Actinohivin,a Broadly Neutralizing Prokaryotic Lectin,Inhibits HIV-1 Infection by Specifically Targeting High-Mannose-Type Glycans on the gp120 Envelope

The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants (K_D) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (α1,2-mannose)₃ oligosaccharide but not by α1,3/α1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of α1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (∼5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4⁺ T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4⁺ T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.The envelope glycoproteins of human immunodeficiency virus (HIV) mediate attachment and viral entry into susceptible target cells. The initial steps in HIV infection include binding of the trimeric gp120 with three CD4 receptor molecules, a number of conformational changes in gp120, and interactions with chemokine receptor CCR5 or CXCR4, followed by the unfolding of gp41, which anchors the virion to the target membrane and which brings the viral and cellular membranes in close contact for further virus-cell fusion (17).The HIV type 1 (HIV-1) gp120 envelope glycoprotein is highly glycosylated. Approximately half of its molecular weight is contributed by its carbohydrate content (22, 36). The recombinant HIV-1(III_B) gp120 expressed in Chinese hamster ovary (CHO) cells is occupied by 11 high-mannose- or hybrid-type glycans and 13 complex-type glycans (29).A variety of carbohydrate-binding agents (CBAs), such as the prokaryotic agent cyanovirin-N (CV-N) (7-10), the plant lectins Hippeastrum hybrid agglutinin (HHA) (1) and Galanthus nivalis agglutinin (GNA) (1), and the antibiotics pradimicin A and S (PRM-A and PRM-S, respectively) (4, 7), have been described to inhibit viral entry, presumably by their interaction with the glycans on HIV gp120. It has indeed been demonstrated that CBAs block virus entry by inhibiting the fusion of cell-free HIV virions with their target cells. They prevent the capture of virus particles by the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage mannose receptor, which are present on cells of the innate immune system. The subsequent transmission of the virus to CD4⁺ T cells is also efficiently hampered by CBAs, pointing to their potential as HIV microbicide drug candidates (6). By the lack of an efficient HIV vaccine, microbicides can evolve to be important tools for the prevention of HIV transmission and infection (23, 37, 38).One CBA, namely, the prokaryotic agent CV-N, has been tested in animal vaginal and rectal virus transmission models (43, 44) and shown to successfully inhibit viral infections in the animals, suggesting that CV-N has the potential to act as an anti-HIV microbicide. However, recently, it was described that CV-N induced the production of a variety of cytokines and cellular activation markers in peripheral blood mononuclear cells (PBMCs), and in addition, the pronounced mitogenic activity of CV-N was also observed (2, 26). These potential side effects can compromise the application of CV-N as an efficient microbicide.Actinohivin (AH) is an anti-HIV protein that has been isolated from a prokaryotic microorganism (the actinomycete Longispora albida K97-0003^T) (14, 27). AH consists of 114 amino acids (12,524.3 Da) and exhibits a unique highly conserved internal sequence triplication (60% identity and ∼70% homology on each segment). It was shown that the three repeats of AH are necessary to block syncytium formation in recombinant cell cultures (HeLa /T env tat and HeLa CD4 lacZ cells) (40, 41). Recently, the crystal structure of AH was solved, and it was revealed that AH has three sugar-binding pockets (41). It was suggested before that AH-gp120 interactions are accomplished by N-linked high-mannose-type carbohydrates that are abundantly present on gp120 (15, 41).We have now performed a detailed investigation of AH regarding its anti-HIV activity spectrum, potential side effects, kinetic interaction with the HIV-1 envelope proteins gp120 and gp41, and resistance spectrum and also added data on other CBAs that are related to AH in terms of their carbohydrate specificities (i.e., HHA and monoclonal antibody [MAb] 2G12) for comparative reasons. Based on its safety profile and unique kinetic/antiviral properties our data revealed that AH qualifies as a potential drug lead for further preclinical investigations.     

ADS-J1 Inhibits HIV-1 Entry by Interacting with gp120 and Does Not Block Fusion-Active gp41 Core Formation

We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone.The essential steps of HIV-1 entry in the host cell offer several potential targets for the development of novel antiviral agents (19, 24, 33, 42). Agents that disrupt gp41-mediated membrane fusion, collectively called fusion inhibitors, were the first entry inhibitors to be approved for the treatment of HIV infection. Enfuvirtide (T20, Fuzeon) is a 36-amino-acid synthetic peptide with a sequence identical to a part of the C-terminal heptad repeat 2 (HR2) region of gp41 that binds to the N-terminal heptad repeat 1 (HR1) in an antiparallel manner, forming a coiled-coil structure during the prefusion step. Mutations in the highly conserved amino acid motif 36 to 45 in the HR1 domain confer resistance to T20 (35), providing strong evidence that HR1 is the site of interaction of T20. However, mutations in other regions of HIV-1 envelope (Env) have been also associated with T20 resistance (26, 27).Several low-molecular-weight (SMW) compounds have been identified as blockers of the initial steps of virus entry, including CCR5 coreceptor (33, 42). However, the identification of SMW compounds targeting gp41 has been elusive. A polyanionic compound, ADS-J1, was previously identified in silico as a potential candidate and shown to bind to gp41 peptides and interfere with the formation of the gp41 coiled-coil domain in an in vitro enzyme-linked immunosorbent assay (ELISA) model of HR1-HR2 interaction (16, 30, 31). Conversely, we had shown that ADS-J1 blocked the binding of HIV particles to lymphoid MT-4 cells and inhibited HIV replication at a time/site of interaction similar to those of the polyanion dextran sulfate (DS), a well-described, nonspecific inhibitor of virus entry (3). Moreover, at least four HIV-1 strains resistant to ADS-J1 were generated. The resistance to ADS-J1 was associated with gp120 based on the fact that the majority of the mutations were located in the gp120 coding sequence, mainly in the V3 loop region. Although three of the resistant strains contained mutations in gp41, one of them, HIV-1 ARA45C, did not (3). In addition, molecular modeling suggested that the gp120 V3 loop was the preferential binding site for ADS-J1 onto HIV-1, and mutations induced by the inhibitor significantly changed the stereoelectronic properties of the gp120 surface, justifying a marked drop in the affinity of ADS-J1 toward an ADS-J1-resistant HIV-1 strain (36). At that time, we considered conclusive the evidence of the mode of action of ADS-J1.More recently, Wang et al. (43) suggested that ADS-J1 could bind directly to a trimeric peptide containing the gp41 pocket region (IQN17) in a surface plasmon resonance (SPR) assay and indicated that ADS-J1 can be used as a lead compound for the design of novel HIV-1 fusion inhibitors (44). Therefore, we thought it relevant to provide further evidence of the mode of action of ADS-J1.     

Identification of a CD36-related Thrombospondin 1–binding Domain in HIV-1 Envelope Glycoprotein gp120: Relationship to HIV-1–specific Inhibitory Factors in Human Saliva

Human and non–human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1–gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1–gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.     

Autoimmune anti-HIV-1gp120 antibody with antiidiotype-like activity in sera and immune complexes of HIV-1-related immunologic thrombocytopenia.

Autoimmune antiidiotype-like antibody (Ab2) directed against anti-HIV-1gp120 (Ab1) was found in high titer in the sera of 10 consecutive homosexual and 11 narcotic addict HIV-1-related immunologic thrombocytopenia (HIV-1-ITP) patients, was barely detectable in 10 nonthrombocytopenic HIV-1 sero-positive individuals, and was not detectable in 5 normal subjects by use of a solid-phase RIA. Reactivity of autologous Ab2 for Ab1 was 4-120-fold greater than Ab2 for homologous Ab1. Affinity-purified Ab2 did not block the binding of affinity-purified Ab1 to its HIV-1gp120 epitopes on immunoblot, indicating the absence of "internal image" antiidiotype. Both Ab1 and Ab2 are precipitable from sera with polyethylene glycol (PEG) and present in a macromolecular complex that is excluded by gel filtration on G200 and contains IgG, IgM, C3, and the anti-F(ab')2 antiidiotype-like complex. PEG-precipitable complexes bind to platelets in a saturation-dependent manner. Neither affinity-purified Ab1 nor Ab2 binds to platelets. However, the combination of Ab1 and Ab2 (preincubated for 2 h at 22 degrees C) binds to platelets in a saturation-dependent manner at an optimum ratio range of 10-20:1. Ab2 reactivity correlates with serum PEG-precipitable immune complex level (r = 0.91; P less than 0.001) and with thrombocytopenia (r = 0.89; P less than 0.001). We suggest that the anti-HIV-1gp120 antiidiotype-like complex contributes to the markedly elevated platelet Ig and C3 level of HIV-1-ITP patients and propose that this may contribute to their thrombocytopenia.     

Mutation in the D1 domain of CD4 receptor modulates the binding affinity to HIV-1 gp120

The gp120 surface subunit of HIV-1 envelope lycoprotein (Env) is the key component for the viral entry process through interaction with the CD4 binding site (CD4bs) of the primary receptor CD4. The point mutant was introduced into SD1, a CD4 D1 variant, to enhance the interaction with HIV-1 gp120.The three-dimensional structures of gp120 and SD1 were determined using homology modeling based on the results previously determined by X-ray crystallography. The binding models were carried out via protein–protein docking tools. The 5 best docking solutions were retained according to the docking scores and were used for structural assessment. Our results demonstrated the consistency between the 3D models of gp120 and SD1 predicted by molecular docking calculations and the co-crystallized data available. We first discovered that most residues in SD1 that interacted with gp120 were located within the region 6–94 of the first N-terminal D1 domain of CD4. SD1 bound to gp120 stably at which 15 residues formed 20 hydrogen bonds with 16 residues of gp120. Five pairs of electrostatic interactions between positively and negatively charged side chains of amino acids were identified in the SD1-gp120 interface, which showed an increased number of electrostatic interactions with gp120. The mutant in the D1 domain of human CD4 receptor could strengthen binding affinity with HIV-1 gp120 and might improve the interaction pattern of the neighboring residues. The sequence analysis of gp120 suggested that Asp186, Asn189, Arg191, Glu293, Phe318 and Tyr319 were located in the variable regions of gp120, which may be HIV-1 AE strain-specific amino acid residues. Together, the results presented in this study contributed to a better understanding of the changes in the interaction between the gp120 protein and the human host CD4 receptor associated with point mutation in the D1 domain. The stabilized derivative of human CD4 D1 should serve as a promising target for therapeutics development in HIV-1 vaccine and viral entry inhibitor and may warrant further investigation.

HIV-1 gp120 is the key component for viral entry through interaction with CD4 D1. The 5 best docking solutions were retained according to the docking scores. SD1 bound to gp120 at which 15 residues formed 20 hydrogen bonds with 16 residues of gp120.     

HIV-1 gp120 modulates the immunological function and expression of accessory and co-stimulatory molecules of monocyte-derived dendritic cells

Initiation of a primary immune response requires antigen specific CD4(+) T helper (T(h)) cells to assist in priming of CD8(+) cytotoxic T cell (CTL) activity. This is optimal when T(h) cells and CTL recognize antigen when presented to them by a dendritic cell (DC) in the context of major histocompatibility complex (MHC) class I and class II complexes. We have hypothesized that human DC exposed to HIV-1 gp120 IIIB envelope glycoprotein may activate or alter the immunological activation of DCs. Our findings have led us to conclude that HIV-1 gp120 LAV/IIIB activates monocyte-derived DC when they are in their immature state while HIV-1 gp120 exhibits highly selective effects on mature DC. We have observed that following maturation of DCs with lipopolysaccharide (LPS) that they are less susceptible to the modulatory effects of gp120. Although HIV-1 gp120 activates immature DC, it does so in a manner that abrogates their normal function in host immune responses and consequently disturbs the homeostatic balance of host immune response to infection. We suggest that HIV-1 gp120 may support sustained productive infection and transinfection of activated T cells that cluster with gp120-activated DC. We believe that these are promoted by mechanisms that are dependent, at least in part, on altered cytokine responses, enhanced expression of cellular adhesion molecules and augmented DC-mediated activation of T cells in nonspecific and antigen-specific immune reactivities. Consequently, HIV-1 gp120 may actively contribute to the immunopathogenesis of AIDS.     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

Coadsorption of HIV-1 p24 and gp120 proteins to surfactant-free anionic PLA nanoparticles preserves antigenicity and immunogenicity.

Biodegradable micro- or nanoparticles with surface adsorbed antigens represent a promising method for in vivo delivery of vaccines. Most vaccines, licensed or under development, are based on combined delivery of multiple antigens. Thus, we investigated the feasibility of combining two vaccine antigens, HIV-1 p24 and gp120 proteins, on the surface of surfactant-free anionic PLA nanoparticles obtained by an improved solvent diffusion method. The analysis of adsorption isotherms has shown that both proteins had similar and high affinities for the nanoparticles. Coadsorption of p24 and gp120 onto the same PLA particle was evidenced by sandwich ELISA, using antibodies directed against one protein for particle capture and the other one for detection. To assess structural integrity, the antigenicity of free and PLA-adsorbed antigens was compared by competition ELISA, using a set of 6 anti-p24 and 7 anti-gp120 antibodies, as well as soluble CD4. The antigenicity of proteins on the nanoparticle surface was well preserved, adsorbed either individually or in combination. Furthermore, both antigens maintained their immunogenicity, since high antibody titres (10(6) for p24 and 10(5) for gp120) were elicited in mice with monovalent and divalent PLA formulations. Taken together our results show that development of multivalent vaccines based on anionic PLA nanoparticles is possible. Moreover, coadsorption of a ligand for cell-specific targeting or of an immunostimulatory molecule will further extend the field of application of delivery systems based on charged micro- and nanoparticles.     

Interaction of Chemokine Receptor CCR5 with its Ligands: Multiple Domains for HIV-1 gp120 Binding and a Single Domain for Chemokine Binding

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α, and MIP-1β, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1α, or MIP-1β. This mAb inhibited most of the RANTES and MIP-1α chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH₂-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1–derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH₂-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.     

Assays to detect and characterize human immunodeficiency virus type 1 (HIV-1) receptor antagonists, compounds that inhibit binding of the HIV-1 surface glycoprotein, gp120, to the CD4 receptor on human T lymphocytes.

Human immunodeficiency virus type 1 infects human helper T lymphocytes by an interaction between gp120, the viral coat protein, and the T-cell receptor CD4. Two microtiter-based immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a particle concentration fluorescence assay, were developed to measure gp120-CD4 binding and were then used to screen a variety of compounds for the inhibition of this interaction. Additional protocols, called "consumption assays," were defined to distinguish inhibitors which functioned by sequestering either gp120 or CD4 to prevent the final effective bimolecular interaction. Monoclonal antibodies of defined specificity and compounds known from other published studies to inhibit gp120-CD4 binding were tested in an attempt to validate the assays used in the study. Once the capacity of these assays to detect known gp120-CD4 inhibitors was confirmed, they were used to screen synthetic agents and fermentation broths for novel compounds that might be used as human immunodeficiency virus receptor antagonists. A 2,4-diaminoquinazoline, CP-101,816-1, was found to inhibit this interaction (50% inhibitory concentration in ELISA, 32.5 micrograms/ml) and to interact more strongly with CD4 than with gp120 in the consumption assays. The identification of a novel inhibitor, a 2,4-diaminoquinazoline, confirmed that such assays are useful for the detection of human immunodeficiency virus type 1 receptor antagonists.     

Safety,Tolerability, and Pharmacokinetics of Ribavirin in Hepatitis C Virus-Infected Patients with Various Degrees of Renal Impairment

Ribavirin (RBV) is an integral part of standard-of-care hepatitis C virus (HCV) treatments and many future regimens under investigation. The pharmacokinetics (PK), safety, and tolerability of RBV in chronically HCV-infected patients with renal impairment are not well defined and were the focus of an open-label PK study in HCV-infected patients receiving RBV plus pegylated interferon. Serial RBV plasma samples were collected over 12 h on day 1 of weeks 1 and 12 from patients with moderate renal impairment (creatinine clearance [CL_CR], 30 to 50 ml/min; RBV, 600 mg daily), severe renal impairment (CL_CR, <30 ml/min; RBV, 400 mg daily), end-stage renal disease (ESRD) (RBV, 200 mg daily), or normal renal function (CL_CR, >80 ml/min; RBV, 800 to 1,200 mg daily). Of the 44 patients, 9 had moderately impaired renal function, 10 had severely impaired renal function, 13 had ESRD, and 12 had normal renal function. The RBV dose was reduced because of adverse events (AEs) in 71% and 53% of severe and moderate renal impairment groups, respectively. Despite this modification, patients with moderate and severe impairment had 12-hour (area under the concentration-time curve from 0 to 12 h [AUC_0–12]) values 36% (38,452 ng · h/ml) and 25% (35,101 ng · h/ml) higher, respectively, than those with normal renal function (28,192 ng · h/ml). Patients with ESRD tolerated a 200-mg daily dose, and AUC_0–12 was 20% lower (22,629 ng · h/ml) than in patients with normal renal function. PK modeling and simulation (M&S) indicated that doses of 200 mg or 400 mg alternating daily for patients with moderate renal impairment and 200 mg daily for patients with severe renal impairment were the most appropriate dose regimens in these patients.     

HIV gp120 H375 is unique to HIV-1 subtype CRF01_AE and confers strong resistance to the entry inhibitor BMS-599793, a candidate microbicide drug

BMS-599793 is a small molecule entry inhibitor that binds to human immunodeficiency virus type 1 (HIV-1) gp120, resulting in the inhibition of CD4-dependent entry into cells. Since BMS-599793 is currently considered a candidate microbicide drug, we evaluated its efficacy against a number of primary patient HIV isolates from different subtypes and circulating recombinant forms (CRFs) and showed that activity varied between ～3 ρM and 7 μM at 50% effective concentrations (EC(50)s). Interestingly, CRF01_AE HIV-1 isolates consistently demonstrated natural resistance against this compound. Genotypic analysis of >1,600 sequences (Los Alamos HIV sequence database) indicated that a single amino acid polymorphism in Env, H375, may account for the observed BMS-599793 resistance in CRF01_AE HIV-1. Results of site-directed mutagenesis experiments confirmed this hypothesis, and in silico drug docking simulations identified a drug resistance mechanism at the molecular level. In addition, CRF01_AE viruses were shown to be resistant to multiple broadly neutralizing monoclonal antibodies. Thus, our results not only provide insight into how Env polymorphisms may contribute to entry inhibitor resistance but also may help to elucidate how HIV can evade some broadly neutralizing antibodies. Furthermore, the high frequency of H375 in CRF01_AE HIV-1, and its apparent nonoccurrence in other subtypes, could serve as a means for rapid identification of CRF01_AE infections.     

Insights into the molecular mechanism underlying CD4-dependency and neutralization sensitivity of HIV-1: a comparative molecular dynamics study on gp120s from isolates with different phenotypes

The envelope (Env) of HIV-1 plays critical roles in viral infection and immune evasion. Although structures of prefusion Env have been determined and phenotypes relevant to the CD4 dependency and the neutralization sensitivity for various HIV-1 isolates have been identified, the detailed structural dynamics and energetics underlying these two phenotypes have remained elusive. In this study, two unliganded structural models of gp120, one from the CD4-dependent, neutralization-resistant isolate H061.14 and the other from the CD4-independent, neutralization-sensitive R2 strain, were constructed, and subsequently were subjected to multiple-replica molecular dynamics (MD) simulations followed by free energy landscape (FEL) construction. Comparative analyses of MD trajectories reveal that during simulations R2-gp120 demonstrated larger structural fluctuations/deviations and higher global conformational flexibility than H061.14-gp120. Close comparison of local conformational flexibility shows that some of the structural regions involving direct interactions with gp41 and adjacent gp120 subunits in the context of the closed trimeric Env exhibit significantly higher flexibility in R2-gp120 than in H061.14-gp120, thus likely increasing the probability for R2-Env to open the trimer crown and prime gp41 fusogenic properties without induction by CD4. Collective motions derived from principal component analysis (PCA) reveal that R2-gp120 is prone to spontaneous transition to the neutralization-sensitive CD4-bound state while H061.14-gp120 tends to maintain the neutralization-resistant unliganded state. Finally, comparison between FELs reveals that R2-gp120 has larger conformational entropy, richer conformational diversity, and lower thermostability than H061.14-gp120, thus explaining why R2-gp120 is more structurally unstable and conformationally flexible, and has a higher propensity to transition to the CD4-bound state than H061.14-gp120. The present results reveal that the differences in dynamics and energetics between R2-gp120 and H061.14-gp120 impart Env trimers with distinct capacities to sample different states (i.e., R2-Env samples more readily the open state while H061.14-Env is more inclined to maintain the closed state), thus shedding light on the molecular mechanism underlying the HIV-1 phenotype associated with CD4 dependency/neutralization sensitivity.

The envelope (Env) of HIV-1 plays critical roles in viral infection and immune evasion.     

Regulation of CXCR4 receptor dimerization by the chemokine SDF-1alpha and the HIV-1 coat protein gp120: a fluorescence resonance energy transfer (FRET) study

Both the chemokine SDF-1alpha and the human immunodeficiency virus-1 (HIV-1) coat protein gp120 can bind to CXCR4 chemokine receptors but with different signaling consequences. To understand the molecular basis for these differences, we tagged the rat CXCR4 receptor with enhanced cyan (ECFP) and yellow (EYFP) derivatives of the green fluorescent protein and investigated CXCR4 receptor dimerization in human embryonic kidney (HEK)-tsA201 cells using fluorescence resonance energy transfer (FRET). Elevated FRET was detected under basal conditions from EYFP-CXCR4 and ECFP-CXCR4 receptor-transfected cells indicating a high level of CXCR4 receptor dimerization. In comparison, EYFP-CXCR4 and ECFP-mu-opioid receptor-cotransfected cells displayed a much lower FRET signal. The FRET signal resulting from EYFP-CXCR4- and ECFP-CXCR4-expressing cells could be attenuated by coexpressing nontagged CXCR4 receptors suggesting competition with fluorophore-tagged receptors in the membrane. Nontagged mu-opioid, kappa-opioid, and muscarinic receptors also decreased the FRET between the tagged CXCR4 receptor pairs but to a lesser extent. Application of the CXCR4 receptor agonist SDF-1alpha (50 nM) further increased the FRET signal from tagged CXCR4 receptors, an effect that was inhibited by the CXCR4 antagonist AMD3100. SDF-1alpha had no effect when EYFP-CXCR4 and ECFP-mu-opioid receptors were coexpressed. The effect of gp120IIIB on CXCR4 FRET was dependent on the coexpression of human CD4 (hCD4) when it increased the FRET signal, and this was decreased by AMD3100 pretreatment. FRET analysis of tagged hCD4 constructs demonstrated that there was significant association of hCD4 and CXCR4, as well as hCD4 dimerization. These data suggest that CXCR4 dimerization is involved in SDF-1alpha- and gp120-induced signaling events.     

Protecting neurons from HIV-1 gp120-induced oxidant stress using both localized intracerebral and generalized intraventricular administration of antioxidant enzymes delivered by SV40-derived vectors

Human immunodeficiency virus-1 (HIV-1) is the most frequent cause of dementia in adults under 40. We sought to use gene delivery to protect from HIV-1-related neuron loss. Because HIV-1 envelope (Env) gp120 elicits oxidant stress and apoptosis in cultured neurons, we established reproducible parameters of Env-mediated neurotoxicity in vivo, then tested neuroprotection using gene delivery of antioxidant enzymes. We injected 100-500 ng mul(-1)gp120 stereotaxically into rat caudate-putamens (CP) and assayed brains for apoptosis by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) 6-h to 14-day post-injection. Peak apoptosis occurred 1 day after injection of 250 and 500 ng microl(-1)gp120. TUNEL-positive cells mostly expressed neuronal markers (NeuroTrace), although some expressed CD68 and so were most likely microglial cells. Finally, we compared neuroprotection from gp120-induced apoptosis provided by localized and generalized intra-central nervous system (CNS) gene delivery. Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) were injected into the CP, where gp120 was administered 4-24 weeks later. Alternatively, we inoculated the vector into the lateral ventricle (LV), with or without prior intraperitoneal (i.p.) administration of mannitol. Intracerebral injection of SV(SOD1) or SV(GPx1) significantly protected neurons from gp120-induced apoptosis throughout the 24-week study. Intraventricular vector administration protected from gp120 neurotoxicity comparably, particularly if preceded by mannitol i.p. Thus, HIV-1 gp120 is neurotoxic in vivo, and intracerebral or intra-ventricular administration of rSV40 vectors carrying antioxidant enzymes is neuroprotective. These findings suggest the potential utility of both localized and widespread gene delivery in treating neuroAIDS and other CNS diseases characterized by excessive oxidative stress.