[Show abstract][Hide abstract] ABSTRACT: Objective:The identification of still unrevealed mechanisms affecting the anti-HIV CD8(+) T-cell response in HIV-1 infection.Design:Starting from the observation that anti-Tat immunization is associated with improved CD8(+) T-cell immunity, we developed both in-vitro and ex-vivo assays to characterize the effects of extra-cellular Tat on the adaptive CD8(+) T-cell response.Methods:The effects of Tat on CD8(+) T-cell activation were assayed using CD8(+) T-cell clones specific for either cellular (MART-1) or viral (HIV-1 Nef) antigens, and HIV-1 Gag-specific CD8(+) T cells from HIV-1 patients.Results:The interaction between CD8(+) T lymphocytes and immobilized Tat, but not its soluble form, inhibits peptide-specific CD8(+) T-lymphocyte activation. The inhibition does not depend on Tat trans-activation activity, but on the interaction of the Tat RGD domain with 51 and v3 integrins. Impaired CD8(+) T-cell activation was also observed in cocultures of CD8(+) T cells with HIV-1-infected cells. Anti-Tat Abs abrogate the inhibitory effect, consistently with the evidence that extracellular Tat accumulates on the cell membrane of virus-producing cells. The Tat-induced inhibition of cell activation associates with increased apoptosis of CD8(+) T cells. Finally, the inhibition of cell activation also takes place in Gag-specific CD8(+) T lymphocytes from HIV-1-infected patients.Conclusion:Our results support the idea that CD8(+) T-cell apoptosis induced by surface-bound extracellular Tat can contribute to the dysregulation of the CD8(+) T-cell adaptive response against HIV as well as other pathogens present in AIDS patients.
AIDS 07/2014; 28(15). DOI:10.1097/QAD.0000000000000389 · 5.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs) via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs). Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.
PLoS ONE 11/2012; 7(11):e48781. DOI:10.1371/journal.pone.0048781 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retroviral transduction of cells is improved upon virus adsorption onto immobilized fibronectin (FN) fragments. Because HIV-1 Tat possesses the same functional domains that lead to increased transduction efficiency in FN by colocalization of bound virus and cells, we hypothesized that Tat could enhance gene transfer by a similar mechanism.
Single-cycle replication retro- or lentivirus carrying green fluorescent protein or cloramphenicol acetyltransferase as reporter genes were added to wells coated with Tat or Tat peptides. Wells were extensively washed to remove unbound virus and levels of transduction were detected by measuring reporter gene expression. Virus adsorption to immobilized Tat was measured using a p24 antigen capture assay.
Immobilized Tat efficiently binds retro- and lentiviral particles and mediates virus transmission at virus input doses that were otherwise unable to transduce susceptible cells. Virus adsorption to Tat is not mediated by envelope glycoprotein (Env) because immobilized Tat binds and retains vesicular stomatitis virus G (VSV-G) pseudotypes as well as envelope-free particles. HIV-1 Env or VSV-G are required for Tat-assisted transduction, which is abrogated by an antibody blocking the HIV-1 Env-CD4 interaction. Tat-assisted transduction is mediated by the cysteine-rich region of Tat, which is known to be essential for Tat transactivation activity. However, Tat transactivation is not required for Tat-assisted transduction, as indicated by the enhancement of transduction by transactivation-silent Tat mutants.
Immobilized Tat promotes virus transduction by a transactiva- tion-independent mechanism, which requires binding of virus to Tat. Recombinant Tat or Tat fragments provide a new method to increase efficiency of retro- and lentiviral based gene transfer and gene therapy.
The Journal of Gene Medicine 11/2009; 11(11):955-65. DOI:10.1002/jgm.1381 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (approximately 20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and beta-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-alpha gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-alpha production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.
The Journal of Immunology 04/2009; 182(5):2888-97. DOI:10.4049/jimmunol.0711406 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary effusion lymphomas (PELs) are invariably infected by the human herpesvirus 8 (HHV8)that is present in most PEL cells as latent virus but replicates in a subset of permissive cells to produce infectious progeny. Here we show that productively infected PEL cells release C-type retrovirus-like particles encoding an Mn++-dependent RT activity, which is typical of endogenous retroviruses. Strikingly, C-type particles are produced only in cells showing advanced HHV8 morphogenesis. Phorbol esters, which induce productive HHV8 replication and morphogenesis in PEL cells, increase RLP production. Phosphonoacetic acid, a blocker of HHV8 late gene expression, inhibits the production of C-type particles, whereas neutralizing anti-alphaIFN antibodies, which are known to increase HHV8 assembly, increases C-type particle production. These data suggest that factors expressed in advanced stages of HHV8 reactivation support endogenous C-type particle morphogenesis in PEL cells.
International journal of immunopathology and pharmacology 10/2008; 21(4):999-1006. · 1.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a cyclopentenonic prostaglandin endowed with powerful anti-inflammatory activities, as shown in animal models of inflammatory/autoimmune diseases, where pharmacological administration of this prostanoid can ameliorate inflammation and local tissue damage via activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and/or covalent modifications of cellular proteins. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily expressed in most of the cells, including those of immune system such as T lymphocytes, in which it is up-regulated upon antigen-specific stimulation. This cytokine plays an important role in regulating various physiological and immunopathological processes, such as immunosurveillance of tumors and tissue destruction associated with different inflammatory and autoimmune diseases. Here, we demonstrate that 15d-PGJ(2) inhibits trail mRNA and protein expression by down-regulating the activity of its promoter in human T lymphocytes. Our data indicate that both the chemically reactive cyclopentenone moiety of 15d-PGJ(2) and the activation of PPARgamma may be involved in this repressive mechanism. We identified nuclear factor kappaB (NF-kappaB) as a direct target of the prostanoid. 15d-PGJ(2) significantly decreases the expression and/or DNA binding of c-rel, RelA, and p50 transcription factors to the NF-kappaB1 site of trail promoter. Moreover, 15d-PGJ(2)-mediated activation of the transcription factor heat shock factor-1 may contribute to inhibit trail promoter activity in transfected Jurkat T cells. These results suggest that modulation of TRAIL gene expression by 15d-PGJ(2) in T cells may provide a novel pharmacological tool to modify the onset and the progression of specific autoimmune and inflammatory disorders.
[Show abstract][Hide abstract] ABSTRACT: Receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK are cell surface proteins abundantly expressed in bone and lymphoid tissues, whose interaction triggers different signaling pathways leading to activation and differentiation of osteoclasts, pivotal actors of the normal bone remodeling cycle. Moreover, RANKL may act as an immunomodulator, representing an important dendritic cell survival factor produced by activated T cells. A large body of research has shown that not only does the RANKL/RANK system regulate the physiology of bone development but also plays an important pathological role in bone destruction mediated by inflammatory disorders or bone metastatic tumors. 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a cyclopentenone-type PG endowed with anti-inflammatory properties and produced by different cells, including those of the immune system. Although 15d-PGJ(2) has been studied as a natural ligand of the peroxisome proliferator-activated receptor-gamma nuclear receptor, relevant peroxisome proliferator-activated receptor-gamma-independent actions mediated by this prostanoid have been described. In this study, we describe the effect of 15d-PGJ(2) on the expression of the rankl gene in T lymphocytes. We show that 15d-PGJ(2) inhibits rankl mRNA expression, protein, and rankl promoter activity by mechanisms mediated by its chemically reactive cyclopentenone moiety. Our data also indicate that 15d-PGJ(2) represses rankl activation by interfering with the expression and/or activity of the transcription factors NF-kappaB, early growth response-2, and early growth response-3, whose altered balancing and transactivation may contribute for the repression of this gene. These results place rankl as a novel molecular target for the different immunoregulatory activities mediated by 15d-PGJ(2). The physiological and pharmacological implications of these observations are discussed.
The Journal of Immunology 05/2007; 178(7):4039-50. DOI:10.4049/jimmunol.178.7.4039 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Tat protein is the transcriptional activator of HIV-1 gene expression, which is not only essential for viral replication, but also important in the complex HIV-induced pathogenesis of AIDS, as both an intracellular and an extracellular released protein. Accordingly, Tat is able to profoundly affect cellular gene expression, regulating several cellular functions, also in non-infected cells. We showed recently that Tat induces modification of immunoproteasomes in that it up-regulates LMP7 (low-molecular-mass polypeptide 7) and MECL1 (multicatalytic endopeptidase complex-like 1) subunits and down-modulates the LMP2 subunit, resulting in a change in the generation and presentation of epitopes in the context of MHC class I. In particular, Tat increases presentation of subdominant and cryptic epitopes. In the present study, we investigated the molecular mechanism responsible for the Tat-induced LMP2 down-regulation and show that intracellular Tat represses transcription of the LMP2 gene by competing with STAT1 (signal transducer and activator of transcription 1) for binding to IRF-1 (interferon-regulatory factor-1) on the overlapping ICS-2 (interferon consensus sequence-2)-GAS (gamma-interferon-activated sequence) present in the LMP2 promoter. This element is constitutively occupied in vivo by the unphosphorylated STAT1-IRF-1 complex, which is responsible for the basal transcription of the gene. Sequestration of IRF-1 by intracellular Tat impairs the formation of the complex resulting in lower LMP2 gene transcription and LMP2 protein expression, which is associated with increased proteolytic activity. On the other hand, extracellular Tat induces the expression of LMP2. These effects of Tat provide another effective mechanism by which HIV-1 affects antigen presentation in the context of the MHC class I complex and may have important implications in the use of Tat for vaccination strategies.
[Show abstract][Hide abstract] ABSTRACT: Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.
Molecular Biology of the Cell 05/2006; 17(4):1985-94. DOI:10.1091/mbc.E05-08-0717 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.
[Show abstract][Hide abstract] ABSTRACT: A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophage-activating lipopeptide-2 (MALP-2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tat-specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-gamma-producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.
European Journal of Immunology 07/2003; 33(6):1548-56. DOI:10.1002/eji.200323954 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.
DNA and Cell Biology 10/2002; 21(9):599-610. DOI:10.1089/104454902760330138 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transcription of the human immunodeficiency virus (HIV)-1 is controlled by the cooperation of virally encoded and host regulatory proteins. The Tat protein is essential for viral replication, however, expression of Tat after virus entry requires HIV-1 promoter activation. A sequence in the 5' HIV-1 LTR, containing a binding site for transcription factors of the interferon regulatory factors (IRF) family has been suggested to be critical for HIV-1 transcription and replication. Here we show that IRF-1 activates HIV-1 LTR transcription in a dose-dependent fashion and in the absence of Tat. This has biological significance since IRF-1 is produced early upon virus entry, both in cell lines and in primary CD4+ T cells, and before expression of Tat. IRF-1 also cooperates with Tat in amplifying virus gene transcription and replication. This cooperation depends upon a physical interaction that is blocked by overexpression of IRF-8, the natural repressor of IRF-1, and, in turn is released by overexpression of IRF-1. These data suggest a key role of IRF-1 in the early phase of viral replication and/or during viral reactivation from latency, when viral transactivators are absent or present at very low levels, and suggest that the interplay between IRF-1 and IRF-8 may play a key role in virus latency.
Journal of Experimental Medicine 06/2002; 195(10):1359-70. · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaccination of cynomolgus monkeys with the biologically active HIV-1 Tat protein induces specific Th1 responses, including CTLs. Similar responses are also induced by vaccination with tat DNA, but not by vaccination with inactivated Tat or Tat peptides. This suggested that the native Tat protein may act differently on APC as compared with inactivated Tat or peptide Ag. In this study, we show that biologically active Tat is very efficiently taken up by monocyte-derived dendritic cells (MDDC) in a time (within minutes)- and dose-dependent (starting from 0.1 ng/ml) fashion, whereas uptake is very poor or absent with other APC, including T cell blasts and B lymphoblastoid cell lines. Although maturation of MDDC reduces their pino/phagocytic activity, mature MDDC take up Tat much more efficiently than immature cells. In addition, Tat uptake is abolished or greatly hampered by oxidation/inactivation of the protein or by performing the experiments at 4 degrees C, suggesting that MDDC take up native Tat by a receptor-mediated endocytosis. After uptake, active Tat protein induces up-regulation of MHC and costimulatory molecules and production of IL-12, TNF-alpha, and beta chemokines, which drive Th1-type immune response. In contrast, these effects are lost by oxidation and inactivation of the protein. Finally, native Tat enhances Ag presentation by MDDC, increasing Ag-specific T cell responses. These data indicate that native Tat selectively targets MDDC, is taken up by these cells via specialized pathways, and promotes their maturation and Ag-presenting functions, driving Th1-type immune responses. Thus, Tat can act as both Ag and adjuvant, capable of driving T cell-mediated immune responses.
The Journal of Immunology 02/2002; 168(1):197-206. DOI:10.4049/jimmunol.168.1.197 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The viral protein Rev is essential for the export of the subset of unspliced and partially spliced lentiviral mRNAs and the production of structural proteins. Rev and its RNA binding site RRE can be replaced in both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) by the constitutive RNA transport element CTE of the simian type D retroviruses. We used neonatal macaques as a sensitive animal model to evaluate the pathogenicity of a pair of SIV mutant strains generated from Rev-independent molecular clones of SIVmac239 which differ only in the presence of the nef open reading frame. After high primary viremia, all animals remained persistently infected at levels below the threshold of detection. All macaques infected as neonates developed normally, and none showed any signs of immune dysfunction or disease during follow-up ranging from 2.3 to 4 years. Therefore, the Rev-RRE regulatory mechanism plays a key role in the maintenance of high levels of virus propagation, which is independent of the presence of nef. These data demonstrate that Rev regulation plays an important role in the pathogenicity of SIV. Replacement of Rev-RRE by the CTE provides a novel approach to dramatically lower the virulence of a pathogenic lentivirus. These data further suggest that antiretroviral strategies leading to even a partial block of Rev function may modulate disease progression in HIV-infected individuals.
Journal of Virology 02/2002; 76(1):96-104. DOI:10.1128/JVI.76.1.96-104.2002 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi's sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases.
The EMBO Journal 11/2001; 20(20):5568-77. DOI:10.1093/emboj/20.20.5568 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous work from our group showed that genetic immunization of mice with HIV-1 tat genes (tat22 and tat22/37), encoding Tat proteins mutated in the transactivation domain and lacking Tat-transactivating activity, evoke an immune response to wild-type Tat, both humoral and cellular. In the present work we report that the mutated Tat proteins localize within the cells, are released and taken up by the cells in a fashion similar to wild-type Tat. Moreover, the exogenous mutated Tat proteins interfere with the transactivating function of extracellular wild-type Tat. These results support the notion that tat22 and tat22/37 genes may represent good candidates for the development of an anti-HIV-1 vaccine, especially for HIV-1 infected patients.
[Show abstract][Hide abstract] ABSTRACT: Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE
RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation
of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses
rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A
functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote
replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its
potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize
RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export fromXenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export
factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways,
these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional
control element that uses a conserved cellular export mechanism.
Journal of Virology 06/2001; 75(10):4558-69. DOI:10.1128/JVI.75.10.4558-4569.2001 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human T helper (Th) cells (Th1- or Th2-oriented memory T cells as well as Th1- or Th2-polarized naive T cells) were infected in vitro with an R5-tropic HIV-1 strain (BaL) and assessed for their profile of cytokine production, CCR5 receptor expression, and HIV-1 p24 antigen (p24 Ag) production. Higher p24 Ag production was found in CCR5-negative Th2-like memory T cells than in CCR5-positive Th1-like memory T cells. By contrast, p24 Ag production was higher in Th1-polarized activated naive T cells in the first 4 days after infection. However, p24 Ag production in Th1-polarized T cells became comparable or even lower than the production in Th2-polarized populations later in infection or when the cells were infected with HIV-1BaL after secondary stimulation. The higher levels of p24 Ag production by Th1-polarized naive T cells soon after infection reflected a higher virus entry, as assessed by the single round infection assay using the HIV-chloramphenicol acetyl transferase (HIV-CAT) R5-tropic virus that contains the envelope protein of HIV-1 YU2 strain. The limitation of viral spread in the Th1-polarized populations, despite the initial higher level of T-cell entry of R5-tropic strains, was due to the ability of Th1 cells to produce greater amounts of beta-chemokines than Th2 cells. In fact, an inverse correlation was observed between Th1-polarized naive T cells and Th1-like memory-activated T cells in regards to p24 Ag production and the release of the following CCR5-binding chemokines: regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta. Moreover, infection with the HIV-1BaL strain of Th1-polarized T cells in the presence of a mixture of anti-RANTES, anti-MIP-1alpha, and anti-MIP-1beta neutralizing antibodies resulted in a significant increase of HIV-1 expression. These findings suggest that Th1-type responses may favor CD4(+) T-cell infection by R5-tropic HIV-1 strains, but HIV-1 spread in Th1 cells is limited by their ability to produce CCR5-binding chemokines. (Blood. 2000;95:1167-1174)
[Show abstract][Hide abstract] ABSTRACT: We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV gag, vif, or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpu F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV vpr, pol, or env gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV vif or nef allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy.
Journal of Virology 06/1998; 72(5):4308-19. · 4.44 Impact Factor