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Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4[sup.-] and CD8[sup.-], but CD44[sup.+], CD25[sup.+] and cytoplasmic CD3[sup.+]. This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-[kappa]B. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics., Author(s): Hideki Hasegawa [1, 2, 8]; Hirofumi Sawa [3, 4, 8]; Martha J Lewis [2, 5]; Yasuko Orba [3]; Noreen Sheehy [2]; Yoshie Yamamoto [6]; Takeshi Ichinohe [1]; Yasuko Tsunetsugu-Yokota [...]

The primary function of the HIV-1 regulatory protein Tat, activation of transcription from the viral LTR, is highly regulated by complex interactions between Tat and a number of host cell proteins. Tat nuclear import, a process mediated by importin [beta], is a prerequisite for its activity. Here, we report and characterize the interaction of the human inhibitor of MyoD family domain-containing protein (I-mfa), HIC, with Tat at a biochemical and a functional level. This interaction was shown to occur in vivo and in vitro and to involve the nuclear localization signal and the transactivation responsive element-binding domains of Tat and the I-tufa domain of HIC. Coexpression of HIC and Tat resulted in the down-regulation of transactivation of the HIV-1 LTR, and colocalization studies revealed the cytoplasmic sequestration of Tat by HIC. Functionally this sequestration appears to be the underlying mechanism of LTR transcriptional repression by HIC and represents a unique mechanism for the control of Tat activity and regulation of HIV-1 replication. NLS | protein-protein interaction | nuclear import

Abstract Background In contrast with human T-cell leukemia virus type 1 (HTLV-1) that causes ATL (adult T-cell leukemia), HTLV-2 has not been causally linked to malignant disease. The minus strand of the HTLV genomes encode the regulatory proteins HTLV-1 bZIP factor (HBZ) for HTLV-1 and antisense protein of HTLV-2 (APH-2) for HTLV-2. Unlike the viral proteins Tax1 and Tax2, both HBZ and APH-2 are constitutively expressed in infected cells suggesting that they may play important roles in the pathogenesis of these viruses. To date, very little is known about the function of APH-2 except that it inhibits Tax2-mediated transcription of HTLV-2 genes. In the present study, we investigated the role of APH-2 in basal and Tax2B-mediated activation of the AP-1 pathway. Results We demonstrate that, unlike HBZ, APH-2 stimulates basal AP-1 transcription by interacting with c-Jun and JunB through its non-conventional bZIP domain. In addition, when Tax2 and APH-2 are co-expressed, they physically interact in vivo and in vitro and APH-2 acts as an inhibitor of Tax2-mediated activation of AP-1 transcription. Conclusions This report is the first to document that HTLV-2 can modulate the AP-1 pathway. Altogether our results reveal that, in contrast with HBZ, APH-2 regulates AP-1 activity in a Tax2-dependant manner. As the AP-1 pathway is involved in numerous cellular functions susceptible to affect the life cycle of the virus, these distinct biological properties between HBZ and APH-2 may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2.

Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

Abstract Background One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. Results Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. Conclusion We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.

Abstract Background The Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are transcriptional activators of both the viral long terminal repeat (LTR) and cellular promoters via the CREB and NFkB pathways. In contrast to HTLV-1, HTLV-2 has been classified into four distinct genetic subtypes A, B, C and D defined by phylogenetic analysis of their nucleotide sequences and the size and amino acid sequence of their Tax proteins. In the present study we have analysed and compared the transactivating activities of three Tax 2A and one Tax 2B proteins using LTR and NFkB reporter assays. Results We found that with the exception of the prototype Tax 2A Mo protein, the other two Tax 2A proteins failed to transactivate either the viral LTR or NFkB promoter in Jurkat and 293T cells. Loss of activity was not associated with either expression levels or an alteration in subcellular distribution as all Tax 2 proteins were predominantly located in the cytoplasm of transfected cells. Analysis of the sequence of the two inactive Tax 2A proteins relative to Mo indicated that one had six amino acid changes and the other had one change in the central region of the protein. Mutations present at the amino and the extreme carboxy termini of Mo resulted in the loss of LTR but not NFkB activation whereas those occurring in the central region of the protein appeared to abolish transactivation of both promoters. Analysis of the transactivation phenotypes of Tax 1, Tax 2A Mo and Tax 2B containing mutations identified in the present study or previously characterised Tax mutations showed that domains required for LTR and NFkB activation are very similar but not identical in all three Tax proteins. Conclusion Our results suggest that loss of activity of two Tax 2A proteins derived from different isolates is associated with multiple amino acid changes relative to Mo in domains required for the activation of the CREB or CREB and NFkB pathways and that these domains are very similar but not identical in Tax 2B and Tax 1. The loss of Tax function in 2A viruses may have implications for their biological and pathogenic properties.

Background: The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results: In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportinmediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions: Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy. [ABSTRACT FROM AUTHOR]

The primary function of the HIV-1 regulatory protein Tat, activation of transcription from the viral LTR, is highly regulated by complex interactions between Tat and a number of host cell proteins. Tat nuclear import, a process mediated by importin β, is a prerequisite for its activity. Here, we report and characterize the interaction of the human inhibitor of MyoD family domain-containing protein (l-mfa), HIC, with Tat at a biochemical and a functional level. This interaction was shown to occur in vivo and in vitro and to involve the nuclear localization signal and the transactivation responsive element-binding domains of Tat and the I-mfa domain of HIC. Coexpression of HIC and Tat resulted in the down-regulation of transactivation of the HIV-1 LTR, and colocalization studies revealed the cytoplasmic sequestration of Tat by HIC. Functionally this sequestration appears to be the underlying mechanism of LTR transcriptional repression by HIC and represents a unique mechanism for the control of Tat activity and regulation of HIV-1 replication. [ABSTRACT FROM AUTHOR]

The divergent clinical outcomes of human T cell leukemia virus type 1 (HTLV-1) and HTLV-2 infections have been attributed to functional differences in their antisense proteins. In contrast to HTLV-1 bZIP factor (HBZ), the role of the antisense protein of HTLV-2 (APH-2) in HTLV-2 infection is poorly understood. In previous studies, we identified the endosomal sorting complex required for transport 0 (ESCRT-0) subunit HRS as a novel interaction partner of APH-2 but not HBZ. HRS is a master regulator of endosomal protein sorting for lysosomal degradation and is hijacked by many viruses to promote replication. However, no studies to date have shown a link between HTLVs and HRS. In this study, we sought to characterize the interaction between HRS and APH-2 and to investigate the impact of HRS on the life cycle of HTLV-2. We confirmed a direct specific interaction between APH-2 and HRS and showed that the CC2 domain of HRS and the N-terminal domain of APH-2 mediate their interaction. We demonstrated that HRS recruits APH-2 to early endosomes, possibly furnishing an entry route into the endosomal/lysosomal pathway. We demonstrated that inhibition of this pathway using either bafilomycin or HRS overexpression substantially extends the half-life of APH-2 and stabilizes Tax2B expression levels. We found that HRS enhances Tax2B-mediated long terminal repeat (LTR) activation, while depletion of HRS enhances HTLV-2 production and release, indicating that HRS may have a negative impact on HTLV-2 replication. Overall, our study provides important new insights into the role of the ESCRT-0 HRS protein, and by extension the ESCRT machinery and the endosomal/lysosomal pathway, in HTLV-2 infection. [ABSTRACT FROM AUTHOR]

The endosomal sorting complex required for transport (ESCRT) pathway plays key roles in multivesicular bodies (MVBs) formation and lysosomal degradation of membrane receptors, viral budding, and midbody abscission during cytokinesis. The epidermal growth factor receptor (EGFR) is regarded as a prototypical cargo of the MVB/ESCRT pathway and following stimulation by epidermal growth factor (EGF) EGFR/EGF complexes are internalized, sorted into MVBs, and degraded by lysosomes or recycled back to the cell membrane. Here, we describe an assay to analyze the effect of human T-cell leukemia (HTLV) regulatory proteins on the functionality of ESCRT-dependent MVB/lysosomal trafficking of EGFR/EGF complexes. This is performed by direct visualization and quantification of the rate of EGF-Alexa595/EGFR internalization and degradation in HeLa cells expressing HTLV regulatory proteins by immunofluorescence and western blot.

The Tax protein encoded by HTLV-1 plays a key role in the development of ATL in infected individuals. Our previous studies showed that tax transgenic mice develop disease that is almost identical to human ATL, with widespread organ invasion by lymphomatous cells and the development of leukemia. The same pathology develops rapidly in SCID mice engrafted with cells from the transgenic animals. In the present study, we used this SCID model to analyze the expression levels of several cytokines, growth factors, and adhesion molecules to determine their possible involvement in the development of disease. We showed that Tax expression was undetectable at the protein level in the tax-transformed cells used to inoculate the SCID mice and that these cells displayed constitutive NF-kappaB and Akt activity. We demonstrated significant differences in the levels of circulating PDGF-BB, TNF-alpha, sICAM-1, and sVCAM-1 in inoculated animals. Cell-surface staining of the tax transgenic cells showed that they do not express receptors for any of the upregulated growth factors. Significant differences were not found in the secreted levels of bFGF, MMP9, VEGF, or E-selectin, whereas IL-2, IL-15, IL-6, IL-1beta, and IFN-gamma expression was undetectable. Even though the number of factors analyzed is limited, our study identified TNF-alpha, PDGF-BB, and the adhesion molecules sICAM-1 and sVCAM-1 as factors that may contribute to the high levels of organ infiltration by leukemic cells in this tax transgenic SCID model.