ALIX Is Recruited Temporarily into HIV-1 Budding Sites at the End of Gag Assembly

Polymerization of Gag on the inner leaflet of the plasma membrane drives the assembly of Human Immunodeficiency Virus 1 (HIV-1). Gag recruits components of the endosomal sorting complexes required for transport (ESCRT) to facilitate membrane fission and virion release. ESCRT assembly is initiated by recruitment of ALIX and TSG101/ESCRT-I, which bind directly to the viral Gag protein and then recruit the downstream ESCRT-III and VPS4 factors to complete the budding process. In contrast to previous models, we show that ALIX is recruited transiently at the end of Gag assembly, and that most ALIX molecules are recycled into the cytosol as the virus buds, although a subset remains within the virion. Our experiments imply that ALIX is recruited to the neck of the assembling virion and is mostly recycled after virion release.     

Clustering and Mobility of HIV-1 Env at Viral Assembly Sites Predict Its Propensity To Induce Cell-Cell Fusion

HIV-1 Env mediates virus attachment to and fusion with target cell membranes, and yet, while Env is still situated at the plasma membrane of the producer cell and before its incorporation into newly formed particles, Env already interacts with the viral receptor CD4 on target cells, thus enabling the formation of transient cell contacts that facilitate the transmission of viral particles. During this first encounter with the receptor, Env must not induce membrane fusion, as this would prevent the producer cell and the target cell from separating upon virus transmission, but how Env''s fusion activity is controlled remains unclear. To gain a better understanding of the Env regulation that precedes viral transmission, we examined the nanoscale organization of Env at the surface of producer cells. Utilizing superresolution microscopy (stochastic optical reconstruction microscopy [STORM]) and fluorescence recovery after photobleaching (FRAP), we quantitatively assessed the clustering and dynamics of Env upon its arrival at the plasma membrane. We found that Gag assembly induced the aggregation of small Env clusters into larger domains and that these domains were completely immobile. Truncation of the cytoplasmic tail (CT) of Env abrogated Gag''s ability to induce Env clustering and restored Env mobility at assembly sites, both of which correlated with increased Env-induced fusion of infected and uninfected cells. Hence, while Env trapping by Gag secures Env incorporation into viral particles, Env clustering and its sequestration at assembly sites likely also leads to the repression of its fusion function, and thus, by preventing the formation of syncytia, Gag helps to secure efficient transfer of viral particles to target cells.     

Visualizing HIV-1 Assembly

The assembly of an HIV-1 particle is a complex, multistep process involving several viral and cellular proteins, RNAs and lipids. While many macroscopic and fixed-cell microscopic techniques have provided important insights into the structure of HIV-1 particles and the mechanisms by which they assemble, analysis of individual particles and their assembly in living cells offers the potential of surmounting many of the limitations inherent in other approaches. In this review, we discuss how the recent application of live-cell microscopic imaging techniques has increased our understanding of the process of HIV-1 particle assembly. In particular, we focus on recent studies that have employed total internal reflection fluorescence microscopy and other single-virion imaging techniques in live cells. These approaches have illuminated the dynamics of Gag protein assembly, viral RNA packaging and ESCRT (endosomal sorting complex required for transport) protein recruitment at the level of individual viral particles. Overall, the particular advantages of individual particle imaging in living cells have yielded findings that would have been difficult or impossible to obtain using macroscopic or fixed-cell microscopic techniques.     

Dynamics of HIV-1 Assembly and Release

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10⁻³ s⁻¹, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.     

Functional Redundancy in HIV-1 Viral Particle Assembly

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.     

Protease Cleavage Sites in HIV-1 gp120 Recognized by Antigen Processing Enzymes Are Conserved and Located at Receptor Binding Sites

The identification of vaccine immunogens able to elicit broadly neutralizing antibodies (bNAbs) is a major goal in HIV vaccine research. Although it has been possible to produce recombinant envelope glycoproteins able to adsorb bNAbs from HIV-positive sera, immunization with these proteins has failed to elicit antibody responses effective against clinical isolates of HIV-1. Thus, the epitopes recognized by bNAbs are present on recombinant proteins, but they are not immunogenic. These results led us to consider the possibility that changes in the pattern of antigen processing might alter the immune response to the envelope glycoprotein to better elicit protective immunity. In these studies, we have defined protease cleavage sites on HIV gp120 recognized by three major human proteases (cathepsins L, S, and D) important for antigen processing and presentation. Remarkably, six of the eight sites identified in gp120 were highly conserved and clustered in regions of the molecule associated with receptor binding and/or the binding of neutralizing antibodies. These results suggested that HIV may have evolved to take advantage of major histocompatibility complex (MHC) class II antigen processing enzymes in order to evade or direct the antiviral immune response.A major goal of HIV vaccine development is the development of immunogens that elicit protective antiviral antibody and cellular immune responses. However, after more than 25 years of research, vaccine immunogens able to elicit protective immunity in humans have yet to be described (11, 31). Although it has been possible to produce recombinant envelope proteins (gp120 and gp140) with many of the features of native virus proteins (e.g., complex glycosylation and the ability to bind CD4, chemokine receptors, and neutralizing antibodies), these antigens have not been able to elicit broadly neutralizing antibodies (bNAbs) or protective immune responses when used as immunogens (11, 32, 43, 50, 56, 74, 79). The fact that recombinant proteins can adsorb virus bNAbs from HIV-1-positive sera (59, 91) indicates that many recombinant envelope proteins are correctly folded but that the epitopes recognized by bNAbs are simply not immunogenic. Over the last decade, several different approaches have been employed to create immunogens able to elicit broadly neutralizing antibodies. These strategies have included efforts to duplicate and/or stabilize the oligomeric structure of HIV envelope proteins (5, 26, 87), the creation of minimal antigenic structures lacking epitopes that conceal important neutralizing sites (27, 46, 70, 89), and prime/boost strategies combining protein immunization with DNA immunization or infection with recombinant viruses in order to stimulate the endogenous synthesis and presentation of HIV immunogens (15, 29, 30, 83). However, none of these approaches has resulted in a clinically significant improvement in antiviral immunity or HIV vaccine efficacy. Efforts to elicit protective cellular immune responses (e.g., cytotoxic lymphocytes) by use of recombinant virus vaccines have likewise been disappointing (10, 61). In fact, such vaccines may have promoted HIV infection rather than inhibiting it (22, 23).In the present study, we describe the first steps in a new approach to reengineering the immunogenicity of HIV envelope proteins in order to improve the potency and specificity of humoral and cellular immune responses. The approach is based on defining the determinants of antigen processing and presentation of HIV envelope glycoproteins. Both humoral and cellular immune responses depend on proteolytic degradation of protein antigens prior to antigen presentation, mediated by professional antigen-presenting cells (APCs) such as macrophages, dendritic cells, and B cells (97). Normally, proteins of intracellular origin are processed by the proteasome, a 14- to 17-subunit protein complex located in the cytosol. Proteins of extracellular origin are processed in lysosomes or late endosomes of APCs. The resulting peptide epitopes are then loaded into major histocompatibility complex (MHC) class I or class II molecules and presented on the surfaces of APCs to CD8 or CD4 T cells. Within the endosomes and lysosomes of APCs, there are cathepsins, acid thiol reductase, and aspartyl endopeptidase. The enzymes perform two activities: degrading endocytosed protein antigens to liberate peptides for MHC class II binding (99) and removing the invariant chain chaperone (6, 94). Although all cathepsins can liberate epitopes from a diverse range of antigens (16), only cathepsins S and L have nonredundant roles in antigen processing in vivo (reviewed by Hsing and Rudensky [45]). Cathepsin L is expressed in thymic cortical epithelial cells but not in B cells or dendritic cells, while cathepsin S is found in all three types of APCs. Unlike cathepsins L and S, which are cysteine proteases and active at neutral pH, cathepsin D is an aspartic protease, is active at acidic pH, and participates in proteolysis and antigen presentation in connection with MHC class I and class II antigen presentation pathways established for CD4 and CD8 T cells. In considering the use of envelope proteins as potential vaccines, the route of immunization, formulation (e.g., adjuvants), protein folding, disulfide bonding, and glycosylation pattern all determine which peptides are available for MHC-restricted presentation.Previous studies provided evidence that gp120 was sensitive to digestion by cathepsins B, D, and L, but the specific cleavage sites were not defined (18). In the present study, we (i) describe the locations of eight protease cleavage sites on HIV-1 gp120 recognized by cathepsins L, S, and D, involved in antigen processing; (ii) determine the extent to which they are conserved; and (iii) evaluate the effect of cathepsin cleavage on the binding of gp120 to CD4-IgG and neutralizing antibodies. The results obtained provide new insights into the basis of envelope immunogenicity that may prove to be useful in the development of HIV vaccine antigens.     

HIV-1 at 25

In 1981, the acquired immunodeficiency syndrome (AIDS) appeared insidiously and mystified doctors and scientists alike. No one could have predicted then that it would become, arguably, the worst plague in human history. Today, 33 million persons are living with infection by the human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, while another 25 million have already died of this disease.     

Defining the DNA Substrate Binding Sites on HIV-1 Integrase

A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.     

Super-Resolution Imaging of Bacteria in a Microfluidics Device

Bacteria have evolved complex, highly-coordinated, multi-component cellular engines to achieve high degrees of efficiency, accuracy, adaptability, and redundancy. Super-resolution fluorescence microscopy methods are ideally suited to investigate the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. These techniques require the long-term stability of samples, high signal-to-noise-ratios, low chromatic aberrations and surface flatness, conditions difficult to meet with traditional immobilization methods. We present a method in which cells are functionalized to a microfluidics device and fluorophores are injected and imaged sequentially. This method has several advantages, as it permits the long-term immobilization of cells and proper correction of drift, avoids chromatic aberrations caused by the use of different filter sets, and allows for the flat immobilization of cells on the surface. In addition, we show that different surface chemistries can be used to image bacteria at different time-scales, and we introduce an automated cell detection and image analysis procedure that can be used to obtain cell-to-cell, single-molecule localization and dynamic heterogeneity as well as average properties at the super-resolution level.     

Extended-Depth 3D Super-Resolution Imaging Using Probe-Refresh STORM

Single-molecule localization microscopy methods for super-resolution fluorescence microscopy such as STORM (stochastic optical reconstruction microscopy) are generally limited to thin three-dimensional (3D) sections (≤600 nm) because of photobleaching of molecules outside the focal plane. Although multiple focal planes may be imaged before photobleaching by focusing progressively deeper within the sample, image quality is compromised in this approach because the total number of measurable localizations is divided between detection planes. Here, we solve this problem on fixed samples by developing an imaging method that we call probe-refresh STORM (prSTORM), which allows bleached fluorophores to be straightforwardly replaced with nonbleached fluorophores. We accomplish this by immunostaining the sample with DNA-conjugated antibodies and then reading out their distribution using fluorescently-labeled DNA-reporter oligonucleotides that can be fully replaced in successive rounds of imaging. We demonstrate that prSTORM can acquire 3D images over extended depths without sacrificing the density of localizations at any given plane. We also show that prSTORM can be adapted to obtain high-quality, 3D multichannel images with extended depth that would be challenging or impossible to achieve using established probe methods.     

Super-Resolution Imaging of Plasmodesmata Using Three-Dimensional Structured Illumination Microscopy

We used three-dimensional structured illumination microscopy (3D-SIM) to obtain subdiffraction (“super-resolution”) images of plasmodesmata (PD) expressing a green fluorescent protein-tagged viral movement protein (MP) in tobacco (Nicotiana tabacum). In leaf parenchyma cells, we were able to resolve individual components of PD (neck and central cavities) at twice the resolution of a confocal microscope. Within the phloem, MP-green fluorescent protein filaments extended outward from the specialized pore-PD that connect sieve elements (SEs) with their companion cells (CCs) along the tubular sieve element reticulum (SER). The SER was shown to interconnect individual pore-PD at the SE-CC interface. 3D-SIM resolved fine (less than 100 nm) endoplasmic reticulum threads running into individual pore-PD as well as strands that crossed sieve plate pores, structurally linking SEs within a file. Our data reveal that MP entering the SE from the CC may remain associated with the SER. Fluorescence recovery after photobleaching experiments revealed that this MP pool is relatively immobile compared with the membrane probe 3,3’-dihexyloxacarbocyanine iodide, suggesting that MP may become sequestered by the SER once it has entered the SE. The advent of 3D-SIM offers considerable potential in the subdiffraction imaging of plant cells, bridging an important gap between confocal and electron microscopy.Fluorescence-based imaging has revolutionized cell biology, allowing the localization of proteins to specific cells and organelles (Shaner et al., 2007; Frigault et al., 2009). However, conventional fluorescence microscopy is limited by the diffraction of light to approximately 200 nm in the lateral (x-y) plane and to about 500 nm in the axial (z) plane (Fernandez-Suarez and Ting, 2008; Huang et al., 2009). This is because light traveling through a lens cannot be focused to a point, only to an airy disc with a diameter of about half the wavelength of the visible emitted light (Huang et al., 2009). Confocal laser scanning microscopy has produced improvements in axial resolution due to the removal of out-of-focus flare, but it is also limited by diffraction (Huang et al., 2009). Thus, objects closer than about 200 nm cannot be resolved but appear merged into one. Many subcellular structures of interest to cell biologists lie below this resolution limit and have remained below the diffraction barrier. Such structures can be seen but not resolved.Recently, major innovations in biological imaging have broken the diffraction barrier. These include photoactivation localization microscopy (PALM) and stimulated emission and depletion (STED; for review, see Fernandez-Suarez and Ting, 2008; Huang et al., 2009). Most subdiffraction or “super-resolution” approaches have improved resolution in either the lateral (x-y) plane or the axial (z) plane, but usually not both (Schermelleh et al., 2008). Many of the structures of interest within plant cells lie some distance from the cell wall, posing problems for some super-resolution approaches (e.g. PALM) where the subject of interest must lie close to the coverslip (Huang et al., 2009). Recently, Schermelleh et al. (2008) described a subdiffraction multicolor imaging protocol using three-dimensional structured illumination microscopy (3D-SIM). In this method, objects beyond the diffraction limit are illuminated with multiple interfering beams of light transmitted through a series of diffraction gratings, producing a resolution of 100 nm in x-y and 200 nm in z (Schermelleh et al., 2008; Huang et al., 2009). These substantial increases in resolution are significant for plant cell imaging. The thickness of the plant cell wall is typically in the region of about 700 nm, allowing limited optical sectioning capacity with a confocal microscope (about 500 nm in z). A further advantage of 3D-SIM is that it permits the imaging of conventional fluorescent reporters and dyes that are compatible with confocal imaging, allowing a direct correlation of 3D-SIM and confocal images (Schermelleh et al., 2008).The phloem of higher plants is a major conduit for the long-distance transport of solutes (Oparka and Turgeon, 1999) and also functions as a “superhighway” for macromolecular trafficking (Lucas and Lee, 2004; Kehr and Buhtz, 2008; Lee and Cui, 2009). However, the phloem is difficult to image with conventional optical microscopy (Knoblauch and van Bel, 1998; Oparka and Turgeon, 1999; van Bel et al., 2002). Sieve elements (SEs), the conducting cells of the phloem, are enucleate yet contain a plethora of proteins and RNAs associated with long-distance signaling and defense (van Bel and Gaupels, 2004; Lee and Cui, 2009). Many of these macromolecules are synthesized in the companion cell (CC) and passed into the SE via the specialized pore-plasmodesmata (PD) that connect the two cell types (Oparka and Turgeon, 1999; van Bel et al., 2002). Pore-PD have been suggested to be a major “lifeline” from CC to SE (van Bel et al., 2002), but the exact nature of this pathway remains unresolved.Our current understanding of PD substructure is derived largely from electron microscope studies (Roberts, 2005). Such methods are time-consuming and do not permit facile protein localization within PD. Recent proteomics approaches have been successful in identifying new proteins associated with PD (Maule, 2008). Localization of these proteins with confocal microscopy results in the appearance of discrete punctae at the cell wall, consistent with the location of pit fields (Faulkner et al., 2008), but does not pinpoint specific protein locations within PD. In general, there is a growing gap between proteomics studies of plant organelles, including PD, and the ability to ascribe accurate addresses to these proteins (Millar et al., 2009; Moore and Murphy, 2009). The advent of 3D-SIM prompted us to explore the potential of subdiffraction imaging in plant cells, with a view to obtaining improved florescence resolution of PD. We used 3D-SIM to examine PD in a transgenic tobacco (Nicotiana tabacum) line expressing the viral movement protein (MP) of Tobacco mosaic virus (TMV) fused to GFP. Using a specific antibody to callose, a wall constituent located at the PD collar, we were able to resolve clearly the structure of single, simple PD in epidermal cells at 100-nm resolution, discriminating between the neck region of the pore and the central cavity to which it connects (Roberts and Oparka, 2003; Faulkner et al., 2008). 3D-SIM also revealed details of the central cavities of complex PD seen previously only with the electron microscope (Ding et al., 1992; Ehlers and Kollmann, 2001; Faulkner et al., 2008).Using 3D-SIM, we were able to image PD sequentially from the epidermis to the phloem within vascular bundles, producing unparalleled images of sieve plate pores and the specialized pore-PD that connect SEs with their CCs. In the SEs, MP was no longer restricted to the central cavities of PD but became distributed along the SE parietal layer, connecting all the pore-PD along the SE-CC interface. We were able to detect fine threads of MP-GFP that extended for up to 40 μm along the SE and also crossed individual sieve plate pores. Fluorescence recovery after photobleaching (FRAP) experiments revealed that this MP-GFP pool was relatively immobile within the SE parietal layer, suggesting that the SE may sequester TMV MP on or within the sieve element reticulum (SER).Our data reveal that 3D-SIM is especially suited to the subdiffraction imaging of plant cells and yields spatial information not previously possible with conventional fluorescence-based imaging. The unique optical sectioning capacity of 3D-SIM and the ability to produce multicolor imaging with conventional fluorophores offer enormous potential in plant cell biology.     

HIV-1整合酶四聚体结构模拟及其活性位点分析

HIV-1复制需要HIV-1整合酶将其环状DNA整合进宿主DNA中,这其中包括2个重要反应,即“3′-加工”和“链转移”,两者均由HIV-1整合酶催化完成.阻断其中的任一反应,都能达到抑制HIV-1复制的目的.因此,了解HIV-1整合酶的完整结构和聚合状态,对深入探讨其作用机理及设计新型抑制剂具有重要的指导作用.然而,迄今为止仅有HIV-1整合酶单独结构域的晶体结构可供参考,而其全酶晶体结构尚未获得解析.本研究利用分子模拟技术,通过蛋白质 蛋白质/DNA分子对接、动力学模拟等方法,构建了全长整合酶四聚体的结构模型、HIV-1 DNA与整合酶复合物的结构模型,进一步从理论上证实HIV-1整合酶是以四聚体形态发挥催化作用,明确“3′-加工”和“链转移”在HIV-1整合酶上的催化位点.同时,通过与作用机理相似的细菌转座子Tn5转座酶等的结构比对,推测HIV-1整合酶的核心结构域中应有第2个Mg^２＋存在,其位置螯合于Asp64与Glu152之间.在HIV-1整合酶结构研究的基础上,有望进一步设计出新的抗艾滋病药物.     

Unclosed HIV-1 Capsids Suggest a Curled Sheet Model of Assembly

The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that ~ 25% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms.     

Assembly and maturation of HIV-1--targets for new anti-HIV compounds

Much progress has been recently made in research of the final stages of the HIV-1 life cycle, i.e. of its assembly, gemmation and maturation. The virus was shown, in particular, to use widely cell mechanisms in its replication and assembly. The TSG 101 cellular endosomal sorting protein interacting with the p6 viral protein is necessary for gemmation. Cyclophilin and HP68 (cell proteins) needed for marphogenesis of the virus were identified. The recently obtained data on the interaction of Vif (a viral protein) and Apobec (a cell protein) showed that HIV-1 has an action mechanism overcoming the cell barriers. The "early" virus phenomenon, which is deprived of any mature structure or the ability to infect and does not contain mature Gag and Env proteins, illustrates that the proteolytic pressing of the Gag p55 precursor is not enough for the maturation of the virus and additional viral or cellular factors are needed for the virus to become infectious. Although the current antiviral therapy has been successful enough, it is far from being effective in all cases; one of the reasons is resistance to chemodrugs developing rapidly in patients. Fast mutations and exceptional plasticity of the viral genome (which helps the virus to develop rapidly resistance to drugs) belong to the major problems. The circulation of persistent virus variants has been quickly increasing. There is an urgent need in developing new antiviral drugs acting on new viral targets; progress in experimental virology would speed it up. Thus, new drugs can be created, which block the activity of Vif, that would make Apobec block the virus replication. Compounds can be developed, which block the interaction of cyclophilin and TSG101 with viral proteins. The recently described importance of cholesterol in the sexual transfer of viruses is expected to bring simple and inexpensive compounds destroying cholesterol in the mucous tunic of genitals into clinical use. The identification of additional factors needed for the maturation of the virus and for its becoming infectious can be a basis for the development of drugs blocking their packaging into virions. Future research is expected to define new targets for the chemotherapy of AIDS and to promote the designing of new chemodrugs.     

Broadband Mid-Infrared Super-Resolution Imaging with Metallic Nanorod-Bridged Dimer Array

We propose a broadband mid-infrared super-resolution imaging system comprising a metallic nanorod-bridged dimer array. The imaging array enables super-resolution imaging of shaped dipole sources in the near field. A charge transfer plasmon (CTP) appears in a metallic nanorod-bridged dimer. By varying the radius of the junction, the plasmon resonance wavelength of CTP mode can be tuned into the mid-infrared region. Here, we investigate the broadband super-resolution imaging of the incoherent and coherent dipole sources at mid-infrared wavelengths. With the array pitch varying, we calculate the cross-sectional field intensity distributions at the source plane and the image plane by using the finite element method. The simulation results indicate that the broadband incoherent and coherent super-resolution imaging can be realized at mid-infrared wavelengths with the imaging array. The image quality is sensitively dependent on the source coherent, the array pitch, and the distance from the image plane to the array. In the same structural parameters, the image quality of coherent source of in-phase is lower than that of incoherent source. Increasing the array pitch improves the image quality but it also increases the size of the array. By reasonably choosing the array pitch of the array, the spatial resolution of ～λ/109 and ～λ/73 is obtained corresponding to the incoherent imaging case and coherent imaging case at the mid-infrared wavelength of 4390 nm. Moreover, the larger image-array distance results in the lower image quality.     

Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.     

Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue

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