HIV Tat induces a prolonged MYC relocalization next to IGH in circulating B-cells.

With combined antiretroviral therapy (cART), the risk for HIV-infected individuals to develop a non-Hodgkin lymphoma is diminished. However, the incidence of Burkitt lymphoma (BL) remains strikingly elevated. Most BL present a t(8;14) chromosomal translocation which must take place at a time of spatial proximity between the translocation partners. The two partner genes, MYC and IGH, were found colocalized only very rarely in the nuclei of normal peripheral blood B-cells examined using 3D-FISH while circulating B-cells from HIV-infected individuals whose exhibited consistently elevated levels of MYC-IGH colocalization. In vitro, incubating normal B-cells from healthy donors with a transcriptionally active form of the HIV-encoded Tat protein rapidly activated transcription of the nuclease-encoding RAG1 gene. This created DNA damage, including in the MYC gene locus which then moved towards the center of the nucleus where it sustainably colocalized with IGH up to 10-fold more frequently than in controls. In vivo, this could be sufficient to account for the elevated risk of BL-specific chromosomal translocations which would occur following DNA double strand breaks triggered by AID in secondary lymph nodes at the final stage of immunoglobulin gene maturation. New therapeutic attitudes can be envisioned to prevent BL in this high risk group.

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.

Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription through its intact core and cysteine-rich domains inhibits Wnt/ß-catenin signaling in astrocytes: relevance to HIV neuropathogenesis.

Wnt/ß-catenin is a neuroprotective pathway regulating cell fate commitment in the CNS and many vital functions of neurons and glia. Its dysregulation is linked to a number of neurodegenerative diseases. Wnt/ß-catenin is also a repressor of HIV transcription in multiple cell types, including astrocytes, which are dysregulated in HIV-associated neurocognitive disorder. Given that HIV proteins can overcome host restriction factors and that perturbations of Wnt/ß-catenin signaling can compromise astrocyte function, we evaluated the impact of HIV transactivator of transcription (Tat) on Wnt/ß-catenin signaling in astrocytes. HIV clade B Tat, in primary progenitor-derived astrocytes and U87MG cells, inhibited Wnt/ß-catenin signaling as demonstrated by its inhibition of active ß-catenin, TOPflash reporter activity, and Axin-2 (a downstream target of Wnt/ß-catenin signaling). Point mutations in either the core region (K41A) or the cysteine-rich region (C30G) of Tat abrogated its ability to inhibit ß-catenin signaling. Clade C Tat, which lacks the dicysteine motif, did not alter ß-catenin signaling, confirming that the dicysteine motif is critical for Tat inhibition of ß-catenin signaling. Tat coprecipitated with TCF-4 (a transcription factor that partners with ß-catenin), suggesting a physical interaction between these two proteins. Furthermore, knockdown of ß-catenin or TCF-4 enhanced docking of Tat at the TAR region of the HIV long terminal repeat. These findings highlight a bidirectional interference between Tat and Wnt/ß-catenin that negatively impacts their cognate target genes. The consequences of this interaction include alleviation of Wnt/ß-catenin-mediated suppression of HIV and possible astrocyte dysregulation contributing to HIV neuropathogenesis.

Inhibition of genotoxic stress induced apoptosis by novel TAT-fused peptides targeting PIDDosome.

Genotoxic stress induced apoptosis is mediated by the formation of PIDDosome, which is a caspase-2 activating complex composed of three protein components, PIDD, RAIDD, and caspase-2. Here, synthetic TAT-fused peptides designed by the structure of PIDD and RAIDD, TAT-Y814A and TAT-R147E, respectively, were produced and tested for their ability to inhibit PIDDosome formation in vitro as well as to attenuate genotoxic stress-induced apoptosis in human renal cancer cells. The results show that TAT-Y814A and TAT-R147E have the potential to inhibit formation of the PIDDosome in a dose-dependent manner. Furthermore, both peptides partially inhibit genotoxic stress mediated apoptosis and activation of caspase2 and caspase3 in Caki cells. These results suggest that TAT-Y814A (also TAT-R147E) is a novel inhibitor of genotoxic stress-induced apoptosis that may serve as a prototype for anti-apoptotic drug development.

HIV-1 Tat upregulates expression of histone deacetylase-2 (HDAC2) in human neurons: implication for HIV-associated neurocognitive disorder (HAND).

Histone deacetylases (HDACs) play a pivotal role in epigenetic regulation of transcription and homeostasis of protein acetylation in histones and other proteins involved in chromatin remodeling. Histone hypoacetylation and transcriptional dysfunction have been shown to be associated with a variety of neurodegenerative diseases. More recently, neuron specific overexpression of HDAC2 has been shown to modulate synaptic plasticity and learning behavior in mice. However, the role of HDAC2 in development of HIV-associated neurocognitive disorders (HAND) is not reported. Herein we report that HIV-1 Tat protein upregulate HDAC2 expression in neuronal cells leading to transcriptional repression of genes involved in synaptic plasticity and neuronal function thereby contributing to the progression of HAND. Our results indicate upregulation of HDAC2 by Tat treatment in dose and time dependant manner by human neuroblastoma SK-N-MC cells and primary human neurons. Further, HDAC2 overexpression was associated with concomitant downregulation in CREB and CaMKIIa genes that are known to regulate neuronal activity. These observed effects were completely blocked by HDAC2 inhibition. These results for the first time suggest the possible role of HDAC2 in development of HAND. Therefore, use of HDAC2 specific inhibitor in combination with HAART may be of therapeutic value in treatment of neurocognitive disorders observed in HIV-1 infected individuals.

The HIV-1 transactivator factor (Tat) induces enterocyte apoptosis through a redox-mediated mechanism.

The intestinal mucosa is an important target of human immunodeficiency virus (HIV) infection. HIV virus induces CD4+ T cell loss and epithelial damage which results in increased intestinal permeability. The mechanisms involved in nutrient malabsorption and alterations of intestinal mucosal architecture are unknown. We previously demonstrated that HIV-1 transactivator factor (Tat) induces an enterotoxic effect on intestinal epithelial cells that could be responsible for HIV-associated diarrhea. Since oxidative stress is implicated in the pathogenesis and morbidity of HIV infection, we evaluated whether Tat induces apoptosis of human enterocytes through oxidative stress, and whether the antioxidant N-acetylcysteine (NAC) could prevent it. Caco-2 and HT29 cells or human intestinal mucosa specimens were exposed to Tat alone or combined with NAC. In an in-vitro cell model, Tat increased the generation of reactive oxygen species and decreased antioxidant defenses as judged by a reduction in catalase activity and a reduced (GSH)/oxidized (GSSG) glutathione ratio. Tat also induced cytochrome c release from mitochondria to cytosol, and caspase-3 activation. Rectal dialysis samples from HIV-infected patients were positive for the oxidative stress marker 8-hydroxy-2'-deoxyguanosine. GSH/GSSG imbalance and apoptosis occurred in jejunal specimens from HIV-positive patients at baseline and from HIV-negative specimens exposed to Tat. Experiments with neutralizing anti-Tat antibodies showed that these effects were direct and specific. Pre-treatment with NAC prevented Tat-induced apoptosis and restored the glutathione balance in both the in-vitro and the ex-vivo model. These findings indicate that oxidative stress is one of the mechanism involved in HIV-intestinal disease.

Cell-penetrating HIV1 TAT peptides float on model lipid bilayers.

Cell-penetrating peptides like the cationic HIV1 TAT peptide are able to translocate across cell membranes and to carry molecular cargoes into the cellular interior. For most of these peptides, the biophysical mechanism of the membrane translocation is still quite unknown. We analyzed HIV1 TAT peptide binding and mobility within biological model membranes. To this end, we generated neutral and anionic giant unilamellar vesicles (GUVs) containing DPPC, DOPC, and cholesterol and containing DPPC, DOPC, cholesterol, and DPPS (DOPS), respectively. First, we characterized the mobility of fluorescently labeled lipids (TR-DHPE) within liquid-ordered and liquid-disordered lipid phases by single-molecule tracking, yielding a D(LO) of 0.6 +/- 0.05 microm(2)/s and a D(LD) of 2.5 +/- 0.05 microm(2)/s, respectively, as a reference. Fluorescently labeled TAT peptides accumulated on neutral GUVs but bound very efficiently to anionic GUVs. Single-molecule tracking revealed that HIV1 TAT peptides move on neutral and anionic GUV surfaces with a D(N,TAT) of 5.3 +/- 0.2 microm(2)/s and a D(A,TAT) of 3.3 +/- 0.2 mum(2)/s, respectively. TAT peptide diffusion was faster than fluorescent lipid diffusion, and also independent of the phase state of the membrane. We concluded that TAT peptides are not incorporated into but rather floating on lipid bilayers, but they immerged deeper into the headgroup domain of anionic lipids. The diffusion constants were not dependent on the TAT concentration ranging from 150 pM to 2 microM, indicating that the peptides were not aggregated on the membrane and not forming any "carpet".

The ups and downs of SIRT1.

Reversible acetylation has emerged as a key post-translational modification of proteins. Although the number of acetylated proteins is rapidly growing, the ways in which protein acetyltransferases and deacetylases connect with extracellular stimuli remain unclear. Recently, a regulatory network has emerged that controls the expression and activity of SIRT1, a mammalian class-III protein deacetylase. SIRT1 is an important regulator of metabolism, senescence, cancer and, possibly, longevity and is connected with crucial stress-responsive signal-transduction pathways. These connections provide important clues about how protein acetylation and deacetylation mediate cellular adaptations to extrinsic stress.

[Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT].

AIM: To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT, laying a foundation for gene therapy research of malignant tumors. METHODS: The Apoptin and HA2-TAT gene were inserted in pUC19/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-Apoptin-HA2-TAT was sub-cloned into the shuttle plasmid of adeno-associated virus; the products were co-transferred into HEK-293 cell line with helper plasmid pAAV/Ad and adeno-plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was measured by quantitative dot blot hybridization. The effect of AAV-NT4-Apoptin -HA2-TAT on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS: The NT4-Apoptin-HA2-TAT was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cells (3.14 x 10(15) pfu/L). AAV-NT4-Apoptin-HA2-TAT had strong deduce apoptosis effect on HepG2 cells. Compared with Adeno-associated mock virus and in normal cell line NIH3T3, Aden-associated virus NT4-Apoptin-HA2-TAT significantly decreased the survival rate of HepG2 cells. CONCLUSION: The recombinant adeno-associated virus vector encoding gene NT4-Apoptin-HA2-TAT has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques, laying a foundation for further research of gene therapy of cancer.

HIV TAT peptide modifies the distribution of DNA nanolipoparticles following convection-enhanced delivery.

We evaluated gene transfer using PEGylated bioresponsive nanolipid particles (NLPs) containing plasmid DNA administered by convection-enhanced delivery (CED) into orthotopically implanted U87-MG tumors in rat brain. We hypothesized that attachment of the human immunodeficiency virus trans-acting transcriptional activator peptide (TATp) to pH-sensitive, reduction-sensitive NLPs would increase gene transfer. TATp was attached either directly to a phospholipid (TATp-lipid) or via a 2-kd polyethylene glycol (PEG) to a lipid (TATp-PEG-lipid). Incorporation of 0.3 mol% TATp-PEG into pH-sensitive NLPs improved transfection 100,000-fold compared to NLPs in culture. In the brain or implanted tumors, the TATp-PEG restricted NLP convection to regions adjacent to the infusion catheter. Gene transfer in the brain from TATp-PEG NLPs, measured by green fluorescent protein (GFP) expression, was substantially greater than from NLPs adjacent to the catheter. Gene transfer using TATp-PEG NLPs, measured by luciferase expression, was 8-12-fold greater than from a 1,2-dioleoyl-3-trimethylammonium-propane/cholesterol cationic lipoplex but 13-27-fold less than from the NLPs. Brain luciferase expression was localized in perivascular macrophages. Thus a cationic ligand, such as the TATp-PEG-lipid, can dramatically increase gene expression in culture, in the normal brain, and in implanted tumors; however, restriction of NLP distribution to the vicinity of the infusion catheter reduces the absolute level of gene transfer.

Intranasal tat alters gene expression in the mouse brain.

Intranasal (IN) delivery of HIV-1 Tat in aging mice was investigated as a possible model for HIV-1 infection in the brain. After IN administration, the distribution of [(125)I]-labeled Tat in the brains of Swiss Webster mice was evaluated by autoradiography and gamma counting. [(125)I]-labeled Tat was detected at the highest concentrations in the olfactory bulb, cervical nodes, and trigeminal nerve tract. In another experiment, APPSw transgenic mice were used to model chronic Tat exposure. The mice were treated intranasally with 6 mug Tat (n = 4) or vehicle (n = 4) three times per week for 4 weeks. Total RNA was isolated from the frontal cortex, and differential gene expression analysis was performed using gene microarrays. Gene ontology profiles indicated innate immunity, inflammatory and apoptotic responses. Five genes of interest in the Tat-treated mice that were significantly elevated in the microarrays were validated by RT-PCR. One gene, the Toll-like receptor 9 (Tlr9), has previously been shown to activate signaling cascades leading to innate immunity and enhanced HIV-1 gene expression. A second gene, Fas, plays a key role in neuroinflammation. Two cysteine-rich cytokines associated with chemotaxis were elevated: MCP-1 (Ccl2), which is chemotactic for monocytes, and Ccl17 (TARC), which is chemotactic for lymphocytes. Finally, the gene sestrin was significantly elevated and has been associated with oxidative stress, in particular amyloid beta-induced oxidative stress. This IN Tat model of neuroinflammation may be useful to study HIV-1-induced neurodegeneration.

TAT transduction: the molecular mechanism and therapeutic prospects.

Research into the mechanism of protein transduction has undergone a renaissance in the past five years as many groups have sought to understand the behavior of transducing peptides to harness their enormous therapeutic and diagnostic potential. The field has benefited greatly from rigorous cell biological and biophysical studies of the mechanism used by cell penetrating peptides to enter cells and deliver their cargo. The recent identification of fluid phase endocytosis as the mode of cellular entry for TAT and other protein transduction domains has enhanced our understanding of how transduction facilitates intracellular delivery. Many outstanding questions and contradictions still remain to be resolved in the field. Nevertheless, the current body of work regarding the mechanism of uptake gives a much clearer picture of how these macromolecules enter cells and how we might enhance the bioavailability to take advantage of them clinically.

Oncogenic properties of HIV-Tat in colorectal cancer cells.

With the advent of Highly-Active-Anti-Retroviral-Therapy (HAART), HIV patients can expect to live beyond 10-15 years following diagnosis. An unexpected result of increased survival is the emergence of opportunistic, oncogenic virus-associated cancers such as Burkitt's lymphoma (Epstein-Barr Virus), cervical cancer (Human Papilloma Virus) and Kaposi's sarcoma (Kaposi's sarcoma-associated herpesvirus) in this immuno-compromised population. Furthermore, there are reports of colorectal cancers (CRC) in long-term HIV-AIDS survivors. Compared to the general, non-immuno-compromised population, long-term AIDS patients have 4 and 3.3-fold increased risk of developing colorectal and anorectal cancer respectively. Unlike oncogenic virus-associated cancers, CRC is not known to have a viral etiology. Our study aimed to investigate one aspect of HIV infection and colorectal carcinogenesis. We proposed that the HIV transactivator protein Tat; a protein with known oncogenic properties that is secreted and can re-enter non-infected cells may have a role in CRC. Using two CRC cell lines, LIM1215 and LIM2537 we found that Tat inhibits epithelial cyto-differentiation, blocks apoptosis in vitro and accelerates tumour formation in vivo. In addition, Tat significantly increases in vitro migration in the absence of foetal calf serum. These properties underpin CRC, and as HIV infection is initiated in the gut lymphoid system, these data provide a basis for the increased incidence of CRC in long term AIDS patients.

Activation of the adenosine A1 receptor inhibits HIV-1 tat-induced apoptosis by reducing nuclear factor-kappaB activation and inducible nitric-oxide synthase.

Human immunodeficiency virus dementia (HIV-D) is a nonfocal central nervous system manifestation characterized by cognitive, behavioral, and motor abnormalities. The pathophysiology of neuronal damage in HIV-D includes a direct toxic effect of viral proteins on neuronal cells and an indirect effect caused by the release of inflammatory mediators and neurotoxins by activated macrophages/microglia and astrocytes, culminating into neuronal apoptosis. Previous studies have documented that the nucleoside adenosine mediates neuroprotection by activating adenosine A(1) receptor subtype (A(1)AR) linked to suppression of neuronal excitability. In this study, we show that A(1)AR activation protects against HIV-1 Tat-induced toxicity in primary cultures of rat cerebellar granule neurons and in rat pheochromocytoma (PC12) cell. In PC12 cells, HIV-1 Tat increased [Ca(2+)](i) levels, release of nitric oxide (NO), and expression of inducible nitric-oxide synthase (iNOS) and A(1)AR. Activation of A(1)AR suppressed Tat-mediated increases in [Ca(2+)](i) and NO. Furthermore, A(1)AR agonists inhibited iNOS expression in a nuclear factor-kappaB (NF-kappaB)-dependent manner. It is noteworthy that activation of the A(1)AR or inhibition of NOS protected against Tat-induced apoptosis in PC12 cells and cerebellar granule cells. Moreover, activation of the A(1)AR-inhibited Tat-induced increases in the levels of proapoptotic proteins Bax and caspase-3. Taken together, our results demonstrate that the A(1)AR protects against HIV-1 toxicity by inhibiting NF-kappaB, thereby reducing the expression of iNOS and NO radicals and neuronal apoptosis.

The Ebola virus VP35 protein is a suppressor of RNA silencing.

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.

Intracellular delivery of Neuroglobin using HIV-1 TAT protein transduction domain fails to protect against oxygen and glucose deprivation.

Neuroglobin (Ngb) is a heme protein that is primarily localised in the retina and the brain. Its physiological role is largely unknown. It has been reported that its overexpression protects neurons from hypoxia in vitro and in vivo, suggesting that the rapid modulation of the Ngb level in the nerve cells may be a promising stroke treatment strategy. In this study, we used a novel approach to overexpress Ngb and evaluate its ability to promote neuronal survival under hypoxic conditions. We constructed a human recombinant Ngb fused to the cell penetrating peptide (CPP) derived from HIV-1 TAT. Purified recombinant TAT-Ngb was able to efficiently transduce CHO and SHSY5Y cells, when added to the culture media. The potential neuroprotective action of Ngb was then examined by using an in vitro model of ischemia. The two neuronal cell lines RGC-5 and SH-SY5Y were subjected to oxygen glucose deprivation (OGD) after pre-treatment with TAT-Ngb. In both cell types, however, the treatment with the TAT-Ngb fusion protein did not show any effect on cell viability. This discrepancy to earlier reports might be due to the experimental model for oxygen glucose deprivation we employed. Alternatively, intracellular delivery of Ngb by the TAT/CPP might not have beneficial effects in the treatment of ischemic pathology.

HIV Tat protein increases Bcl-2 expression in monocytes which inhibits monocyte apoptosis induced by tumor necrosis factor-alpha-related apoptosis-induced ligand.

OBJECTIVE: To investigate the effect of HIV Tat protein on Bcl-2 expression in human monocytes, and observe apoptosis of Tat-stimulated monocytes induced by TNF-alpha-related apoptosis-induced ligand (TRAIL). METHODS: Western blot was used to detect Bcl-2 expression in monocytes stimulated by HIV Tat protein, and Annexin V and 7-AAD staining were used to detect apoptosis of monocytes induced by TRAIL. RESULTS: HIV Tat protein increased Bcl-2 expression in human monocytes in a dose-dependent manner. Annexin V staining showed that 51.54% of monocytes underwent apoptosis after being treated with 100 ng/ml recombinant TRAIL. When monocytes were prestimulated with HIV Tat, only 15.46% of monocytes underwent apoptosis. This effect can be inhibited by polyclonal anti-Tat serum. 7-AAD staining showed similar results. CONCLUSION: HIV Tat protein increases Bcl-2 expression in monocytes which inhibited apoptosis induced by TRAIL. HIV Tat protein may play an important role in the mechanisms of HIV-persistent infection in monocytes.

Human immunodeficiency virus type 1 subtype C Tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes.

Over 50% of all human immunodeficiency virus type 1 (HIV-1) infections worldwide are caused by subtype C strains, yet most research to date focuses on subtype B, the subtype most commonly found in North America and Europe. The HIV-1 trans-acting regulatory protein (Tat) is essential for regulating productive replication of HIV-1. Tat is secreted by HIV-infected cells and alters several functions of uninfected bystander cells. One such function is that, by acting at the cell membrane, subtype B Tat stimulates the production of tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) from human monocytes and can act as a chemoattractant. In this study, we show that the mutation of a cysteine to a serine at residue 31 of Tat commonly found in subtype C variants significantly inhibits the abilities of the protein to bind to chemokine (C-C motif) receptor 2 (CCR2), induce intracellular calcium flux, stimulate TNF and CCL2 production, and inhibit its chemoattractant properties. We also show that TNF is important in mediating some effects of extracellular Tat. This report therefore demonstrates the important functional differences between subtype C and subtype B Tat and highlights the need for further investigation into the different strains of HIV-1.

Herpesvirus saimiri terminal membrane proteins modulate HIV-1 replication by altering Nef and Tat functions.

Herpesvirus saimiri (HVS)-transformed human T cells expressing terminal membrane proteins (TMPs) tyrosine kinase interacting protein (Tip) and saimiri transformation associated protein strain C (StpC) are highly permissive for R5 and X4 strains of HIV-1. StpC expression enhances replication of R5 and X4 strains of HIV-1 and induces latent reservoirs of replication competent HIV-1 in cell lines derived from T cells or monocytes. Paradoxically Tip expression restricts replication and cytopathic effects of R5 and X4 strains of HIV-1 in T cells and monocytes post-retrotransposition. Understanding the canonical pathways whereby Tip and StpC alter HIV-1 replication may uncover novel therapeutic approaches to HIV-1 infection. Here we show Tip inhibits Tat-mediated transcriptional activation of the long terminal repeat (LTR). Tip mediated inhibition of Tat transactivation is reversed by Nef. Tip also mediates restriction of late-stage replication of HIV-1 by disrupting Nef interaction with lymphocyte-specific protein-tyrosine kinase (Lck) in lipid rafts. Specifically, in the presence of Tip, Lck does not localize to lipid rafts reducing Nef interaction with Lck within the lipid rafts. Finally, the permissive phenotype conferred by StpC is the result of synergy with Tat during transcriptional activation of the HIV-1 LTR. This transcriptional synergy between StpC and Tat requires Lck and NF-kappaB consensus binding sequences. These findings demonstrate that the HVS TMPs influence transcriptional and post-transcriptional stages in HIV-1 replication. We propose that HVS-encoded TMPs associated with T cell transformation have evolved ability to modulate the replication of competing retroviruses. Gene based approaches utilizing Tip and StpC may provide therapeutic models for treating acute and latent HIV-1 infections, respectively.