Plasmodium's sticky fingers

The life cycle of the malaria parasite (Plasmodium) is remarkably complex. Malaria parasites must engage in highly specific and varied interactions with cell types of both the mammalian host and the mosquito vector. In this issue of Cell, report detailed molecular insights into an intimate interaction between a malaria parasite protein and its host cell receptor that enables the parasite to invade erythrocytes.     

Primary haemostasis: sticky fingers cement the relationship

Platelet aggregation to form a haemostatic plug, or thrombus, plays a key role in preventing bleeding from a wound. Recent studies have provided new insights into how platelet receptors are deployed during the interactions with the vascular subendothelial matrix that lead to haemostatic plug formation.     

Peripheral membrane proteins: FYVE sticky fingers

The recently determined structure of the lipid-binding 'FYVE' domain provides several clues to the mode of interaction for this class of peripheral membrane proteins. However, the application of traditional modes of structural analysis to diffusible membrane-binding proteins exposes some limitations of these techniques.     

Targeting the dimerization interface for irreversible inhibition of HIV-1 protease

A novel strategy was used to irreversibly inhibit HIV-1 protease. The inhibitor was designed to form a disulfide bond with Cys95, present at the dimerization interface of HIV-1 protease. The inhibitor was shown to be active against HIV-1 protease with K(inact) = 3.7 microM and V(inact) = 0.012 min(-1).     

Role of the zinc fingers of HIV-1 nucleocapsid protein in maturation of genomic RNA

The nucleocapsid protein of HIV-1 consists of two basic amino acid regions and two zinc fingers. We investigated the requirement of these domains for the structural conversion of a 39mer RNA covering the dimerization initiation site by using three peptides; wild-type NCp7, a mutant in which the two zinc fingers are mutated, and another mutant in which the two zinc fingers are deleted. The two mutants exhibited similar conversion activities to each other, which were lower than that of the wild-type, indicating that the two basic regions exhibit some activity for RNA chaperone, as we suggested before, and the zinc fingers enhance the efficiency of this activity.     

Suppression of HIV-1 Integration by Targeting HIV-1 Integrase for Degradation with A Chimeric Ubiquitin Ligase

Human immunodeficiency virus(HIV) attacks human immune system and causes life-threatening acquired immune deficiency syndrome(AIDS). Treatment with combination antiretroviral therapy(cART) could inhibit virus growth and slow progression of the disease, however, at the same time posing various adverse effects. Host ubiquitin-proteasome pathway(UPP) plays important roles in host immunity against pathogens including viruses by inducing degradation of viral proteins. Previously a series of methods for retargeting substrates for ubiquitin-proteasome degradation have been successfully established. In this study, we attempted to design and construct artificial chimeric ubiquitin ligases(E3 s) based on known human E3 s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation.Herein, a series of prototypical chimeric E3 s have been designed and constructed, and original substrate-binding domains of these E3 s were replaced with host protein domains which interacted with viral proteins. After functional assessment screening, 146 LI was identified as a functional chimeric E3 for HIV-1 NL4-3 integrase. 146 LI was then further optimized to generate 146 LIS(146 LI short) which has been shown to induce Lys48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells. Lymphocyte cells with 146 LIS knock-in generated by CRISPR/Cas-mediated homology-directed repair(HDR) showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity. Our study successfully obtained an artificial chimeric E3 which can induce Lys48-specific polyubiquitination and proteasome-mediated degradation of HIV-1 NL4-3 integrase, thus effectively inhibiting viral DNA integration and viral replication upon virus infection.     

MxB Restricts HIV-1 by Targeting the Tri-hexamer Interface of the Viral Capsid

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Targeting human langerin promotes HIV-1 specific humoral immune responses

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4⁺ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.     

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

The matrix domain (MA) of the HIV-1 precursor Gag (PrGag) protein directs PrGag proteins to assembly sites at the plasma membrane by virtue of its affinity to the phospholipid, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). MA also binds to RNA at a site that overlaps its PI(4,5)P₂ site, suggesting that RNA binding may protect MA from associating with inappropriate cellular membranes prior to PrGag delivery to the PM. Based on this, we have developed an assay in which small molecule competitors to MA-RNA binding can be characterized, with the assumption that such compounds might interfere with essential MA functions and help elucidate additional features of MA binding. Following this approach, we have identified four compounds, including three thiadiazolanes, that compete with RNA for MA binding. We also have identified MA residues involved in thiadiazolane binding and found that they overlap the MA PI(4,5)P₂ and RNA sites. Cell culture studies demonstrated that thiadiazolanes inhibit HIV-1 replication but are associated with significant levels of toxicity. Nevertheless, these observations provide new insights into MA binding and pave the way for the development of antivirals that target the HIV-1 matrix domain.     

Targeting of HIV-1 Nef to the centrosome: implications for antigen processing

To gain a better understanding of the intracellular sites of antigen processing we have looked at the localization of human immunodeficiency virus (HIV)-1 Nef protein by confocal microscopic and biochemical means. We found that ubiquitin (Ub)-Nef fusion proteins were localized to the centrosome in transfected COS-7 cells, and that the colocalization was inhibited by the microtubule-disrupting agent, nocodazole. Interestingly, we found that Ub-Nef trafficking to the centrosome was not dependent upon the metabolic stability of Ub-Nef nor on the inhibition of proteasome activity. We also analyzed the MHC class I antigen processing of a reporter epitope linked to the Ub-Nef fusion proteins and found that Ub-Nef was processed in COS-7 cells. In addition, we show that this processing was inhibited by nocodazole. We suggest that the centrosome may serve as a site of antigen processing in vivo.     

HIV-1整合酶链转移反应抑制剂的荧光筛选方法

整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。     

HIV-1 Matrix Protein Interactions with tRNA: Implications for Membrane Targeting

The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNA^Lys3), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNA^Lys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNA^Lys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. Unexpectedly, the affinity of MA for tRNA^Lys3 (K_d = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P₂-enriched liposomes (K_d(apparent) = 10.2 ± 2.1 μM), and NMR studies indicate that tRNA^Lys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P₂, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNA^Lys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag–Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled.     

The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers

The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.