Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/ or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and Cterminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn136 in V1 (T138N mutation) inconjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons inV1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection

Mechanistic insights from targeted molecular profiling of repolarization alternans in the intact human heart

Aims: Action potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological properties relevant to arrhythmia exist between experimental models and the diseased in vivo human heart. To investigate mechanisms of APD alternans using a novel approach combining intact heart and cellular cardiac electrophysiology in human in vivo. Methods and results: We developed a novel approach combining intact heart electrophysiological mapping during cardiac surgery with rapid on-site data analysis to guide myocardial biopsies for laboratory analysis, thereby linking repolarization dynamics observed at the organ level with underlying ion channel expression. Alternans-susceptible and alternans-resistant regions were identified by an incremental pacing protocol. Biopsies from these sites (n = 13) demonstrated greater RNA expression in Calsequestrin (CSQN) and Ryanodine (RyR) and ion channels underlying IK1 and Ito at alternans-susceptible sites. Electrical restitution properties (n = 7) showed no difference between alternans-susceptible and resistant sites, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites (P = 0.001). The degree of histological fibrosis between alternans-susceptible and resistant sites was equivalent. Mathematical modelling of these changes indicated that both CSQN and RyR up-regulation are key determinants of APD alternans. Conclusion: Combined intact heart and cellular electrophysiology show that regions of myocardium in the in vivo human heart exhibiting APD alternans are associated with greater expression of CSQN and RyR and show no difference in restitution properties compared to non-alternans regions. In silico modelling identifies up-regulation and interaction of CSQN with RyR as a major mechanism underlying APD alternans

Epidemiology and pathophysiology of cancer-associated thrombosis

Venous thromboembolism (VTE) is a common complication in patients with malignant disease. First recognised by Bouillard in 1823 and later described by Trousseau in 1844, multiple studies have since provided considerable evidence for a clinical association between VTE and cancer. Across all cancers, the risk for VTE is elevated 7-fold; in certain malignancies, the risk for VTE may be increased up to 28-fold. Venous thromboembolism is the second leading cause of death in patients with cancer; among survivors, complications commonly include recurrent VTE and post-thrombotic syndrome, and (more rarely) chronic thromboembolic pulmonary hypertension, which are costly, and have a profound impact on the patient's quality of life. Tumour cells can activate blood coagulation through multiple mechanisms, including production of procoagulant, fibrinolytic, and proaggregating activities, release of proinflammatory and proangiogenic cytokines, and interacting directly with host vascular and blood cells (e.g., endothelial cells, leukocytes, and platelets) through adhesion molecules. Increasing evidence suggests that elements of the haemostatic system also have a direct role in eliciting or enhancing angiogenesis, cell survival, and metastasis. Despite the problem posed by VTE in the setting of cancer, it is evident that a significant number of oncologists do not recognise the link between cancer, its treatment, and thrombogenesis. On 22 May 2009, a group of UK-based physicians met in London, UK, to evaluate recent data on cancer thrombosis. This article (1 of 4) briefly reviews key data on the epidemiology and pathophysiology of VTE as a context for a discussion and consensus statement developed by meeting attendees, on the implications of this information for UK clinical practice

Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance

B Cell Recognition of the Conserved HIV-1 Co-Receptor Binding Site Is Altered by Endogenous Primate CD4

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein

Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins