Clinical outcome of concomitant vs interrupted BRAF inhibitor therapy during radiotherapy in melanoma patients.

This corrects the article DOI: 10.1038/bjc.2017.85.

Lectin Affinity Plasmapheresis for Middle East Respiratory Syndrome-Coronavirus and Marburg Virus Glycoprotein Elimination.

BACKGROUND/AIMS: Middle East respiratory syndrome coronavirus (MERS-CoV) and Marburg virus (MARV) are among the World Health Organization's top 8 emerging pathogens. Both zoonoses share nonspecific early symptoms, a high lethality rate, and a reduced number of specific treatment options. Therefore, we evaluated extracorporeal virus and glycoprotein (GP) elimination by lectin affinity plasmapheresis (LAP). METHODS: For both MERS-CoV (pseudovirus) as well as MARV (GPs), 4 LAP devices (Mini Hemopurifiers, Aethlon Medical, San Diego, CA, USA) and 4 negative controls were tested. Samples were collected every 30 min and analyzed for reduction in virus infectivity by a flow cytometry-based infectivity assay (MERS-CoV) and in soluble GP content (MARV) by an immunoassay. RESULTS: The experiments show a time-dependent clearance of MERS-CoV of up to 80% within 3 h (pseudovirus). Up to 70% of MARV-soluble GPs were eliminated at the same time. Substantial saturation of the binding resins was detected within the first treatment hour. CONCLUSION: MERS-CoV (pseudovirus) and MARV soluble GPs are eliminated by LAP in vitro. Considering the high lethality and missing established treatment options, LAP should be evaluated in vivo. Especially early initiation, continuous therapy, and timed cartridge exchanges could be of importance.

Systemic therapy of metastatic melanoma.

For patients with metastatic melanoma, there are currently several effective therapeutic options. The BRAF inhibitors vemurafenib and dabrafenib are characterized by rapid tumor control and high response rates. In combination with one of the two MEK inhibitors trametinib and cobimetinib, they achieve response rates (CR + PR, complete plus partial remissions) of 70%, while delaying the development of treatment resistance, as well as a median overall survival of > 2 years with tolerable side effects. Showing long-term survival rates of approximately 20%, the anti-CTLA-4 antibody ipilimumab is the first substance that has led to a significant prolongation of overall survival in patients with metastatic melanoma. However, delayed treatment response and severe immune-mediated side effects may pose limitations to its therapeutic benefit. Usually well tolerated, anti-PD-1 antibody monotherapy using nivolumab and pembrolizumab has yielded response rates (CR + PR) of up to 45% and one-year survival rates of > 70%. The combination of ipilimumab and nivolumab has shown response rates of up to 58% and a median progression-free survival of > 11 months. While this combination is expected to result in a rapid and long-lasting response, this potential benefit comes at the expense of a high level of toxicity. Strategies for treatment sequencing and treatment combinations are currently being investigated in clinical studies. Overall, the prognosis for patients with metastatic melanoma has significantly improved. With long-term survival a possibility, not only acute but also long-term therapeutic side effects must be taken into account.

Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers.

BACKGROUND: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure. RESULTS: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction. CONCLUSIONS: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library.

HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIV-neutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development.

Influenza A virus replicates productively in primary human kidney cells and induces factors and mechanisms related to regulated cell death and renal pathology observed in virus-infected patients.

Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.

Phage display-directed discovery of LEDGF/p75 binding cyclic peptide inhibitors of HIV replication.

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.

Comparison of three humanized mouse models for adoptive T cell transfer.

BACKGROUND: Humanized mouse models for adoptive T cell transfer are important for preclinical efficacy and toxicity studies. However, common xenograft models using immunodeficient mice have so far failed to efficiently support the homing of human T cells to secondary lymphoid tissues. METHODS: We established a new mouse model for the adoptive transfer of genetically-modified (gm) T cells using conditioned BALB/c mice. Conditioning involved cyclophosphamide injections, lethal irradiation and radioprotection with bone marrow from immunodeficient mice. We compared repopulation kinetics and the quality of grafts in these modified Trimera (mT3) mice with immunodeficient BALB/c Rag2(-/-) interleukin (IL)2 receptor gamma (rg) knockout (DKO) and NOD/LtSz-scid IL2rg(-/-) (NSG) recipient mouse strains. RESULTS: DKO mice showed only marginal engraftment until onset of graft-versus-host disease, whereas mT3 and NSG were repopulated with comparable kinetics. However, T cell repertoire and human cytokine profiles suggest a xenoreactivity-driven gm T cell expansion in mT3 mice, whereas NSG mice were characterized by an initial homeostatic proliferation. Morphological analysis revealed high levels of human gm T cell infiltration in the spleen and liver of all three mouse strains. However, mT3 mice provided the strongest homing of human gm T cells to mucosal sites. Additionally, mT3 mice were the only model with macroscopically visible superficial inguinal lymph nodes. These lymph nodes strongly supported the homing of gm T cells. CONCLUSIONS: In the present study, we give proof-of-concept that wild-type mice can accept gm T cell grafts while providing secondary lymphoid structures. Despite limitations, mT3 mice are a valid alternative for applications that specifically rely on improved secondary lymphoid structures.

Pseudotyping vesicular stomatitis virus with lymphocytic choriomeningitis virus glycoproteins enhances infectivity for glioma cells and minimizes neurotropism.

Vesicular stomatitis virus (VSV)-based oncolytic virotherapy has the potential to significantly improve the prognosis of aggressive malignancies such as brain cancer. However, VSV's inherent neurotoxicity has hindered clinical development so far. Given that this neurotropism is attributed to the glycoprotein VSV-G, VSV was pseudotyped with the nonneurotropic envelope glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP→VSV-GP). Compared to VSV, VSV-GP showed enhanced infectivity for brain cancer cells in vitro while sparing primary human and rat neurons in vitro and in vivo, respectively. In conclusion, VSV-GP has a much wider therapeutic window than VSV and is thus more suitable for clinical applications, especially in the brain.

Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.

The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC(4) -glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions.

Tumours involving the retrobulbar space in cats: 37 cases.

OBJECTIVES: The aim of this multicentre retrospective study was to describe the clinical presentation, imaging findings, diagnosis and outcomes of cats with retrobulbar neoplasia. METHODS: A total of 37 cats that were diagnosed with retrobulbar neoplasia and underwent advanced imaging were recruited from searches of the clinical records of two referral hospitals. All cats had neoplasia confirmed via cytology or histopathology. Data relating to the signalment, presentation, results of investigations, treatment and outcome were recorded. A review of imaging studies was performed where possible. RESULTS: In total, 23 cases (62%) were presented with respiratory signs. Exophthalmos was the most common ophthalmological examination finding, present in 18 cases (49%). Thirty-two cases (86%) had secondary extension of neoplasia to the retrobulbar space (most commonly from the nasal cavities), present in 20 cases (54%), of which 12 were lymphoma. In cases where contrast was administered, 28/35 (80%) had contrast-enhancing masses. Orbital extension was detected in 21 cases (57%), exophthalmos in 22 (59%), globe deformation in 12 (32%) and local lymphadenomegaly in 22 (61%). In total, 36 (97%) retrobulbar tumours were malignant. Thoracic imaging, where it was performed, was concerning for metastasis in 8/25 cases (31%), with abdominal imaging suggestive of metastasis in 5/12 (42%). The most common diagnosis was lymphoma with 19 cases (51%), with nasal lymphoma representing 12 of these, followed by carcinoma in 10 (27%). The median survival time, for cases where death was recorded, was 85 days (range 1-263 days). CONCLUSIONS AND RELEVANCE: To the authors' knowledge, this is the largest study of neoplasia affecting the feline retrobulbar space. Retrobulbar tumours in cats are overwhelmingly malignant, and commonly due to secondary extension of tumours originating elsewhere. Lymphoma, particularly arising from the nasal cavities, was the most common cause. Cats presenting with signs suggestive of retrobulbar disease should be assessed for disease affecting any of the structures of the head.

HIV-1 Infection of Long-Lived Hematopoietic Precursors In Vitro and In Vivo.

Latent reservoirs in human-immunodeficiency-virus-1 (HIV-1)-infected individuals represent a major obstacle in finding a cure for HIV-1. Hematopoietic stem and progenitor cells (HSPCs) have been described as potential HIV-1 targets, but their roles as HIV-1 reservoirs remain controversial. Here we provide additional evidence for the susceptibility of several distinct HSPC subpopulations to HIV-1 infection in vitro and in vivo. In vitro infection experiments of HSPCs were performed with different HIV-1 Env-pseudotyped lentiviral particles and with replication-competent HIV-1. Low-level infection/transduction of HSPCs, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPP), was observed, preferentially via CXCR4, but also via CCR5-mediated entry. Multi-lineage colony formation in methylcellulose assays and repetitive replating of transduced cells provided functional proof of susceptibility of primitive HSPCs to HIV-1 infection. Further, the access to bone marrow samples from HIV-positive individuals facilitated the detection of HIV-1 gag cDNA copies in CD34+ cells from eight (out of eleven) individuals, with at least six of them infected with CCR5-tropic HIV-1 strains. In summary, our data confirm that primitive HSPC subpopulations are susceptible to CXCR4- and CCR5-mediated HIV-1 infection in vitro and in vivo, which qualifies these cells to contribute to the HIV-1 reservoir in patients.

Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.

During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.

Repulsive axon guidance molecule Slit3 is a novel angiogenic factor.

Slits are large, secreted repulsive axon guidance molecules. Recent genetic studies revealed that the Slit3 is dispensable for neural development but required for non-neuron-related developmental processes, such as the genesis of the diaphragm and kidney. Here we report that Slit3 potently promotes angiogenesis, a process essential for proper organogenesis during embryonic development. We observed that Slit3 is expressed and secreted by both endothelial cells and vascular smooth muscle cells in vasculature and that the Slit cognate receptors Robo1 and Robo4 are universally expressed by endothelial cells, suggesting that Slit3 may act in paracrine and autocrine manners to regulate endothelial cells. Cellular function studies revealed that Slit3 stimulates endothelial-cell proliferation, promotes endothelial-cell motility and chemotaxis via interaction with Robo4, and accelerates endothelial-cell vascular network formation in vitro with a specific activity comparable with vascular endothelial growth factor. Furthermore, Slit3 stimulates neovessel sprouting ex vivo and new blood vessel growth in vivo. Consistent with these observations, the Slit3 knockout mice display disrupted angiogenesis during embryogenesis. Taken together, our studies reveal that the repulsive axon guidance molecule Slit3 is a novel and potent angiogenic factor and functions to promote angiogenesis in coordinating organogenesis during embryonic development.

Changes in retinal vascular diameters in senior and geriatric cats in association with variation in systemic blood pressure.

OBJECTIVES: Early diagnosis of arterial hypertension is essential to prevent target organ damage. In humans, retinal arteriolar narrowing predicts hypertension. This blinded prospective observational study investigated the retinal vessel diameters in senior and geriatric cats of varying systolic blood pressure (SBP) status and evaluated retinal vascular changes in hypertensive cats after treatment. METHODS: Cats with a median age of 14 years (range 9.1-22 years) were categorised into five groups: group 1, healthy normotensive (SBP <140 mmHg; n = 40) cats; group 2, pre-hypertensive (SBP 140-160 mmHg; n = 14) cats; group 3, cats with chronic kidney disease (CKD) and normotensive (n = 26); group 4, cats with CKD and pre-hypertensive (n = 13); and group 5, hypertensive cats (SBP >160 mmHg, n = 15). Colour fundus images (Optibrand ClearView) were assessed for hypertensive lesions. Retinal vascular diameters and bifurcation angles were annotated and calculated using the Vascular Assessment and Measurement Platform for Images of the Retina annotation tool (VAMPIRE-AT). When available, measurements were obtained at 3 and 6 months after amlodipine besylate treatment. RESULTS: Ten hypertensive cats had retinal lesions, most commonly intraretinal haemorrhages and retinal exudates. Arteriole and venule diameters decreased significantly with increasing age (-0.17 ± 0.05 pixels/year [P = 0.0004]; -0.19 ± 0.05 pixels/year). Adjusted means ± SEM for arteriole and venule diameter (pixels) were 6.3 ± 0.2 and 8.9 ± 0.2 (group 1); 7.6 ± 0.3 and 10.1 ± 0.4 (group 2); 6.9 ± 0.2 and 9.5 ± 0.3 (group 3); 7.4 ± 0.3 and 10.0 ± 0.4 (group 4); and 7.0 ± 0.3 and 9.8 ± 0.4 (group 5). Group 1 arteriole and venule diameters were significantly lower than those of groups 2 and 4. Group 2 arteriole bifurcation angle was significantly narrower than those of groups 1 and 3. Post-treatment, vessel diameters decreased significantly at 3 and 6 months in seven hypertensive cats. CONCLUSIONS AND RELEVANCE: Increased age was associated with reduced vascular diameters. Longitudinal studies are required to assess if vessel diameters are a risk indicator for hypertension in cats.

Inhibition of HIV-1 replication by small interfering RNAs directed against glioma pathogenesis related protein (GliPR) expression.

BACKGROUND: Previously, we showed that glioma pathogenesis related protein (GliPR) is induced in CEM T cells upon HIV-1 infection in vitro. To examine whether GliPR plays a role as HIV dependency factor (HDF), we tested the effect of GliPR suppression by siRNA on HIV-1 replication. RESULTS: Induction of GliPR expression by HIV-1 was confirmed in P4-CCR5 cells. When GliPR was suppressed by siRNA, HIV-1 replication was significantly reduced as measured by HIV-1 transcript levels, HIV-1 p24 protein levels, and HIV-1 LTR-driven reporter gene expression, suggesting that GliPR is a cellular co-factor of HIV-1. Microarray analysis of uninfected HeLa cells following knockdown of GliPR revealed, among a multitude of gene expression alterations, a down-regulation of syndecan-1, syndecan-2, protein kinase C alpha (PRKCA), the catalytic subunit beta of cAMP-dependent protein kinase (PRKACB), nuclear receptor co-activator 3 (NCOA3), and cell surface protein CD59 (protectin), all genes having relevance for HIV-1 pathology. CONCLUSIONS: The up-regulation of GliPR by HIV-1 and the early significant inhibition of HIV-1 replication mediated by knockdown of GliPR reveal GliPR as an important HIV-1 dependency factor (HDF), which may be exploited for HIV-1 inhibition.

Mutations in gp120 contribute to the resistance of human immunodeficiency virus type 1 to membrane-anchored C-peptide maC46.

Binding of the human immunodeficiency virus (HIV) envelope glycoprotein (Env) to the cellular CD4 receptor and a chemokine coreceptor initiates a series of conformational changes in the Env subunits gp120 and gp41. Eventually, the trimeric gp41 folds into a six-helix bundle, thereby inducing fusion of the viral and cellular membranes. C peptides derived from the C-terminal heptad repeat (CHR) of gp41 are efficient entry inhibitors as they block the six-helix bundle formation. Previously, we developed a membrane-anchored C peptide (maC46) expressed from a retroviral vector that also shows high activity against virus strains resistant to enfuvirtide (T-20), an antiviral C peptide approved for clinical use. Here, we present a systematic analysis of mutations in Env that confer resistance of HIV type 1 (HIV-1) to maC46. We selected an HIV-1 BaL strain with 10-fold reduced sensitivity to maC46 (BaL_C46) by passaging virus for nearly 200 days in the presence of gradually increasing concentrations of maC46. In comparison to wild-type BaL, BaL_C46 had five mutations at highly conserved positions in Env, three in gp120, one in the N-terminal heptad-repeat (NHR), and one in the CHR of gp41. No mutations were found in the NHR domain around the GIV motif that are known to cause resistance to enfuvirtide. Instead, maC46 resistance was found to depend on complementary mutations in the NHR and CHR that considerably favor binding of the mutated NHR to the mutated CHR over binding to maC46. In addition, resistance was highly dependent on mutations in gp120 that accelerated entry. Taken together, resistance to maC46 did not develop readily and required multiple cooperating mutations at conserved positions of the viral envelope glycoproteins gp120 and gp41.

A peptide inhibitor of HIV-1 assembly in vitro.

Formation of infectious HIV-1 involves assembly of Gag polyproteins into immature particles and subsequent assembly of mature capsids after proteolytic disassembly of the Gag shell. We report a 12-mer peptide, capsid assembly inhibitor (CAI), that binds the capsid (CA) domain of Gag and inhibits assembly of immature- and mature-like capsid particles in vitro. CAI was identified by phage display screening among a group of peptides with similar sequences that bind to a single reactive site in CA. Its binding site was mapped to CA residues 169-191, with an additional contribution from the last helix of CA. This result was confirmed by a separate X-ray structure analysis showing that CAI inserts into a conserved hydrophobic groove and alters the CA dimer interface. The CAI binding site is a new target for antiviral development, and CAI is the first known inhibitor directed against assembly of immature HIV-1.

Molecular dynamics and DFT study on HIV-1 nucleocapsid protein-7 in complex with viral genome.

The HIV-1 nucleocapsid protein-7 (NCp7) is a highly basic, small zinc-binding protein involved in both deoxyribonucleic (DNA) and ribonucleic (RNA) acids annealing and in viral particle maturation including genome encapsidation, with an additional chaperoning activity toward reverse transcriptase by promoting the two obligatory strand transfers during reverse transcription. Because of its interaction with highly conserved sequences of the HIV-1 genome, NCp7 is being considered a new potential drug target, resistant to mutation, for antiviral activity. The high flexibility of this protein has, however, limited the identification of structural determinants involved in the interaction with stranded sequences of DNA and RNA. Here, we provide a quantum mechanics (density functional theory) study of the zinc-binding motifs and a molecular dynamics simulation of the protein in complex with RNA and DNA, starting from available nuclear magnetic resonance (NMR) structures. Results show that the interaction between the NCp7 and the viral genome is probably based on electrostatic interactions due to a cluster of basic residues, which is reinforced by the exploitation of nonelectrostatic contacts that further stabilize the complexes. Moreover, a possible mechanism for DNA destabilization that involves amino acids T24 and R26 is also hypothesized. Finally, a network of hydrophobic and hydrogen-bond interactions for the stabilization of complexes with DNA and, especially, with RNA is described here for the first time. The complexes between NCp7 and both DNA and RNA, resulting from computer simulations, showed structural properties that are in agreement with most of the currently available molecular biology evidence and could be considered as reliable models (better than NMR structures currently available) for subsequent structure-based ligand design approaches.