Sébastien Alain Chevalier

Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the hosts miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.

NRP/Optineurin Cooperates with TAX1BP1 to potentiate the activation of NF-kappaB by human T-lymphotropic virus type 1 tax protein.

Nuclear factor (NF)-κB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-κB occurs predominantly in the cytoplasm, where Tax1 binds NF-κB Essential Modulator (NEMO/IKKγ) and triggers the activation of IκB kinases. Several independent studies have shown that Tax1-mediated NF-κB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-κB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-κB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-κB activation.

Human T-Cell Lymphotropic Virus Type 3: Complete Nucleotide Sequence and Characterization of the Human Tax3 Protein

ABSTRACT We and others have recently uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3), the third member of the HTLV family. We have now sequenced the full-length HTLV-3Pyl43 provirus. As expected, HTLV-3Pyl43 contains open reading frames corresponding to the gag, pol, env, tax, and rex genes. Interestingly, its long terminal repeat (LTR) includes only two Tax-responsive elements, as is the case for type 3 simian T-cell lymphotropic viruses (STLV-3). Phylogenetic analyses reveal that HTLV-3Pyl43 is closely related to central African STLV-3. Unexpectedly, the proximal pX region of HTLV-3Pyl43 lacks 366 bp compared to its STLV-3 counterpart. Because of this deletion, the previously described RorfII sequence is lacking. At the amino acid level, Tax3Pyl43 displays strong similarities with HTLV-1 Tax, including the sequence of a PDZ class I binding motif. In transient-transfection assays, Tax3Pyl43 activates the transcriptions from HTLV-3, HTLV-1, and HTLV-2 LTRs. Mutational analysis indicates that two functional domains (M22 and M47) important for transactivation through the CREB/ATF or NF-κB pathway are similar but not identical in Tax1 and Tax3Pyl43. We also show that Tax3Pyl43 transactivates the human interleukin-8 and Bcl-XL promoters through the induction of the NF-κB pathway. On the other hand, Tax3Pyl43 represses the transcriptional activity of the p53 tumor suppressor protein as well as the c-Myb promoter. Altogether, these results demonstrate that although HTLV-3 and HTLV-1 have only 60% identity, Tax3Pyl43 is functionally closely related to the transforming protein Tax1 and suggest that HTLV-3, like HTLV-1, might be pathogenic in vivo.

Direct Detection of Diverse Metabolic Changes in Virally Transformed and Tax-Expressing Cells by Mass Spectrometry

Background Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells. Methods and Principal Findings Here, we report the detection of key changes in metabolites and lipids directly from human T-lymphotropic virus type 1 and type 3 (HTLV1 and HTLV3) transformed, as well as Tax1 and Tax3 expressing cell lines by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Comparing LAESI-MS spectra of non-HTLV1 transformed and HTLV1 transformed cells revealed that glycerophosphocholine (PC) lipid components were dominant in the non-HTLV1 transformed cells, and PC(O-32∶1) and PC(O-34∶1) plasmalogens were displaced by PC(30∶0) and PC(32∶0) species in the HTLV1 transformed cells. In HTLV1 transformed cells, choline, phosphocholine, spermine and glutathione, among others, were downregulated, whereas creatine, dopamine, arginine and AMP were present at higher levels. When comparing metabolite levels between HTLV3 and Tax3 transfected 293T cells, there were a number of common changes observed, including decreased choline, phosphocholine, spermine, homovanillic acid, and glycerophosphocholine and increased spermidine and N-acetyl aspartic acid. These results indicate that the lipid metabolism pathway as well as the creatine and polyamine biosynthesis pathways are commonly deregulated after expression of HTLV3 and Tax3, indicating that the noted changes are likely due to Tax3 expression. N-acetyl aspartic acid is a novel metabolite that is upregulated in all cell types and all conditions tested. Conclusions and Significance We demonstrate the high throughput in situ metabolite profiling of HTLV transformed and Tax expressing cells, which facilitates the identification of virus-induced perturbations in the biochemical processes of the host cells. We found virus type-specific (HTLV1 vs. HTLV3), expression-specific (Tax1 vs. Tax3) and cell-type–specific (T lymphocytes vs. kidney epithelial cells) changes in the metabolite profiles. The new insight on the affected metabolic pathways can be used to better understand the molecular mechanisms of HTLV induced transformation, which in turn can result in new treatment strategies.

The Transcription Profile of Tax-3 Is More Similar to Tax-1 than Tax-2: Insights into HTLV-3 Potential Leukemogenic Properties

Human T-cell Lymphotropic Viruses type 1 (HTLV-1) is the etiological agent of Adult T-cell Leukemia/Lymphoma. Although associated with lymphocytosis, HTLV-2 infection is not associated with any malignant hematological disease. Similarly, no infection-related symptom has been detected in HTLV-3-infected individuals studied so far. Differences in individual Tax transcriptional activity might account for these distinct physiopathological outcomes. Tax-1 and Tax-3 possess a PDZ binding motif in their sequence. Interestingly, this motif, which is critical for Tax-1 transforming activity, is absent from Tax-2. We used the DNA microarray technology to analyze and compare the global gene expression profiles of different T- and non T-cell types expressing Tax-1, Tax-2 or Tax-3 viral transactivators. In a T-cell line, this analysis allowed us to identify 48 genes whose expression is commonly affected by all Tax proteins and are hence characteristic of the HTLV infection, independently of the virus type. Importantly, we also identified a subset of genes (n = 70) which are specifically up-regulated by Tax-1 and Tax-3, while Tax-1 and Tax-2 shared only 1 gene and Tax-2 and Tax-3 shared 8 genes. These results demonstrate that Tax-3 and Tax-1 are closely related in terms of cellular gene deregulation. Analysis of the molecular interactions existing between those Tax-1/Tax-3 deregulated genes then allowed us to highlight biological networks of genes characteristic of HTLV-1 and HTLV-3 infection. The majority of those up-regulated genes are functionally linked in biological processes characteristic of HTLV-1-infected T-cells expressing Tax such as regulation of transcription and apoptosis, activation of the NF-κB cascade, T-cell mediated immunity and induction of cell proliferation and differentiation. In conclusion, our results demonstrate for the first time that, in T- and non T-cells types, Tax-3 is a functional analogue of Tax-1 in terms of transcriptional activation and suggest that HTLV-3 might share pathogenic features with HTLV-1 in vivo.

Alpha Interferon Restricts Human T-Lymphotropic Virus Type 1 and 2 De Novo Infection through PKR Activation

ABSTRACT Type I interferon (IFN-I) inhibits the replication of different viruses. However, the effect of IFN-I on the human T-lymphotropic virus type 1 (HTLV-1) viral cycle is controversial. Here, we investigated the consequences of IFN-α addition for different steps of HTLV-1 and HTLV-2 infection. We first show that alpha interferon (IFN-α) efficiently impairs HTLV-1 and HTLV-2 de novo infection in a T cell line and in primary lymphocytes. Using pseudotyped viruses expressing HTLV-1 envelope, we then show that cell-free infection is insensitive to IFN-α, demonstrating that the cytokine does not affect the early stages of the viral cycle. In contrast, intracellular levels of Gag, Env, or Tax protein are affected by IFN-α treatment in T cells, primary lymphocytes, or 293T cells transfected with HTLV-1 or HTLV-2 molecular clones, demonstrating that IFN-α acts during the late stages of infection. We show that IFN-α does not affect Tax-mediated transcription and acts at a posttranscriptional level. Using either small interfering RNA (siRNA) directed against PKR or a PKR inhibitor, we demonstrate that PKR, whose expression is induced by interferon, plays a major role in IFN-α-induced HTLV-1/2 inhibition. These results indicate that IFN-α has a strong repressive effect on the HTLV-1 and HTLV-2 viral cycle during de novo infection of cells that are natural targets of the viruses.

ADAR1 enhances HTLV-1 and HTLV-2 replication through inhibition of PKR activity

BackgroundThe role of innate immunity in general and of type I interferon (IFN-I) in particular in HTLV-1 pathogenesis is still a matter of debate. ADAR1-p150 is an Interferon Stimulated Gene (ISG) induced by IFN-I that can edit viral RNAs. We therefore investigated whether it could play the role of an anti-HTLV factor.ResultsWe demonstrate here that ADAR1 is also expressed in the absence of IFN stimulation in activated primary T-lymphocytes that are the natural target of this virus and in HTLV-1 or HTLV-2 chronically infected T-cells. ADAR1 expression is also increased in primary lymphocytes obtained from HTLV-1 infected individuals. We show that ADAR1 enhances HTLV-1 and HTLV-2 infection in T-lymphocytes and that this proviral effect is independent from its editing activity. ADAR1 expression suppresses IFN-α inhibitory effect on HTLV-1 and HTLV-2 and acts through the repression of PKR phosphorylation.DiscussionThis study demonstrates that two interferon stimulated genes, i.e. PKR and ADAR1 have opposite effects on HTLV replication in vivo. The balanced expression of those proteins could determine the fate of the viral cycle in the course of infection.

Construction and Characterization of a Full-Length Infectious Simian T-Cell Lymphotropic Virus Type 3 Molecular Clone

ABSTRACT Together with their simian T-cell lymphotropic virus (STLV) equivalent, human T-cell lymphotropic virus type 1 (HTLV-1), HTLV-2, and HTLV-3 form the primate T-cell lymphotropic virus (PTLV) group. Over the years, understanding the biology and pathogenesis of HTLV-1 and HTLV-2 has been widely improved by the creation of molecular clones. In contrast, so far, PTLV-3 experimental studies have been restricted to the overexpression of the tax gene using reporter assays. We have therefore decided to construct an STLV-3 molecular clone. We generated a full-length STLV-3 proviral clone (8,891 bp) by PCR amplification of overlapping fragments. This STLV-3 molecular clone was then transfected into 293T cells. Reverse transcriptase PCR experiments followed by sequence analysis of the amplified products allowed us to establish that both gag and tax/rex mRNAs were transcribed. Western blotting further demonstrated the presence of the STLV-3 p24gag protein in the cell culture supernatant from transfected cells. Transient transfection of 293T cells and of 293T-long terminal repeat-green fluorescent protein cells with the STLV-3 clone promoted syncytium formation, a hallmark of PTLV Env expression, as well as the appearance of fluorescent cells, also demonstrating that the Tax3 protein was expressed. Virus particles were visible by electron microscopy. These particles are infectious, as demonstrated by our cell-free-infection experiments with purified virions. All together, our data demonstrate that the STLV-3 molecular clone is functional and infectious. This clone will give us a unique opportunity to study in vitro the different pX transcripts and the putative presence of antisense transcripts and to evaluate the PTLV-3 pathogenicity in vivo.

Construction and Characterization of a Human T-Cell Lymphotropic Virus Type 3 Infectious Molecular Clone

ABSTRACT We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24gag protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

Human T-Cell Leukemia/Lymphoma Virus Type 1 p30, but Not p12/p8, Counteracts Toll-Like Receptor 3 (TLR3) and TLR4 Signaling in Human Monocytes and Dendritic Cells

ABSTRACT The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p30 protein, essential for virus infectivity in vivo, is required for efficient infection of human dendritic cells (DCs) but not B and T cells in vitro. We used a human monocytic cell line, THP-1, and dendritic cells to study the mechanism of p30 and p12/p8 requirements in these cell types. p30 inhibited the expression of interferon (IFN)-responsive genes (ISG) following stimulation by lipopolysaccharide (LPS) of Toll-like receptor 4 (TLR4) and by poly(I·C) of TLR3 but not of TLR7/8 with imiquimod. Results with THP-1 mirrored those for ex vivo human primary monocytes and monocyte-derived dendritic cells (Mo-mDC). The effect of p30 on TLR signaling was also demonstrated by ablating its expression within a molecular clone of HTLV-1. HTLV-1 infection of monocytes inhibited TLR3- and TLR4-induced ISG expression by 50 to 90% depending on the genes, whereas the isogenic clone p30 knockout virus was less effective at inhibiting TLR3 and TRL4 signaling and displayed lower infectivity. Viral expression and inhibition of ISG transcription was, however, rescued by restoration of p30 expression. A chromatin immunoprecipitation assay demonstrated that p30 inhibits initiation and elongation of PU.1-dependent transcription of IFN-α1, IFN-β, and TLR4 genes upon TLR stimulation. In contrast, experiments conducted with p12/p8 did not demonstrate an effect on ISG expression. These results provide a mechanistic explanation of the requirement of p30 for HTLV-1 infectivity in vivo, suggest that dampening interferon responses in monocytes and DCs is specific for p30, and represent an essential early step for permissive HTLV-1 infection and persistence.