Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the plasma membrane. Inhibition of endosomal traffic did not prevent viral release. However, inhibiting lysosomal degradation induced an accumulation of Gag in endosomes and increased viral production 7-fold, indicating that transport of Gag to lysosomes negatively regulates budding. This also suggested that endosomal Gag-RNA complexes could access retrograde pathways to the cell surface and indeed, depleting cells of TiVamp-reduced viral production. Moreover, inhibition of endosomal transport prevented the accumulation of Gag at sites of cellular contact. HIV-1 Gag could thus generate virions using two pathways, either directly from the plasma membrane or through an endosome-dependent route. Endosomal Gag-RNA complexes may be delivered at specific sites to facilitate cell-to-cell viral transmission.The production of infectious retroviral particles is an ordered process that includes many steps (for review see Refs. 1–3). In particular, three major viral components, Gag, the envelope, and genomic RNAs have to traffic inside the cell to reach their assembly site. Viral biogenesis is driven by the polyprotein Gag, which is able to make viral-like particles when expressed alone (4). Upon release, HIV-1⁴ Gag is processed by the viral protease into matrix (MA(p17)), capsid (CA(p24)), nucleocapsid (NC(p7)), p6, and smaller peptides SP1 and SP2. Gag contains several domains that are essential for viral assembly: a membrane binding domain (M) in MA; a Gag-Gag interaction domain in CA; an assembly domain (I) in NC; and a late domain (L) in p6, which recruits the cellular budding machinery. Genomic RNAs are specifically recognized by NC, and they play fundamental roles in viral biogenesis by acting as a scaffold for Gag multimerization (5).It has been demonstrated that retroviruses bud by hijacking the endosomal machinery that sorts proteins into internal vesicles of multivesicular bodies (for review, see Refs. 6, 7). Indeed, these vesicles bud with the same topology as viral particles. Proteins sorted into this pathway are usually destined for degradation in lysosomes, but some can also recycle to the plasma membrane (for review see Refs. 8, 9). They are also frequently ubiquitinated on their cytoplasmic domain (10, 11), allowing their recognition by ESCRT complexes. ESCRT-0 and ESCRT-I recognize ubiquitinated cargo present at the surface of endosomes and recruit other ESCRT complexes (12–14). ESCRT-III is believed to function directly in the formation of multivesicular body intralumenal vesicles (12), even though its mechanism of action is currently not understood. Remarkably, Gag L domains interact directly with components of the multivesicular body-sorting machinery (for review see Ref. 15). HIV-1 Gag uses a PTAP motif to bind Tsg101, a component of ESCRT-I (16–19), and a YPLTSL motif to interact with Alix, a protein linked to ESCRT-I and -III (20–22). Finally, various ubiquitin ligases are also required directly or indirectly during HIV-1 biogenesis (23, 24; for review see Ref. 25).In many cell lines, Gag is found both at the plasma membrane and in endosomes. This has led to the hypothesis that there are several assembly sites for HIV-1 (1, 3). First, Gag can initiate and complete assembly at the plasma membrane. This is thought to occur predominantly in T lymphocytes, and this process is supported by several lines of evidences: (i) disruption of endosomal trafficking with drugs does not prevent viral production (26, 27); (ii) ESCRT complexes can be recruited at the plasma membrane, at sites where Gag accumulates (28–30); (iii) Gag can be seen multimerizing and budding from the plasma membrane in live cells (31). Second, Gag could initiate assembly in endosomes, and then traffic to the cell surface to be released. This is mainly supported by the presence of Gag in endosomes in several cell lines (32–34), including T cells and more strikingly macrophages (32, 35, 36–39). However, we are currently lacking functional experiments addressing the role of this endosomal pool of Gag, and it is still not clear to what extent it contributes to the production of viral particles. Nevertheless, the presence of Gag in endosomes might facilitate recruitment of ESCRT complexes (34, 40), packaging of viral genomic RNAs (32, 41), and incorporation of the envelope (42). It may also be important for polarized budding (43, 44) and to create a viral reservoir in infected cells (45, 46).Despite great progress, the traffic of HIV-1 components is still not fully elucidated. In particular, the transport of the genomic RNAs is poorly understood. In this study, we have used single molecule techniques to investigate the trafficking of HIV-1 RNAs in fixed and live cells, and we show that they are transported on endosomal vesicles. We also obtained functional evidence that Gag and viral RNAs can use at least two trafficking pathways to produce virions, one going directly from the plasma membrane and another one passing through endosomes.     

First evidence of miniature transposable elements in sponges (Porifera)

Transposable elements play a vital role in genome evolution and may have been important for the formation of the early metazoan genome, but only little is known about transposons at this interface between unicellular opisthokonts and Metazoa. Here, we describe the first miniature transposable elements (MITEs, Queen1 and Queen2) in sponges. Queen1 and Queen2 are probably derived from Tc1/mariner-like MITE families and are represented in more than 3,800 and 1,700 copies, respectively, in the Amphimedon queenslandica genome. Queen elements are located in intergenic regions as well as in introns, providing the potential to induce new splicing sites and termination signals in the genes. Further possible impacts of MITEs on the evolution of the metazoan genome are discussed.     

Conditioned Medium from Bone Marrow-Derived Mesenchymal Stem Cells Improves Recovery after Spinal Cord Injury in Rats: An Original Strategy to Avoid Cell Transplantation

Spinal cord injury triggers irreversible loss of motor and sensory functions. Numerous strategies aiming at repairing the injured spinal cord have been studied. Among them, the use of bone marrow-derived mesenchymal stem cells (BMSCs) is promising. Indeed, these cells possess interesting properties to modulate CNS environment and allow axon regeneration and functional recovery. Unfortunately, BMSC survival and differentiation within the host spinal cord remain poor, and these cells have been found to have various adverse effects when grafted in other pathological contexts. Moreover, paracrine-mediated actions have been proposed to explain the beneficial effects of BMSC transplantation after spinal cord injury. We thus decided to deliver BMSC-released factors to spinal cord injured rats and to study, in parallel, their properties in vitro. We show that, in vitro, BMSC-conditioned medium (BMSC-CM) protects neurons from apoptosis, activates macrophages and is pro-angiogenic. In vivo, BMSC-CM administered after spinal cord contusion improves motor recovery. Histological analysis confirms the pro-angiogenic action of BMSC-CM, as well as a tissue protection effect. Finally, the characterization of BMSC-CM by cytokine array and ELISA identified trophic factors as well as cytokines likely involved in the beneficial observed effects. In conclusion, our results support the paracrine-mediated mode of action of BMSCs and raise the possibility to develop a cell-free therapeutic approach.     

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.     

New oral anticoagulant drugs: dabigatran, rivaroxaban and apixaban. Present and future

For years, prevention and treatment of thromboembolic events have been restricted to the use of heparins and vitamin K antagonists. These treatments, in spite of their unquestioned efficacy, present numerous limits (hemorrhagic risk, need for regular laboratory controls). These limits call for the development of new antithrombotic drugs. This review briefly reports on three new molecules, in very advanced phases of clinical research: dabigatran (Pradaxa?), rivaroxaban (Xarelto?) and apixaban. These molecules represent new oral anticoagulants, which directly inhibit a coagulation factor (thrombin for dabigatran, factor Xa for rivaroxaban and apixaban) and do not need regular anticoagulant monitoring or dose adjustment. The approval is still restricted in France to the prophylaxis of venous thromboembolism in orthopaedics. Dabigratran will be soon available in the prevention of stroke in atrial fibrillation. With the forthcoming phase III studies to prevent and treat venous thromboembolism, anticoagulant therapy management will be most probably improved in the coming years.     

The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization

Magnetotactic bacteria contain nanometre-sized, membrane-bound organelles, called magnetosomes, which are tasked with the biomineralization of small crystals of the iron oxide magnetite allowing the organism to use geomagnetic field lines for navigation. A key player in this process is the HtrA/DegP family protease MamE. In its absence, Magnetospirillum magneticum str AMB-1 is able to form magnetosome membranes but not magnetite crystals, a defect previously linked to the mislocalization of magnetosome proteins. In this work we use a directed genetic approach to find that MamE, and another predicted magnetosome-associated protease, MamO, likely function as proteases in vivo. However, as opposed to the complete loss of mamE where no biomineralization is observed, the protease-deficient variant of this protein still supports the initiation and formation of small, 20 nm-sized crystals of magnetite, too small to hold a permanent magnetic dipole moment. This analysis also reveals that MamE is a bifunctional protein with a protease-independent role in magnetosome protein localization and a protease-dependent role in maturation of small magnetite crystals. Together, these results imply the existence of a previously unrecognized 'checkpoint' in biomineralization where MamE moderates the completion of magnetite formation and thus committal to magneto-aerotaxis as the organism's dominant mode of navigating the environment.     

Aedesin: Structure and Antimicrobial Activity against Multidrug Resistant Bacterial Strains

Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics and have determined its antimicrobial effects against a large panel of multidrug resistant bacterial strains, directly isolated from infected patients. Based the results from nuclear magnetic resonance spectroscopy analysis, Aedesin has a helix-bend-helix structure typical for a member of the family of α-helix anti-microbial peptides. Aedesin efficiently killed Gram-negative bacterial strains that display the most worrisome resistance mechanisms encountered in the clinic, including resistance to carbapenems, aminoglycosides, cephalosporins, 4^th generation fluoroquinolones, folate inhibitors and monobactams. In contrast, Gram-positive strains were insensitive to the lytic effects of the peptide. The anti-bacterial activity of Aedesin was found to be salt-resistant, indicating that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all types of clinically relevant resistance mechanisms known today, Aedesin might be an interesting candidate for the development of alternative treatment for infections caused by these types of bacteria.     

High‐resolution array‐CGH in patients with oculocutaneous albinism identifies new deletions of the TYR,OCA2, and SLC45A2 genes and a complex rearrangement of the OCA2 gene

Oculocutaneous albinism (OCA) is caused by mutations in six different genes, and their molecular diagnosis encompasses the search for point mutations and intragenic rearrangements. Here, we used high‐resolution array‐comparative genome hybridization (CGH) to search for rearrangements across exons, introns and regulatory sequences of four OCA genes: TYR, OCA2, TYRP1, and SLC45A2. We identified a total of ten new deletions in TYR, OCA2, and SLC45A2. A complex rearrangement of OCA2 was found in two unrelated patients. Whole‐genome sequencing showed deletion of a 184‐kb fragment (identical to a deletion previously found in Polish patients), whereby a large portion of the deleted sequence was re‐inserted after severe reshuffling into intron 1 of OCA2. The high‐resolution array‐CGH presented here is a powerful tool to detect gene rearrangements. Finally, we review all known deletions of the OCA1–4 genes reported so far in the literature and show that deletions or duplications account for 5.6% of all mutations identified in the OCA1–4 genes.     

Mesenchymal stem cell graft improves recovery after spinal cord injury in adult rats through neurotrophic and pro-angiogenic actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue.     

Visual agnosia and posterior cerebral artery infarcts: an anatomical-clinical study

Background

To evaluate systematically the cognitive deficits following posterior cerebral artery (PCA) strokes, especially agnosic visual disorders, and to study anatomical-clinical correlations.

Methods and Findings

We investigated 31 patients at the chronic stage (mean duration of 29.1 months post infarct) with standardized cognitive tests. New experimental tests were used to assess visual impairments for words, faces, houses, and objects. Forty-one healthy subjects participated as controls. Brain lesions were normalized, combined, and related to occipitotemporal areas responsive to specific visual categories, including words (VWFA), faces (FFA and OFA), houses (PPA) and common objects (LOC). Lesions were located in the left hemisphere in 15 patients, in the right in 13, and bilaterally in 3. Visual field defects were found in 23 patients. Twenty patients had a visual disorder in at least one of the experimental tests (9 with faces, 10 with houses, 7 with phones, 3 with words). Six patients had a deficit just for a single category of stimulus. The regions of maximum overlap of brain lesions associated with a deficit for a given category of stimuli were contiguous to the peaks of the corresponding functional areas as identified in normal subjects. However, the strength of anatomical-clinical correlations was greater for words than for faces or houses, probably due to the stronger lateralization of the VWFA, as compared to the FFA or the PPA.

Conclusions

Agnosic visual disorders following PCA infarcts are more frequent than previously reported. Dedicated batteries of tests, such as those developed here, are required to identify such deficits, which may escape clinical notice. The spatial relationships of lesions and of regions activated in normal subjects predict the nature of the deficits, although individual variability and bilaterally represented systems may blur those correlations.