Antisense Protein Research Articles

Cuboplex-Mediated Nonviral Delivery of Functional siRNA to Chinese Hamster Ovary (CHO) Cells.

Lipid nanoparticles of internal cubic symmetry, termed cuboplexes, are potential nonviral delivery vehicles for gene therapy due to their "topologically active" nature, which may enhance endosomal escape and improve delivery outcomes. In this study, we have used cationic cuboplexes, based on monoolein (MO) doped with a cationic lipid, for the encapsulation and delivery of antisense green fluorescent protein (GFP)-small interfering RNA (siRNA) into Chinese Hamster Ovary (CHO)-GFP cells. Agarose gel electrophoresis has confirmed the successful encapsulation of siRNA within cationic cubosomes, while synchrotron small-angle X-ray scattering (SAXS) demonstrated that the underlying cubic nanostructure of the particles was retained following encapsulation. The cationic cubosomes were shown to be reasonably nontoxic against the CHO-GFP cell line. Fluorescence-activated cell sorting (FACS) provided evidence of the successful transfection to CHO-GFP cells. Knockdown efficiency was strongly linked to the type of cationic lipid used, although all cubosomes had essentially the same internal nanostructure. The gene knockdown efficiency for some cationic cubosomes was shown to be higher than lipofectamine, which is a commercially available liposome-based formulation, while the controlled release of the siRNA from the cubosomes over a 72 h period was observed using confocal microscopy. This combination exemplifies the potential of cationic cuboplexes as a novel, nonviral, controlled-release delivery vector for siRNA.

Radiosensitivity Effect of Epidermal Growth Factor Receptor Nanoparticles on Head and Neck Squamous Cell Carcinoma.

The effect of antisense epidermal growth factor receptor (EGFR) nanoparticles on the radiosensitivity of squamous cell carcinoma in mice was studied. Antisense EGFR oligonucleotide nanoparticles were introduced into the SCCVII cell line, and the expression of the antisense EGFR in the SCCVII cell line was detected using western blot analysis. After radiotherapy intervention, a cell cloning test and MTT test were used to detect the radiosensitivity of the cells, and flow cytometry was used to detect the cell cycle distribution and apoptosis to establish a model of head and neck cancer in mice. Antisense EGFR nanoparticles were injected into the tumors and treated with 4 Gy radiation therapy to observe their inhibition on the growth of tumors. SCCVII cell line nanoparticles could significantly inhibit the expression of antisense EGFR proteins. The combination of antisense EGFR nanoparticles and radiotherapy could reduce the differentiation and division ability of cancer cells (P < 0.05). It could arrest cancer cells in the G1 phase and significantly increase the apoptotic rate (P <0.05). The growth of tumors in the antisense EGFR nanoparticles group was significantly delayed. Antisense EGFR nanoparticles could induce G1 phase arrest by down-regulating the expression of EGFRs, which had a radiosensitization effect.

Are Antisense Proteins in Prokaryotes Functional?

Many prokaryotic RNAs are transcribed from loci outside of annotated protein coding genes. Across bacterial species hundreds of short open reading frames antisense to annotated genes show evidence of both transcription and translation, for instance in ribosome profiling data. Determining the functional fraction of these protein products awaits further research, including insights from studies of molecular interactions and detailed evolutionary analysis. There are multiple lines of evidence, however, that many of these newly discovered proteins are of use to the organism. Condition-specific phenotypes have been characterized for a few. These proteins should be added to genome annotations, and the methods for predicting them standardized. Evolutionary analysis of these typically young sequences also may provide important insights into gene evolution. This research should be prioritized for its exciting potential to uncover large numbers of novel proteins with extremely diverse potential practical uses, including applications in synthetic biology and responding to pathogens.

A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP) in HIV-1-Infected Patients.

The existence of an antisense Open Reading Frame (ORF) that encodes a putative AntiSense Protein (ASP) on the proviral genome of Human Immunodeficiency Virus type 1 (HIV-1) was a source of debate for 30 years. During the last years, some progresses have been made to characterize the cellular immune response against ASP in HIV-1 seropositive patients. However, no tools were available for the detection of antibodies to ASP in the plasma of HIV-1-infected patients during the natural course of the infection. The aim of our study was to develop a Luciferase Immuno-Precipitation System (LIPS) to monitor the quantitative detection of ASP-specific antibodies in the plasma of HIV-1-infected patients [antiretroviral therapy (ART) naive-patients, patients under ART and HIV-1 controllers], patients who discontinued antiretroviral drugs (ARV). We further used this approach to delineate the epitopes of ASP targeted by antibodies. Antibodies directed against ASP were detected in 3 out of 19 patients who discontinued ARV (15%) and in 1 out of 10 ART-naive patients (10%), but were neither detected in HIV-1 infected patients under ART nor in HIV-1 controllers. Individual variations in levels of ASP-specific antibodies were detected overtime. Both the conserved prolin-rich motif and the core 60–189 region of ASP were found to be essential for antibody recognition in the four patients tested positive for anti-ASP antibodies, who were all untreated at the time of sampling. Moreover, for two of these patients, increased levels of ASP-specific antibodies were observed concomitantly to viremia declines. Overall, our method may represent a useful tool to detect a humoral response to ASP in HIV-1-infected patients, which allowed us to confirm the expression of ASP during the course of HIV-1 infection. Further studies will be needed to fully characterize the humoral response to ASP in HIV-1-infected patients.

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR.

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3' end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

The HIV-1 Antisense Protein ASP Is a Transmembrane Protein of the Cell Surface and an Integral Protein of the Viral Envelope.

The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target.IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.

Novel Interactions between the Human T-Cell Leukemia Virus Type 1 Antisense Protein HBZ and the SWI/SNF Chromatin Remodeling Family: Implications for Viral Life Cycle.

The human T-cell leukemia virus type 1 (HTLV-1) regulatory proteins Tax and HBZ play indispensable roles in regulating viral and cellular gene expression. BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex, has been demonstrated to be essential not only for Tax transactivation but also for viral replication. We sought to investigate the physical interaction between HBZ and BRG1 and to determine the effect of these interactions on Tax-mediated long terminal repeat (LTR) activation. We reveal that HTLV-1 cell lines and adult T-cell leukemia (ATL) cells harbor high levels of BRG1. Using glutathione S-transferase (GST) pulldown and coimmunoprecipitation assays, we have demonstrated physical interactions between BRG1 and HBZ and characterized the protein domains involved. Moreover, we have identified the PBAF signature subunits BAF200 and BAF180 as novel interaction partners of HBZ, suggesting that the PBAF complex may be required for HTLV-1 transcriptional repression by HBZ. Additionally, we found that BRG1 expression translocates HBZ into distinct nuclear foci. We show that HBZ substantially represses HTLV-1 LTR activation by Tax/BRG1. Interestingly, we found that Tax stabilizes the expression of exogenous and endogenous BRG1 and that HBZ reverses this effect. Finally, using a chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay, we illustrate that HBZ facilitates the downregulation of HTLV-1 transcription by deregulating the recruitment of SWI/SNF complexes to the promoter. Overall, we conclude that SWI/SNF complexes, in addition to other cellular transcription factors, are involved in HBZ-mediated suppression of HTLV-1 viral gene expression.IMPORTANCE The pathogenic potential of HTLV-1 is linked to the indispensable multifaceted functions of the viral regulatory proteins Tax and HBZ, encoded by the sense and antisense viral transcripts, respectively. The interaction between Tax and the SWI/SNF family of chromatin remodeling complexes has been associated with HTLV-1 transcriptional activation. To date, the relationship between the SWI/SNF chromatin remodeling family and HBZ, the only viral protein that is consistently expressed in infected cells and ATL cells, has not been elucidated. Here, we have characterized the biological significance of the SWI/SNF family in regard to viral transcriptional repression by HBZ. This is important because it provides a better understanding of the function and role of HBZ in downregulating viral transcription and, hence, its contribution to viral latency and persistence in vivo, a process that may ultimately lead to the development of ATL.

TRAF3 Is Required for NF-κB Pathway Activation Mediated by HTLV Tax Proteins.

Human T-cell leukemia viruses type 1 (HTLV-1) and type 2 (HTLV-2) share a common genome organization and expression strategy but have distinct pathological properties. HTLV-1 is the etiological agent of Adult T-cell Leukemia (ATL) and of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), whereas HTLV-2 does not cause hematological disorders and is only sporadically associated with cases of subacute myelopathy. Both HTLV genomes encode two regulatory proteins that play a pivotal role in pathogenesis: the transactivating Tax-1 and Tax-2 proteins and the antisense proteins HBZ and APH-2, respectively. We recently reported that Tax-1 and Tax-2 form complexes with the TNF-receptor associated factor 3, TRAF3, a negative regulator of the non-canonical NF-κB pathway. The NF-κB pathway is constitutively activated by the Tax proteins, whereas it is inhibited by HBZ and APH-2. The antagonistic effects of Tax and antisense proteins on NF-κB activation have not yet been fully clarified. Here, we investigated the effect of TRAF3 interaction with HTLV regulatory proteins and in particular its consequence on the subcellular distribution of the effector p65/RelA protein. We demonstrated that Tax-1 and Tax-2 efficiency on NF-κB activation is impaired in TRAF3 deficient cells obtained by CRISPR/Cas9 editing. We also found that APH-2 is more effective than HBZ in preventing Tax-dependent NF-κB activation. We further observed that TRAF3 co-localizes with Tax-2 and APH-2 in cytoplasmic complexes together with NF-κB essential modulator NEMO and TAB2, differently from HBZ and TRAF3. These results contribute to untangle the mechanism of NF-κB inhibition by HBZ and APH-2, highlighting the different role of the HTLV-1 and HTLV-2 regulatory proteins in the NF-κB activation.

PJ-1 The HIV-1 antisense protein ASP is a structural protein of the viral envelope

Introduction: The antisense strand of the HIV-1 genome encodes a 189-aa, highly hydrophobic protein (ASP) with no known homologs. Humoral and cellular immune responses against ASP in HIV-1 infected individuals demonstrate its expression invivo. However, whether ASP plays a role in viral replication is still unknown. We studied the expression and subcellular localization of ASP in various chronically infected cell lines. In addition, we investigated the impact of ASP expression on viral fitness. Methods: For intracellular and nuclear stainings, we used BD Cytofix/Cytoperm and eBioscience FoxP3 kits. Flow cytometry data were acquired on a Millipore Guava and analyzed with FlowJo. Confocal and super-resolution microscopy images were acquired on Zeiss LSM 800 and Nikon N-STORMTi-E microscopes, and analyzed with Zen Blue and Nikon Elements Software Version 3.1. For virion-capture assays, antibodies against ASP and gp120 were immobilized on Protein G-dynabeads. For Fluorescence Correlation Spectroscopy (FCS) we used an ISS Q2 confocal microscope and the data were analyzed with ISS VistaVision. Results/Discussion: We generated an in-house monoclonal antibody 324.6 against an epitope located between 2 putative transmembrane domains in the ASP protein. Using this antibody in flow cytometry and confocal microscopy, we found thatASP is expressed in the nuclei of chronically infected myeloid (U1andOM-10.1), and lymphoid (H9-IIIB, H9-CC, H9-MN, H9-RF, ACH2 and 8E5) celllines, but not their parental uninfected lines. However, ASP was not detectable neither on the cell surface or in the cytoplasm of infected cell lines. ASP expression was higher in cells cultured at low density compared to cells cultured at high density, suggesting that the rate of cell division may influence ASP expression levels. Confocal microscopy showed a polarized nuclear distribution of ASP, preferentially in areas of the nucleus containing actively transcribed chromatin. However, after reactivation of HIV-1 expression with PMA, we found that ASP translocated to the cytoplasm and to the cell membrane of all infected cell lines. In addition, the ability to detect ASP on the cell surface without permeabilization indicates that the ASP epitope recognized by324.6 antibody is extracellular. Confocal microscopy showed that ASP and gp120 co-localize on the cell surface of productively infected cells, and we determined that the Manders overlap coefficient is 78%. Super-resolution microscopy analyses provided further evidence of ASP and gp-120 co-localization. These results suggest that ASP might be incorporated in the membrane of budding virions upon their release from infected cells. Indeed, the anti-ASP 324.6 antibody was able to capture HIV-1 particles with an efficiency similar to the anti-gp120 VRC01 antibody. Moreover, FCS showed that the binding efficiency of 324.6 antibody to single virions in solution was ∼28%. Altogether, these 2 assays demonstrate the presence of ASP on the surface of mature HIV-1 particles released from infected cells. To investigate whether ASP expression impacts viral replication, we mutated single nucleotides in the HIV-1NL4-3 genome thus introducing early stop codons in the ASP open reading frame without affecting the amino acid sequence of Env encoded on the opposite strand. We found that these ASP-deficient viruses displayed a ∼50% reduction in replication rate compared to wildtype virus. Conclusions: Our results indicate that ASP is expressed in the nuclei of non-productively infected cell lines. Upon viral reactivation, ASP translocates to the surface where it co-localizes with gp120. Moreover, ASP is present on the membrane of viral particles, indicating that ASP is a structural protein in the envelope of mature HIV-1 virions. Further, ASP expression promotes viral replication. Overall, our results suggest that ASP may represent a new target for therapeutic or preventive vaccine.

Detection of antisense protein (ASP) RNA transcripts in individuals infected with human immunodeficiency virus type 1 (HIV-1).

The detection of antisense RNA is hampered by reverse transcription (RT) non-specific priming, due to the ability of RNA secondary structures to prime RT in the absence of specific primers. The detection of antisense RNA by conventional RT-PCR does not allow assessment of the polarity of the initial RNA template, causing the amplification of non-specific cDNAs. In this study we have developed a modified protocol for the detection of human immunodeficiency virus type 1 (HIV-1) antisense protein (ASP) RNA. Using this approach, we have identified ASP transcripts in CD4+ T cells isolated from five HIV-infected individuals, either untreated or under suppressive therapy. We show that ASP RNA can be detected in stimulated CD4+ T cells from both groups of patients, but not in unstimulated cells. We also show that in untreated patients, the patterns of expression of ASP and env are very similar, with the levels of ASP RNA being markedly lower than those of env. Treatment of cells from one viraemic patient with α-amanitin greatly reduces the rate of ASP RNA synthesis, suggesting that it is associated with RNA polymerase II, the central enzyme in the transcription of protein-coding genes. Our data represent the first nucleotide sequences obtained in patients for ASP, demonstrating that its transcription indeed occurs in those HIV-1 lineages in which the ASP open reading frame is present.

HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62.

The existence of the antisense transcript-encoded HIV-1 antisense protein (ASP) was recently reinforced by in silico analyses providing evidence for recent appearance of this gene in the viral genome. Our previous studies led to the detection of ASP in various cell lines by Western blotting, flow cytometry, and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. Here, our goals were to assess autophagy induction by ASP from different clades and to identify the implicated autophagy factors. We first demonstrated that ASP formed multimers, partly through its amino-terminal region and cysteine residues. Removal of this region was further associated with lower induction of autophagy, as assessed by autophagosome formation. ASPs from different clades (A, B, C, D, and G) were tested next and were detected in monomeric and multimeric forms at various levels, and all induced autophagy (clade A ASP was less efficient), as determined by LC3-II and p62 (SQSTM1) levels. Furthermore, CRISPR-based knockout of ATG5, ATG7, and p62 genes led to increased ASP levels. Confocal microscopy analyses showed that ASP colocalized with p62 and LC3-II in autophagosome-like structures. Coimmunoprecipitation experiments further demonstrated that p62 associated with ASP through its PB1 domain. Interestingly, immunoprecipitation experiments supported the idea that ASP is ubiquitinated and that ubiquitination was modulating its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy, in which ubiquitin plays an important role. Understanding the mechanisms underlying ASP degradation is essential to better assess its function.IMPORTANCE In the present study, we provide the first evidence that a new HIV-1 protein termed ASP derived from different clades acts similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective cellular material. We have gained further knowledge on the mechanism mediating the activation of autophagy. Our studies have important ramifications in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated neurological disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies hence demonstrate the importance in conducting further studies on this protein as it could represent a new interesting target for antiretroviral therapies and vaccine design.

Expression analysis of AFAP1-AS1 and AFAP1 in breast cancer.

Long non-coding RNAs (lncRNA) constitute a significant percentage of RNAs with no translation to proteins. Their participation in fundamental aspects of cell physiology as well as their dysregulation in a number of pathologic conditions such as cancer have been documented. Among lncRNAs is actin filament associated protein 1 antisense RNA1 (AFAP1-AS1) whose elevated expression levels have been demonstrated in different cancers. In the in the present study we evaluated expression levels of AFAP1-AS1 and its antisense protein coding gene AFAP1 in breast cancer samples compare with adjacent non-cancerous tissues (ANCTs) as well as breast cancer cell lines with special focus on the assessment of the association between their transcript levels and patients' clinicopathological data. AFAP1-AS1 has shown significant up-regulation in both MDA-MB-231 and MCF-7 compared with control sample. AFAP1-AS1 has been shown to be expressed in all of tumor tissues but 76% (39 out of 51) ANCTs. AFAP1 expression was not significantly different between tumor samples and ANCTs. AFAP1-AS1 has been demonstrated to be significantly up-regulated in tumor tissues compared with ANCTs (fold change = 4.65, P= 0.028). No significant correlation has been detected between the levels of these two transcripts in tumor tissues (R=2 0.081) or ANCTs (R=2 0.115). No significant associations have been found between expression levels of these genes and patients' characteristics. However, both genes were significantly down-regulated in Ki-67 negative tumor samples. The observed up-regulation of AFAP1-AS1 in tumor samples compared with ANCTs implies its involvement in breast cancer pathogenesis and potentiates it as a biomarker or therapeutic target.

Repeat Problems: Combinatorial Effect of C9orf72‐Derived Dipeptide Repeat Proteins

The most common genetic cause of both Amyotrophic Lateral Sclerosis and Frontotemporal Dementia is a microsatellite expansion mutation in the 5′ UTR region of C9orf72. Expansion of the hexanucleotide repeat region of C9orf72 leads to loss of function, RNA foci, and five species of non‐AUG RAN translated dipeptide repeat proteins (GA, GP, GR, PA, &amp; PR). It has been observed that the sense and antisense dipeptide repeat proteins can be translated simultaneously in one cell, however the interplay between the different species has not been heavily investigated. Here we determined that the antisense repeat protein PR is localized to the nucleus, causes cytotoxicity, disrupts nuclear transport, and triggers PERK phosphorylation and p62 puncta accumulation. However, when PR and its sense partner GA were co‐expressed, PR's localization and cytotoxic outputs were significantly altered. In addition, we showed that GA and PR interact through direct binding that leads to structural and morphological changes. We also demonstrate the ability of dipeptide repeat protein species to be released from one cell and taken up by another. Importantly, we demonstrate that co‐expression of distinct DPRs enhances the release of individual peptides. Thus, the combined expression of distinct C9orf72 derived dipeptide repeat species produces cellular outcomes and structural differences that are unique compared to the expression of a single species.Support or Funding InformationNot fundedThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P-G2 Development of engineered T-cell immmunotherapy for treating Adult-T cell leukemia caused by HTLV-1

HTLV-1 is perhaps the most oncogenic virus in humans. Five percent to ten percent of individuals infected by HTLV-1 develop a fatal T-cell leukemia (ATL) after 2-3 decades of latency. Conventional chemotherapy only creates escape mutants in ATL. Novel treatments including retroviral drugs, Arsenic Trioxide/IFNa combination, or Allogeneic stem cell transplant have increased the 5-year survival, but there is still need to establish a cure. We chose an immunotherapy approach because all leukemic cells express some HTLV-1 viral proteins allowing to distinguish normal and HTLV-1 infected cells by the host immune system. In addition, anti-HTLV-1 immunity is suppressed in ATL patients. We aim to target HBZ (an antisense protein of HTLV-1) for this purpose because; 1) the frequency of T cells recognizing HBZ correlates well with low HTLV-1 proviral loads in contrast to T cells against Tax-1 or Env. 2) Unlike Tax-1, HBZ is constantly expressed by all stages of ATL cells. The challenge is that HBZ is an extremely weak immunogen. We identified a few epitopes of HBZ that stably find to class I HLA molecules. We tested them using an in vivo model of ATL in humanized mice. Immunization by HBZ showed little therapeutic effects in these mice. DC immunization showed prolonged survival, but did not protect mice from ATL. Thus, we grew anti-HBZ CD8 T cells by immunizing normal T cells ex vivo, enriched them by HBZ-tetramer sorting. T-cell therapy involving anti-HBZ CD8 T cells successfully protected host mice, prompting us to rely on an “engineered T cell” strategy as novel treatment of acute/lymphomatous ATL cases.

Stability of HTLV-2 antisense protein is controlled by PML nuclear bodies in a SUMO-dependent manner.

Since the identification of the antisense protein of HTLV-2 (APH-2) and the demonstration that APH-2 mRNA is expressed in vivo in most HTLV-2 carriers, much effort has been dedicated to the elucidation of similarities and/or differences between APH-2 and HBZ, the antisense protein of HTLV-1. Similar to HBZ, APH-2 negatively regulates HTLV-2 transcription. However, it does not promote cell proliferation. In contrast to HBZ, APH-2 half-life is very short. Here, we show that APH-2 is addressed to PML nuclear bodies in T-cells, as well as in different cell types. Covalent SUMOylation of APH-2 is readily detected, indicating that APH-2 might be addressed to the PML nuclear bodies in a SUMO-dependent manner. We further show that silencing of PML increases expression of APH-2, while expression of HBZ is unaffected. On the other hand, SUMO-1 overexpression leads to a specific loss of APH-2 expression that is restored upon proteasome inhibition. Furthermore, the carboxy-terminal LAGLL motif of APH-2 is responsible for both the targeting of the protein to PML nuclear bodies and its short half-life. Taken together, these observations indicate that natural APH-2 targeting to PML nuclear bodies induces proteasomal degradation of the viral protein in a SUMO-dependent manner. Hence, this study deciphers the molecular and cellular bases of APH-2 short half-life in comparison to HBZ and highlights key differences in the post-translational mechanisms that control the expression of both proteins.

HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4+/CD25+ T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins.

Erratum for Narulla et al., "Positive and Negative Regulation of Type I Interferons by the Human T Cell Leukemia Virus Antisense Protein HBZ".

Erratum for Narulla et al., "Positive and Negative Regulation of Type I Interferons by the Human T Cell Leukemia Virus Antisense Protein HBZ".

The expressions of Caveolin-2 mRNAs, natural antisense transcripts and proteins in mouse liver development

Caveolin-2 (Cav2) has been shown to be a major component of the inner surface of caveolae and be involved in lipid metabolism, development and cancer. Natural Antisense Transcripts (NATs) are transcribed from a large number of genes in various species, including humans and mice. In the present study, we investigated the expressions and localization of Cav2 mRNAs, NATs and proteins in the mouse liver at various developmental stages. The expressions of Cav2 mRNAs and proteins in the liver were higher at the new-born stage (NB) than at days 14, 17, and 19 of the embryonic stage (E) and at day 3 after birth. The expressions of Cav2 NATs were lower than that of Cav2 mRNAs at each stage. On tissues, the expressions of Cav2 mRNAs and NATs were uniformly low in the liver at E14. The expressions of Cav2 mRNAs were predominantly strong in the hepatocytes at NB, whereas the expressions of Cav2 NATs were low in the hepatocytes at NB but strongly detected in specific cells. In conclusions, Cav2 mRNAs and NATs did not co-localized in the liver at NB. These results suggest that Cav2 NATs may be not involved in the regulation of Cav2 mRNA expressions at NB.

Positive and Negative Regulation of Type I Interferons by the Human T Cell Leukemia Virus Antisense Protein HBZ.

The pathogenesis of human T cell leukemia virus type 1 (HTLV-1) is strongly linked to the viral regulatory proteins Tax1 and HBZ, whose opposing functions contribute to the clinical outcome of infection. Type I interferons alpha and beta (IFN-α and IFN-β) are key cytokines involved in innate immunity, and IFN-α, in combination with other antivirals, is extensively used in the treatment of HTLV-1 infection. The relationship between HTLV-1 and IFN signaling is unclear, and to date the effect of HBZ on this pathway has not been examined. Here we report that HBZ significantly enhances interferon regulatory factor 7 (IRF7)-induced IFN-α- and IFN-stimulated response element (ISRE) promoter activities and IFN-α production and can counteract the inhibitory effect of Tax1. In contrast to this, we show that HBZ and Tax1 cooperate to inhibit the induction of IFN-β and ISRE promoters by IRF3 and IFN-β production. In addition, we reveal that HBZ enhances ISRE activation by IFN-α. We further show that HBZ enhances IRF7 and suppresses IRF3 activation by TBK1 and IKKε. We demonstrate that HBZ has no effect on virus-induced nuclear accumulation of IRF3, suggesting that it may inhibit IRF3 activity at a transcriptional level. We show that HBZ physically interacts with IRF7 and IKKε but not with IRF3 or TBK1. Overall, our findings suggest that both HBZ and Tax1 are negative regulators of immediate early IFN-β innate immune responses, while HBZ but not Tax1 positively regulates the induction of IFN-α and downstream IFN-α signaling.IMPORTANCE Type I interferons are powerful antiviral cytokines and are used extensively in the treatment of HTLV-1-induced adult T cell leukemia (ATL). To date, the relationship between HTLV-1 and the IFN pathway is poorly understood, and studies so far have focused on Tax1. Our study is unique in that it examined the effect of HBZ, alone or in combination with Tax1, on type I IFN signaling. This is important because HBZ is frequently the only viral protein expressed in infected cells, particularly at later stages of infection. A better understanding of the how HBZ regulates IFN signaling may lead to the development of therapeutics that can modify such responses and improve the clinical outcome for infected individuals.