NECAP 1 regulates AP-2 interactions to control vesicle size, number, and cargo during clathrin-mediated endocytosis.

AP-2 is the core-organizing element in clathrin-mediated endocytosis. During the formation of clathrin-coated vesicles, clathrin and endocytic accessory proteins interact with AP-2 in a temporally and spatially controlled manner, yet it remains elusive as to how these interactions are regulated. Here, we demonstrate that the endocytic protein NECAP 1, which binds to the α-ear of AP-2 through a C-terminal WxxF motif, uses an N-terminal PH-like domain to compete with clathrin for access to the AP-2 ß2-linker, revealing a means to allow AP-2-mediated coordination of accessory protein recruitment and clathrin polymerization at sites of vesicle formation. Knockdown and functional rescue studies demonstrate that through these interactions, NECAP 1 and AP-2 cooperate to increase the probability of clathrin-coated vesicle formation and to control the number, size, and cargo content of the vesicles. Together, our data demonstrate that NECAP 1 modulates the AP-2 interactome and reveal a new layer of organizational control within the endocytic machinery.

Detection of the HIV-1 minus-strand-encoded antisense protein and its association with autophagy.

HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also led to the detection of optimized ASP in transfected cells but also provided evidence of its instability and high multimerization potential. ASP was mainly distributed in the cytoplasm in a punctate manner, which was reminiscent of autophagosomes. In agreement with this observation, a significant increase in ASP-positive cells and loss of its punctate distribution was observed in transfected cells when autophagy was inhibited at early steps. Induction of autophagy was confirmed by Western blot analyses that showed an ASP-mediated increase in levels of LC3b-II and Beclin 1, as well as colocalization and interaction between ASP and LC3. Interestingly, Myc-tagged ASP was detected in the context of proviral DNA following autophagy inhibition with a concomitant increase in the level and punctate distribution of LC3b-II. Finally, 3-methyladenine treatment of transfected or infected U937 cells decreased extracellular p24 levels in wild-type proviral DNA and to a much lesser extent in ASP-mutated proviral DNA. This study provides the first detection of ASP in mammalian cells by Western blotting. ASP-induced autophagy might explain the inherent difficulty in detecting this viral protein and might justify its presumed low abundance in infected cells.

Human T-cell lymphotropic virus type 3 (HTLV-3)- and HTLV-4-derived antisense transcripts encode proteins with similar Tax-inhibiting functions but distinct subcellular localization.

The human T-cell lymphotropic virus (HTLV) retrovirus family is composed of the well-known HTLV type 1 (HTLV-1) and HTLV-2 and the most recently discovered HTLV-3 and HTLV-4. Like other retroviruses, HTLV-1 and HTLV-2 gene expression has been thought to be orchestrated through a single transcript. However, recent reports have demonstrated the unique potential of both HTLV-1 and HTLV-2 to produce an antisense transcript. Furthermore, these unexpected and newly identified transcripts lead to the synthesis of viral proteins termed HBZ (HTLV-1 basic leucine zipper) and APH-2 (antisense protein of HTLV-2), respectively. As potential open reading frames are present on the antisense strand of HTLV-3 and HTLV-4, we tested whether in vitro antisense transcription occurred in these viruses and whether these transcripts had a coding potential. Using HTLV-3 and HTLV-4 proviral DNA constructs, antisense transcripts were detected by reverse transcriptase PCR. These transcripts are spliced and polyadenylated and initiate at multiple sites from the 3' long terminal repeat (LTR). The resulting proteins, termed APH-3 and APH-4, are devoid of a typical basic leucine zipper domain but contain basic amino acid-rich regions. Confocal microscopy and Western blotting experiments demonstrated a nucleus-restricted pattern for APH-4, while APH-3 was localized both in the cytoplasm and in the nucleus. Both proteins showed partial colocalization with nucleoli and HBZ-associated structures. Finally, both proteins inhibited Tax1- and Tax3-mediated HTLV-1 and HTLV-3 LTR activation. These results further demonstrate that retroviral antisense transcription is not exclusive to HTLV-1 and HTLV-2 and that APH-3 and APH-4 could impact HTLV-3 and HTLV-4 replication.

Human T-cell leukemia virus type 2 produces a spliced antisense transcript encoding a protein that lacks a classic bZIP domain but still inhibits Tax2-mediated transcription.

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.

Upregulation of human T-cell leukemia virus type 1 antisense transcription by the viral tax protein.

Several studies have recently demonstrated the existence of human T-cell leukemia virus type 1 (HTLV-1) antisense transcripts, which allow the synthesis of the newly described HBZ protein. Although previous reports have been aimed at understanding the potential role of the HBZ protein in HTLV-1 pathogenesis, little is known as to how this viral gene is regulated. Here, using our K30-3'asLuc reporter construct, we show that the viral Tax protein upregulates antisense transcription through its action on the TRE sequences located in the 3' long terminal repeat. Generation of stable clones in 293T cells demonstrated that Tax-induced HBZ expression is importantly influenced by the integration site in the host genome. The cellular DNA context could thus affect the level of HBZ mRNA expression in infected cells.

Detection, characterization and regulation of antisense transcripts in HIV-1.

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.