TGFßRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment.

BACKGROUND: HIV-1-associated neurocognitive disorders (HAND) progression is related to continued inflammation despite undetectable viral loads and may be caused by early viral proteins expressed by latently infected cells. Astrocytes represent an HIV reservoir in the brain where the early viral neurotoxin negative factor (Nef) is produced. We previously demonstrated that astrocytic expression of Nef in the hippocampus of rats causes inflammation, macrophage infiltration, and memory impairment. Since these processes are affected by TGFß signaling pathways, and TGFß-1 is found at higher levels in the central nervous system of HIV-1+ individuals and is released by astrocytes, we hypothesized a role for TGFß-1 in our model of Nef neurotoxicity. METHODS: To test this hypothesis, we compared cytokine gene expression by cultured astrocytes expressing Nef or green fluorescent protein. To determine the role of Nef and a TGFßRI inhibitor on memory and learning, we infused astrocytes expressing Nef into the hippocampus of rats and then treated them daily with an oral dose of SD208 (10 mg/kg) or placebo for 7 days. During this time, locomotor activity was recorded in an open field and spatial learning tested in the novel location recognition paradigm. Postmortem tissue analyses of inflammatory and signaling molecules were conducted using immunohistochemistry and immunofluorescence. RESULTS: TGFß-1 was induced in cultures expressing Nef at 24 h followed by CCL2 induction which was prevented by blocking TGFßRI with SD208 (competitive inhibitor). Interestingly, Nef seems to change the TGFßRI localization as suggested by the distribution of the immunoreactivity. Nef caused a deficit in spatial learning that was recovered upon co-administration of SD208. Brain tissue from Nef-treated rats given SD208 showed reduced CCL2, phospho-SMAD2, cluster of differentiation 163 (CD163), and GFAP immunoreactivity compared to the placebo group. CONCLUSIONS: Consistent with our previous findings, rats treated with Nef showed deficits in spatial learning and memory in the novel location recognition task. In contrast, rats treated with Nef + SD208 showed better spatial learning suggesting that Nef disrupts memory formation in a TGFß-1-dependent manner. The TGFßRI inhibitor further reduced the induction of inflammation by Nef which was concomitant with decreased TGFß signaling. Our findings suggest that TGFß-1 signaling is an intriguing target to reduce neuroHIV.

Short Communication: Accumulation of Neutral Lipids in Liver and Aorta of Nef-Transgenic Mice.

HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements.

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the "6ATRi" element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function.

Transcriptomic Analysis of mRNAs in Human Monocytic Cells Expressing the HIV-1 Nef Protein and Their Exosomes.

The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression to AIDS. Besides its well-studied effects on intracellular signaling, Nef also functions through its secretion in exosomes, which are nanovesicles containing proteins, microRNAs, and mRNAs and are important for intercellular communication. Nef expression enhances exosome secretion and these exosomes can enter uninfected CD4 T cells leading to apoptotic death. We have recently reported the first miRNome analysis of exosomes secreted from Nef-expressing U937monocytic cells. Here we show genome-wide transcriptome analysis of Nef-expressing U937 cells and their exosomes. We identified four key mRNAs preferentially retained in Nef-expressing cells; these code for MECP2, HMOX1, AARSD1, and ATF2 and are important for chromatin modification and gene expression. Interestingly, their target miRNAs are exported out in exosomes. We also identified three key mRNAs selectively secreted in exosomes from Nef-expressing U937 cells and their corresponding miRNAs being preferentially retained in cells. These are AATK, SLC27A1, and CDKAL and are important in apoptosis and fatty acid transport. Thus, our study identifies selectively expressed mRNAs in Nef-expressing U937 cells and their exosomes and supports a new mode on intercellular regulation by the HIV-1 Nef protein.

Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61) and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.

Identification of essential cis element in 5'UTR of Nef mRNA for Nef translation.

Nef is one of the accessory proteins of the human immunodeficiency virus type 1 (HIV-1). Nef is translated from multiple-spliced mRNAs transcribed from the viral genome, whose mRNAs have a relatively long 5' untranslated region (5'UTR). Here, we identified a cis element in the 5'UTR of Nef mRNA essential for efficient Nef translation, which was named the Nef-translation essential region (NER). Mutants with a deleted NER in the 5'UTR of the HIV-1 NL4-3 strain showed an almost undetectable Nef expression owing to a low Nef translation efficiency. The NER of the NL4-3 strain was predicted to form putative stem loops. Although the 5'UTR showed significant but relatively low internal ribosome entry site (IRES) activity, the mechanism of 5'cap-dependent translation mainly contributed to the Nef translation from its Nef mRNA. Altogether, it was clarified that not only the 5' cap but also the NER in the 5'UTR is an essential cis element for efficient Nef translation, which is not a typical 5'-cap-dependent mechanism, and that there must be an as yet unknown mechanism using the NER for efficient Nef translation.

HIV Nef expression favors the relative preservation of CD4+ T regulatory cells that retain some important suppressive functions.

HIV-1 infection causes depletion and/or dysfunction of distinct CD4(+) T cell subsets and may affect these differently. Using the CD4C/HIV-1(Nef) transgenic (Tg) mice as a model, we report that HIV-1 Nef causes depletion of total CD4(+) T cells, but preserves and relatively enriches CD4(+) regulatory T cells (Treg). We found that Nef-mediated CD4(+) Treg enrichment is the direct result of Nef expression in CD4(+) T cells, occurs independently of Nef-induced lymphopenia, and most likely results from multiple mechanisms: lower apoptosis, enhanced cell division, and increased generation from precursors. Interestingly, Tg Treg relative enrichment could be reversed by enhancing Lck activity. Most importantly, we show that, in contrast to Tg helper CD4(+) T cells that have lost their function, Nef-expressing CD4(+) Treg retain their regulatory function in vitro and also in vivo, under some settings. In particular, we found that Treg prevent expansion of Tg B and non-Treg T cells in vivo. Our study reveals that Nef affects distinct CD4(+) T cell subsets differently and uncovers the high proliferative potential of B and non-Treg T cells in this mouse model.

A consensus surface activation marker signature is partially dependent on human immunodeficiency virus type 1 Nef expression within productively infected macrophages.

BACKGROUND: The high prevalence of HIV-associated comorbidities including neurocognitive disorder, high levels of residual inflammatory mediators in the plasma and cerebrospinal fluid and the resurgence of HIV replication upon interruption of antiviral treatment in HIV-1 infected individuals, strongly suggests that despite therapy HIV persists in its cellular targets which include T-lymphocytes and cells of the myeloid lineage. These reservoirs present a major barrier against eradication efforts. Knowledge of the molecular mechanisms used by HIV to modulate innate macrophage immune responses and impair viral clearance is quite limited. To explore the role of HIV in potentially modulating macrophage function through changes in protein expression, we used single-cell analyses with flow cytometry to determine whether, in unpolarized cultures, macrophage surface marker phenotype was altered by HIV infection in a manner that was independent of host genetic background. RESULTS: These analyses revealed that at several time points post-infection, GFP + HIV-infected macrophages were significantly enriched in the CD14+ fraction (3 to 5-fold, p = .0001) compared to bystander, or uninfected cells in the same culture. However, the enrichment and higher levels of CD14 on HIV expressing macrophages did not depend on the production of HIV Nef. Sixty to eighty percent of macrophages productively infected with HIV after day 28 post-infection were also enriched in the population of cells expressing the activation markers CD69 (2 to 4-fold, p < .0001) and CD86 (2 to 4-fold, p < .0001 ) but suppressed amounts of CD68 (3 to 10-fold, p < .0001) compared to bystander cells. Interestingly, there was no enrichment of CD69 on the surface of HIV producing cells that lacked Nef or expressed a variant of Nef mutated in its SH3-binding domain. CONCLUSIONS: These findings suggest that HIV actively regulates the expression of a subset of surface molecules involved in innate and inflammatory immune signaling in primary human macrophages through Nef-dependent and Nef-independent mechanisms acting within productively infected cells.

Normal development and function but impaired memory phenotype of CD8⁺ T cells in transgenic mice expressing HIV-1 Nef in its natural target cells.

We studied the impact of HIV Nef on CD8(+) T cells in a mouse model of AIDS, the CD4C/HIV(Nef) transgenic (Tg) mice. We found that negative and positive thymic selections of CD8(+) T cells proceeded normally in Nef Tg mice bred respectively with HY or OT-1 TCR Tg mice. Tg peripheral CD8(+) T cells showed an activated phenotype and enhanced cell division in vivo and proliferated efficiently when stimulated in vitro with antigenic peptide. When challenged with LCMV(Armstrong), Nef Tg mice developed a strong acute CD8(+) T cell response and cleared the virus as efficiently as wild-type mice. However, maintenance of LCMV-specific CD8(+) memory T cells was impaired in Nef Tg mice, a defect partially rescued by adoptive transfer of non-Tg naïve CD4(+) T cells. Thus, despite severe abnormalities of their precursors, the double-positive CD4(+)CD8(+) thymocytes, Tg CD8(+) T cells have conserved important functions.

Validation of a generation 4 phosphorus-containing polycationic dendrimer for gene delivery against HIV-1.

Gene therapy, in which oligomeric genetic material is carried into cells by nano-sized gene delivery vehicles to interfere with gene expression, represents a promising approach for preventive therapy against HIV/AIDS pandemic. Herein, we evaluate the usefulness of a phosphorus-containing dendrimer G4(NH+Et2Cl-)96 as a delivery agent of ODNs and siRNAs. G4(NH+Et2Cl-)96 formed stable complexes with ODNs or siRNAs and exhibited very low cytotoxicity in Sup T1 cells or PBMC. Functional validation was performed by using specific siRNA against HIV-1 Nef, siNEF to interfere in HIV-1 replication. G4(NH+Et2Cl-)96/siNEF dendriplex showed a high efficiency in Nef silencing. Furthermore, in vitro treatment of HIV-infected PBMC with G4(NH+Et2Cl-)96/siNEF dendriplex significantly reduced the viral replication. Our results prove the usefulness of phosphorus-containing dendrimers to deliver and transfect siRNA into CD4-T cells as a potential alternative therapy in the HIV-1 infection.

High-affinity target binding engineered via fusion of a single-domain antibody fragment with a ligand-tailored SH3 domain.

Monoclonal and recombinant antibodies are ubiquitous tools in diagnostics, therapeutics, and biotechnology. However, their biochemical properties lack optimal robustness, their bacterial production is not easy, and possibilities to create multifunctional fusion proteins based on them are limited. Moreover, the binding affinities of antibodies towards their antigens are suboptimal for many applications where they are commonly used. To address these issues we have made use of the concept of creating high binding affinity based on multivalent target recognition via exploiting some of the best features of immunoglobulins (Ig) and non-Ig-derived ligand-binding domains. We have constructed a small protein, named Neffin, comprised of a 118 aa llama Ig heavy chain variable domain fragment (VHH) fused to a ligand-tailored 57 aa SH3 domain. Neffin could be readily produced in large amounts (>18 mg/L) in the cytoplasm of E. coli, and bound with a subpicomolar affinity (K(d) 0.54 pM) to its target, the HIV-1 Nef protein. When expressed in human cells Neffin could potently inhibit Nef function. Similar VHH-SH3 fusion proteins could be targeted against many other proteins of interest and could have widespread use in diverse medical and biotechnology applications where biochemical robustness and strong binding affinity are required.

Altering an artificial Gagpolnef polyprotein and mode of ENV co-administration affects the immunogenicity of a clade C HIV DNA vaccine.

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.

Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K.

HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology.

Quantitation of parvalbumin+ neurons and human immunodeficiency virus type 1 (HIV-1) regulatory gene expression in the HIV-1 transgenic rat: effects of vitamin A deficiency and morphine.

Vitamin A (VA) deficiency in human immunodeficiency virus (HIV) infection has been associated with more progressive HIV disease, which may be enhanced by opioid use. In these studies, we examined the effects of VA deficiency and morphine on frontal cortex neuronal numbers in the HIV-1 transgenic (Tg) rat. These studies showed that total numbers of neurons were similar for rats on the VA-deficient diet as for rats on the normal diet and these numbers were not affected by treatment with morphine. In contrast, numbers of neurons that expressed the calcium-binding protein parvalbumin, which is a marker interneurons that express the inhibitory neurotransmitter gamma-aminobutyric acid (GABAergic neurons) were decreased for wild-type (Wt) rats on the VA-deficient diet and for Wt rats treated with morphine. In addition, parvalbumin+ neurons were also decreased for Tg rats on a normal diet but increased to normal levels when these animals were placed on the VA-deficient diet and treated with morphine. Analysis of expression of the genes that code for the HIV regulatory proteins vif, vpr, nef, and tat in frontal cortex and adjacent subcortical white matter showed that tat expression was increased in the morphine-treated Tg rat on the VA-deficient diet as compared to untreated Tg rats on the normal diet and untreated VA-deficient rats. These studies therefore suggest that VA deficiency, opioid exposure, and HIV infection alone and in combination may potentially alter neuronal metabolic activity and induce cellular stress, resulting in the observed changes in levels of parvalbumin expression. The specific mechanisms that underlie these effects require further study.

HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells.

The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879-887). Extracellular Nef protein has deleterious effects on CD4(+) T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4(+) T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099-3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302-310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro. Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4(+) T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.

BACKGROUND: In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. RESULTS: The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. CONCLUSION: We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study.

The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.

Functional T-cell responses generated by dendritic cells expressing the early HIV-1 proteins Tat, Rev and Nef.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.

Compartmentalization of the gut viral reservoir in HIV-1 infected patients.

BACKGROUND: Recently there has been an increasing interest and appreciation for the gut as both a viral reservoir as well as an important host-pathogen interface in human immunodefiency virus type 1 (HIV-1) infection. The gut associated lymphoid tissue (GALT) is the largest lymphoid organ infected by HIV-1. In this study we examined if different HIV-1 quasispecies are found in different parts of the gut of HIV-1 infected individuals. RESULTS: Gut biopsies (esophagus, stomach, duodenum and colorectum) were obtained from eight HIV-1 infected preHAART (highly active antiretroviral therapy) patients. HIV-1 Nef and Reverse transcriptase (RT) encoding sequences were obtained through nested PCR amplification from DNA isolated from the gut biopsy tissues. The PCR fragments were cloned and sequenced. The resulting sequences were subjected to various phylogenetic analyses. Expression of the nef gene and viral RNA in the different gut tissues was determined using real-time RT-PCR. Phylogenetic analysis of the Nef protein-encoding region revealed compartmentalization of viral replication in the gut within patients. Viral diversity in both the Nef and RT encoding region varied in different parts of the gut. Moreover, increased nef gene expression (p < 0.05) and higher levels of viral genome were observed in the colorectum (p < 0.05). These differences could reflect an adaptation of HIV-1 to the various tissues. CONCLUSION: Our results indicated that different HIV-1 quasispecies populate different parts of the gut, and that viral replication in the gut is compartmentalized. These observations underscore the importance of the gut as a host-pathogen interface in HIV-1 infection.