Human Immunodeficiency Virus Type 1 P24 Research Articles

Glutamylation of an HIV-1 protein inhibits the immune response by hijacking STING

Cyclic GMP-AMP synthase (cGAS) recognizes Y-form cDNA of human immunodeficiency virus type 1 (HIV-1) and initiates antiviral immune response through cGAS-stimulator of interferon genes (STING)-TBK1-IRF3-type I interferon (IFN-I) signalingcascade. Here, we report that the HIV-1 p6 protein suppresses HIV-1-stimulated expression of IFN-I and promotes immune evasion. Mechanistically, the glutamylated p6 at residue Glu6 inhibits the interaction between STING and tripartite motif protein 32 (TRIM32) or autocrine motility factor receptor (AMFR). This subsequently suppresses the K27- and K63-linked polyubiquitination of STING at K337, therefore inhibiting STING activation, whereas mutation of the Glu6 residue partially reverses the inhibitory effect. However, CoCl2, an agonist of cytosolic carboxypeptidases (CCPs), counteracts the glutamylation of p6 at the Glu6 residue and inhibits HIV-1 immune evasion. These findings reveal a mechanism through which an HIV-1 protein mediates immune evasion and provides a therapeutic drug candidate to treat HIV-1 infection.

Consistency of spatial dynamics of HIV-1 and HCV among HIV-1/HCV coinfected drug users in China

BackgroundAs the transmission routes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are similar, previous studies based on separate research on HIV-1 and HCV assumed a similar transmission pattern. However, few studies have focused on the possible correlation of the spatial dynamics of HIV-1 and HCV among HIV-1/HCV coinfected patients.MethodsA total of 310 HIV-1/HCV coinfected drug users were recruited in Yingjiang and Kaiyuan prefectures, Yunnan Province, China. HIV-1 env, p17, pol and HCV C/E2, NS5B fragments were amplified and sequenced from serum samples. The genetic characteristics and spatial dynamics of HIV-1 and HCV were explored by phylogenetic, bootscanning, and phylogeographic analyses.ResultsAmong HIV-1/HCV coinfected drug users, eight HCV subtypes (1a, 1b, 3a, 3b, 6a, 6n, 6v, and 6u) and two HIV-1 subtypes (subtype B and subtype C), three HIV-1 circulating recombinant forms (CRF01_AE, CRF07_BC and CRF08_BC), and four unique recombinant forms (URF_BC, URF_01B, URF_01C and URF_01BC) were identified. HCV subtype 3b was the most predominant subtype in both Yingjiang and Kaiyuan prefectures. The dominant circulating HIV-1 subtypes for drug users among the two areas were CRF08_BC and URF_BC. Maximum clade credibility trees revealed that both HIV-1 and HCV were transmitted from Yingjiang to Kaiyuan.ConclusionsThe spatial dynamics of HIV-1 and HCV among HIV-1/HCV coinfected drug users seem to have high consistency, providing theoretical evidence for the prevention of HIV-1 and HCV simultaneously.

Ultra-low HIV-1 p24 detection limits with a bioelectronic sensor

Early diagnosis of the infection caused by human immunodeficiency virus type-1 (HIV-1) is vital to achieve efficient therapeutic treatment and limit the disease spreading when the viremia is at its highest level. To this end, a point-of-care HIV-1 detection carried out with label-free, low-cost, and ultra-sensitive screening technologies would be of great relevance. Herein, a label-free single molecule detection of HIV-1 p24 capsid protein with a large (wide-field) single-molecule transistor (SiMoT) sensor is proposed. The system is based on an electrolyte-gated field-effect transistor whose gate is bio-functionalized with the antibody against the HIV-1 p24 capsid protein. The device exhibits a limit of detection of a single protein and a limit of quantification in the 10 molecule range. This study paves the way for a low-cost technology that can quantify, with single-molecule precision, the transition of a biological organism from being “healthy” to being “diseased” by tracking a target biomarker. This can open to the possibility of performing the earliest possible diagnosis.

Naturally Derived Anti-HIV Polysaccharide Peptide (PSP) Triggers a Toll-Like Receptor 4-Dependent Antiviral Immune Response.

Aim Intense interest remains in the identification of compounds to reduce human immunodeficiency virus type 1 (HIV-1) replication. Coriolus versicolor's polysaccharide peptide (PSP) has been demonstrated to possess immunomodulatory properties with the ability to activate an innate immune response through Toll-like receptor 4 (TLR4) showing insignificant toxicity. This study sought to determine the potential use of PSP as an anti-HIV agent and whether its antiviral immune response was TLR4 dependent. Materials and Methods HIV-1 p24 and anti-HIV chemokine release was assessed in HIV-positive (HIV+) THP1 cells and validated in HIV+ peripheral blood mononuclear cells (PBMCs), to determine PSP antiviral activity. The involvement of TLR4 activation in PSP anti-HIV activity was evaluated by inhibition. Results PSP showed a promising potential as an anti-HIV agent, by downregulating viral replication and promoting the upregulation of specific antiviral chemokines (RANTES, MIP-1α/β, and SDF-1α) known to block HIV-1 coreceptors in THP1 cells and human PBMCs. PSP produced a 61% viral inhibition after PSP treatment in HIV-1-infected THP1 cells. Additionally, PSP upregulated the expression of TLR4 and TLR4 inhibition led to countereffects in chemokine expression and HIV-1 replication. Conclusion Taken together, these findings put forward the first evidence that PSP exerts an anti-HIV activity mediated by TLR4 and key antiviral chemokines. Elucidating these new molecular mediators may reveal additional drug targets and open novel therapeutic avenues for HIV-1 infection.

Development of a Live Recombinant BCG Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Gag Using a pMyong2 Vector System: Potential Use As a Novel HIV-1 Vaccine.

Even though the rate of new human immunodeficiency virus type 1 (HIV-1) infections is gradually decreasing worldwide, an effective preventive vaccine for HIV-1 is still urgently needed. The recombinant Mycobacterium bovis BCG (rBCG) is promising for the development of an HIV-1 vaccine. Recently, we showed that a recombinant Mycobacterium smegmatis expressing HIV-1 gag in a pMyong2 vector system (rSmeg-pMyong2-p24) increased the efficacy of a vaccine against HIV-1 in mice. Here, we evaluated the potential of an rBCG expressing HIV-1 p24 antigen Gag in pMyong2 (rBCG-pMyong2-p24) in a vaccine application for HIV-1 infection. We found that rBCG-pMyong2-p24 elicited an enhanced HIV-1 p24 Gag expression in rBCG and infected antigen-presenting cells. We also found that compared to rBCG-pAL-p24 in a pAL5000 derived vector system, rBCG-pMyong2-p24 elicited enhanced p24-specific immune responses in vaccinated mice as evidenced by higher levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, gamma interferon ELISPOT cell induction, antibody production, and cytotoxic T lymphocytes (CTL) responses. Furthermore, rBCG-pMyong2-p24 showed a higher level of p24-specific Ab production than rSmeg-pMyong2-p24 in the same pMyong2 vector system. In conclusion, our data indicated that a live recombinant BCG expressing HIV-1 Gag using a pMyong2 vector system, rBCG-pMyong2-p24 elicited an enhanced immune response against HIV-1 infections in a mouse model system. So, rBCG-pMyong2-p24 may have the potential as a prime vaccine in a heterologous prime-boost vaccine strategy for HIV-1 infection.

Expression of Human Immunodeficiency Virus type 1 (HIV-1) coat protein genes in plants using cotton leaf curl Multan betasatellite-based vector.

It has already been demonstrated that a betasatellite associated with cotton leaf curl Multan virus (CLCuMB) can be used as a plant and animal gene delivery vector to plants. To examine the ability of CLCuMB as a tool to transfer coat protein genes of HIV-1 to plants, two recombinant CLCuMB constructs in which the CLCuMB βC1 ORF was replaced with two HIV-1 genes fractions including a 696 bp DNA fragment related to the HIV-1 p24 gene and a 1501 bp DNA fragment related to the HIV-1 gag gene were constructed. Gag is the HIV-1 coat protein gene and p24 is a component of the particle capsid. Gag and p24 are used for vaccine production. Recombinant constructs were inoculated to Nicotiana glutinosa and N. benthamiana plants in the presence of an Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]) as a helper virus. PCR analysis of inoculated plants indicated that p24 gene was successfully replicated in inoculated plants, but the gag gene was not. Real-time PCR and ELISA analysis of N. glutinosa and N. benthamiana plants containing the replicative forms of recombinant construct of CLCuMB/p24 indicated that p24 was expressed in these plants. This CLCuMB-based expression system offers the possibility of mass production of recombinant HIV-1 p24 protein in plants.

Sulfonate-ended carbosilane dendrimers with a flexible scaffold cause inactivation of HIV-1 virions and gp120 shedding.

Infection with human immunodeficiency virus type 1 (HIV-1) continues to be a global public health issue, especially in low-resource countries. Sexual transmission is responsible for the majority of HIV-1 infections worldwide. Women are more susceptible to HIV-1 acquisition than men and represent nearly 50% of the HIV-infected population. Topical vaginal microbicides that act at the earlier stages of infection offer a prevention strategy to reduce the acquisition of HIV-1. Dendrimers are nano-sized, radially symmetric molecules with a well-defined and monodisperse structure consisting of tree-like arms or branches. We perform a TZM.bl cell line-based screening of two families of carbosilane dendrimers (6 nanocompounds: G1-S12P, G2-S24P, G3-S48P, G1-C12P, G2-C24P and G3-C48P) that we have previously synthesized, containing 12, 24 or 48 sulfonate (or carboxylate) end-groups and a polyphenolic core. This work shows that second- and third-generation sulfonate-ended carbosilane dendrimers with a polyphenolic core (G2-S24P and G3-S48P, respectively) display low cytotoxicity (CC50 > 300 μM) with virucidal anti-R5-HIV-1 activity (EC50 < 50 nM; therapeutic index >6000) causing irreversible HIV-1 inactivation (80-90%) by loss of HIV-1 RNA (40%), gp120 shedding (70-80%) and p24 capsid protein release (45-60%). Herein, we demonstrate that sulfonate end-groups and a flexible scaffold from carbosilane dendrimers strongly influence their properties acting as potent virucides.

Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection.

Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection.

Antigen-presenting phagocytic cells ingest malaria parasites and increase HIV replication in a tumor necrosis factor α-dependent manner.

Plasmodium falciparum infection induces human immunodeficiency virus (HIV) replication and accelerates a decline in CD4(+) T-cell count. The mechanisms contributing to these interactions have not been fully elucidated. We infected peripheral blood mononuclear cells (PBMCs) with HIV type 1 (HIV-1) and then cocultured them with P. falciparum-infected red blood cells (iRBCs) or uninfected RBCs (uRBCs). Levels of HIV-1 p24 antigen and activation-associated cytokines were measured in culture supernatants. T-cell surface activation was assessed by flow cytometry. It has been reported that iRBCs increase HIV replication, compared with uRBCs; that neutralizing tumor necrosis factor α (TNF-α) abrogates this increase; and that hemozoin enhances HIV production. In this study, we confirmed that TNF-α plays an important role in this interaction. We show that iRBCs increased CD4(+) T-cell expression of HLA-DR(+)/CD38(+) (P = .001), that monocyte/macrophage depletion reduced HIV production by 40%-50% (P < .001), and that hemozoin-laden monocytes/macrophages that were preincubated with iRBCs also stimulated HIV production. iRBCs activate CD4(+) T cells and stimulate HIV replication in a TNF-α-dependent manner following malarial antigen processing by monocytes/macrophages. These results suggest that the persistent elevation of HIV replication during and after acute bouts of P. falciparum malaria may be due, at least in part, to ongoing stimulation of CD4(+) T cells by hemozoin-loaded antigen-presenting cells within lymphoid tissues.

Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4+ T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. Therefore, immediate protection at these surfaces is of high priority for the development of an HIV-1 vaccine. Thus, transgenic plants expressing HIV-1 antigens, which are exposed to immune competent cells in the GALT during oral administration, can be interesting as potential vaccine candidates. In the present study, we used two HIV-1 p24 antigen-expressing transgenic plant systems, Arabidopsis thaliana and Daucus carota, in oral immunization experiments. Both transgenic plant systems showed a priming effect in mice and induced humoral immune responses, which could be detected as anti-p24-specific IgG in sera after an intramuscular p24 protein boost. Dose-dependent antigen analyses using transgenic A. thaliana indicated that low p24 antigen doses were superior to high p24 antigen doses.

Infection of human pericytes by HIV‐1 disrupts the integrity of the blood–brain barrier

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) affects cross-talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. Although the toxicity of HIV-1 on neurons, astrocytes and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV-1 on pericytes. Therefore, the purpose of this study was to evaluate whether or not pericytes can be infected with HIV-1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV-1 receptor CD4 and co-receptors CXCR4 and CCR5. We also determined that HIV-1 can replicate, although at a low level, in human brain pericytes as detected by HIV-1 p24 ELISA. Pericytes were susceptible to infection with both the X4-tropic NL4-3 and R5-tropic JR-CSF HIV-1 strains. Moreover, HIV-1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV-1 and suggest that infected pericytes are involved in the progression of HIV-1-induced CNS damage.

HIV-1 p6 — a structured to flexible multifunctional membrane-interacting protein

The human immunodeficiency virus type 1 (HIV-1) p6 protein has recently been recognized as a docking site for several cellular and viral binding partners and is important for the formation of infectious viruses. Most of its known functions are suggested to occur under hydrophobic conditions near the cytoplasmic membrane, where the protein is presumed to exist in its most structured state. Although p6 is involved in manifold specific interactions, the protein has previously been considered to possess a random structure in aqueous solution. We show that p6 exhibits a defined structure with N- and C-terminal helical domains, connected by a flexible hinge region in 100mM dodecylphosphocholine micelle solution at pH 7 devoid of any organic co-solvents, indicating that this is a genuine limiting structural feature of the molecule in a hydrophobic environment. Furthermore, we show that p6 directly interacts with a cytoplasmic model membrane through both N-terminal and C-terminal regions by use of surface plasmon resonance (SPR) spectroscopy. Phosphorylation of Ser-40 located in the center of the C-terminal α-helix does not alter the secondary structure of the protein but amplifies the interaction with membranes significantly, indicating that p6 binds to the polar head groups at the surface of the cytoplasmic membrane. The increased hydrophobic membrane interaction of p623-52 S40F correlated with the observed increased amount of the polyprotein Gag in the RIPA insoluble fraction when Ser40 of p6 was mutated with Phe indicating that p6 modulates the membrane interactions of HIV-1 Gag.

Inhibition of HIV-1 replication by RNA with a microRNA-like function.

Human immunodeficiency virus type1 (HIV-1) replication is suppressed by a small guide RNA (sgRNA) that targets the packaging signal of HIV-1 RNA. We unintentionally produced a plasmid with the reverse sequence of the sgRNA and its terminator (pR-Ψ-sgRNA-ter). Both sgRNA and R-Ψ-sgRNA suppress HIV-1, but the mechanism by which R-Ψ-sgRNA suppresses HIV is not clear. To evaluate whether the suppressive effect is caused by an RNA interference or microRNA (miRNA)-like mechanism, R-Ψ-sgRNA was synthesized invitro and treated with the Dicer enzyme, an important enzyme for RNA interference and miRNA. The RNA was cleaved into fragments of approximately 24 nucleotides (nt). We analyzed the sequence of the RNA fragments and predicted the RNA secondary structure of R-Ψ-sgRNA to determine the region recognized by the Dicer enzyme. The lengths of the R-Ψ-sgRNA fragments ranged from 48 to 140nt, and were predicted to form double strands, including mismatches, in this region. An HIV-1 p24 assay indicated that the R-Ψ-sgRNA fragments suppressed HIV-1 replication. These findings suggest that R-Ψ-sgRNA acts as a miRNA to inhibit HIV-1.

Toxicology, biodistribution and shedding profile of a recombinant measles vaccine vector expressing HIV-1 antigens, in cynomolgus macaques

As a new human immunodeficiency virus type 1 (HIV-1) vaccine approach, the live-attenuated measles virus (MV) Schwarz vaccine strain was genetically engineered to express the F4 antigen (MV1-F4). F4 is a fusion protein comprising HIV-1 antigens p17 and p24, reverse transcriptase and Nef. This study assessed the toxicity, biodistribution and shedding profiles of MV1-F4. Cynomolgus macaques were intramuscularly immunized one or three times with the highest dose of MV1-F4 intended for clinical use, the reference (Schwarz) measles vaccine or saline, and monitored clinically for 11 or 85 days. Toxicological parameters included local and systemic clinical signs, organ weights, haematology, clinical and gross pathology and histopathology. Both vaccines were well tolerated, with no morbidity, clinical signs or gross pathological findings observed. Mean spleen weights were increased after three doses of either vaccine, which corresponded with increased numbers and/or sizes of germinal centers. This was likely a result of the immune response to the vaccines. Either vaccine virus replicated preferentially in secondary lymphoid organs and to a lesser extent in epithelium-rich tissues (e.g., intestine, urinary bladder and trachea) and the liver. At the expected peak of viremia, viral RNA was detected in some biological fluid samples from few animals immunized with either vaccine, but none of these samples contained infectious virus. In conclusion, no shedding of infectious viral particles was identified in cynomolgus monkeys after injection of MV1-F4 or Schwarz measles vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines in this study.

Oral delivery of transgenic plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to higher doses

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4+ T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. There ...

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

BackgroundHIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen.MethodA pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified.ResultsThe in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml.ConclusionsWhen combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

HIV-1 p6—Another viral interaction partner to the host cellular protein cyclophilin A

The 52-amino acid human immunodeficiency virus type 1 (HIV-1) p6 protein has previously been recognized as a docking site for several cellular and viral binding factors and is important for the formation of infectious viruses. A particular structural feature of p6 is the notably high relative content of proline residues, located at positions 5, 7, 10, 11, 24, 30, 37 and 49 in the sequence. Proline cis/trans isomerism was detected for all these proline residues to such an extent that more than 40% of all p6 molecules contain at least one proline in a cis conformation. 2D 1H nuclear magnetic resonance analysis of full-length HIV-1 p6 and p6 peptides established that cyclophilin A (CypA) interacts as a peptidyl–prolyl cis/trans isomerase with all proline residues of p6. Only catalytic amounts of CypA were necessary for the interaction with p6 to occur, strongly suggesting that the observed interaction is highly relevant in vivo. In addition, surface plasmon resonance studies revealed binding of full-length p6 to CypA, and that this binding was significantly stronger than any of its N- or C-terminal peptides. This study demonstrates the first identification of an interaction between HIV-1 p6 and the host cellular protein CypA. The mode of interaction involves both transient enzyme–substrate interactions and a more stable binding. The binding motifs of p6 to Tsg-101, ALIX and Vpr coincide with binding regions and catalytic sites of p6 to CypA, suggesting a potential role of CypA in modulating functional interactions of HIV-1.

Enhanced HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women correlates with increased inflammatory responses

AbstractKnowledge about early innate immune responses at the mucosal surfaces of the female genital tract is important in understanding the pathogenesis of heterosexual transmission of human immunodeficiency virus type-1 (HIV-1). As estradiol decreases inflammatory responses, we postulated that an estradiol-deficient state such as post-menopause could enhance expression of inflammatory factors that stimulate HIV-1 replication. We compare HIV-1 integration, transcription, and viral p24 release levels among ectocervical tissues obtained from pre- and post-menopausal donors. We detected enhanced HIV-1 p24 release levels in post- compared with pre-menopausal tissues (P<0.0001), but saw no difference in HIV-1 integration. Overall, 100% of post-menopausal tissues exhibited levels of HIV-1 transcription above background compared with only 60% of pre-menopausal tissues. Increased HIV-1 transcription was associated with enhanced interleukin (IL)-1β, IL-6, monocyte chemotactic protein-1, growth-regulated oncogene-α, and interferon-γ-inducible protein-10 expression. Neutralization and nuclear factor-κB-targeting small-interfering RNA experiments both decreased HIV-1 transcription, suggesting that the early inflammatory response may facilitate HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women.

Fine Epitope Specificity of Anti-erythropoietin Antibodies Reveals Molecular Mimicry With HIV-1 p17 Protein: A Pathogenetic Mechanism for HIV-1–Related Anemia

Circulating autoantibodies to endogenous erythropoietin (anti-Epo) are detected in human immunodeficiency virus type 1 (HIV-1)-infected patients and represent a risk factor for anemia. The aim of this study was to map the B-cell epitopes on the Epo molecule. Serum samples from HIV-1-positive patients and healthy individuals were tested against overlapping peptides covering the entire sequence of Epo. Serum samples from anti-Epo-positive patients exhibited significant binding to Epo epitopes spanning the following sequences: amino acids 1-20 (Ep1), amino acids 54-72 (Ep5), and amino acids 147-166 (Ep12). Structural analysis of erythropoietin revealed that the immunodominant epitopes, Ep1 and Ep12, comprise the interaction interface with Epo receptor (EpoR). Autoantibodies binding to this specific region are anticipated to inhibit the Epo-EpoR interaction, resulting in blunted erythropoiesis; this phenomenon is indicated by the significantly higher Epo levels and lower hemoglobin levels of anti-Ep1-positive patients compared with anti-Ep1-negative individuals. The region corresponding to the Ep1 epitope exhibited a 63% sequence homology with the ³⁴LVCASRELERFAVNPGLLE⁵² fragment of the HIV-1 p17 matrix protein. These results suggest that the main body of anti-Epo is directed against a functional domain of Epo, and that the presence of anti-Epo can be considered to be a result of a molecular mimicry mechanism, which is caused by the similarity between the Ep1 region and the p17 protein.

Evidence of a Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Machinery in HIV-1 Assembly and Release

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.