell line and pNL4.3-transfected 293T cells confirmed the existence of antisense transcription in HIV-1. A more adapted RT-PCR protocol with limited RT-PCR artefacts also led to a successful detection of antisense transcripts in several infected cell lines. RACE analyses demonstrated the existence of several transcription initiation sites mapping near the 5' border of the 3'LTR (in the antisense strand). Interestingly, a new polyA signal was identified on the antisense strand and harboured the polyA signal consensus sequence. Transfection experiments in 293T and Jurkat cells with an antisense luciferase-expressing NL4.3 proviral DNA showed luciferase reporter gene expression, which was further induced by various T-cell activators. In addition, the viral Tat protein was found to be a positive modulator of antisense transcription by transient and stable transfections of this proviral DNA construct. RT-PCR analyses in 293T cells stably transfected with a pNL4.3-derived construct further confirmed these results. Infection of 293T, Jurkat, SupT1, U937 and CEMT4 cells with pseudotyped virions produced from the antisense luciferase-expressing NL4.3 DNA clone led to the production of an AZT-sensitive luciferase signal, which was however less pronounced than the signal from NL4.3Luc-infected cells.Conclusion: These results demonstrate for the first time that antisense transcription exists in HIV-1 in the context of infection. Possible translation of the predicted antisense ORF in this transcript should thus be re-examined.

Human endogenous retroviruses (ERVs) represent 8% of the total human genome. Although the majority of these ancient proviral sequences have only retained non-coding long terminal repeats (LTRs), a number of “endogenized” retroviral genes encode functional proteins. Previous studies have underlined the implication of these ERV-derived proteins in the development and the function of the placenta. In this review, we summarize recent findings showing that two ERV genes, termed Syncytin-1 and Syncytin-2, which encode former envelope (Env) proteins, trigger fusion events between villous cytotrophoblasts and the peripheral multinucleated syncytiotrophoblast layer. Such fusion events maintain the stability of this latter cell structure, which plays an important role in fetal development by the active secretion of various soluble factors, gas exchange and regulation of fetomaternal immunotolerance. We also highlight new studies showing that these ERV proteins, in addition to their localization at the cell surface of cytotrophoblasts, are also incorporated on the surface of various extracellular microvesicles, including exosomes. Such exosome-associated proteins could be involved in the various functions attributed to these vesicles and could provide a form of tropism. Additionally, through their immunosuppressive domains, these ERV proteins could also contribute to fetomaternal immunotolerance in a local and more distal manner. These various aspects of the implication of Syncytin-1 and -2 in placental function are also addressed in the context of the placenta-related disorder, preeclampsia.

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) retroviruses infect T lymphocytes. The minus strand of the HTLV-1 genome encodes HBZ, a protein that could play a role in the development of leukemia in infected patients. Herein, we demonstrate that the complementary strand of the HTLV-2 genome also encodes a protein that we named APH-2 for “antisense protein of HTLV-2.” APH-2 mRNA is spliced, polyadenylated, and initiates in the 3′-long terminal repeat at different positions. This transcript was detected in all HTLV-2–infected cell lines and short-term culture of lymphocytes obtained from HTLV-2 African patients tested and in 4 of 15 HTLV-2–infected blood donors. The APH-2 protein is 183 amino acids long, is localized in the cell nucleus, and is detected in vivo. Despite the lack of a consensus basic leucine zipper domain, APH-2 interacts with cyclic adenosine monophosphate-response element binding protein (CREB) and represses Tax2-mediated transcription in Tax2-expressing cells and in cells transfected with an HTLV-2 molecular clone. Altogether, our results demonstrate the existence of an antisense strand–encoded protein in HTLV-2, which could represent an important player in the development of disorders, such as lymphocytosis, which is frequently observed in HTLV-2 patients.