Andrew Michael Lever

Wrapping up the bad news – HIV assembly and release

The late Nobel Laureate Sir Peter Medawar once memorably described viruses as ‘bad news wrapped in protein’. Virus assembly in HIV is a remarkably well coordinated process in which the virus achieves extracellular budding using primarily intracellular budding machinery and also the unusual phenomenon of export from the cell of an RNA. Recruitment of the ESCRT system by HIV is one of the best documented examples of the comprehensive way in which a virus hijacks a normal cellular process. This review is a summary of our current understanding of the budding process of HIV, from genomic RNA capture through budding and on to viral maturation, but centering on the proteins of the ESCRT pathway and highlighting some recent advances in our understanding of the cellular components involved and the complex interplay between the Gag protein and the genomic RNA.

Dimerisation of HIV-2 genomic RNA is linked to efficient RNA packaging, normal particle maturation and viral infectivity

BackgroundRetroviruses selectively encapsidate two copies of their genomic RNA, the Gag protein binding a specific RNA motif in the 5 UTR of the genome. In human immunodeficiency virus type 2 (HIV-2), the principal packaging signal (Psi) is upstream of the major splice donor and hence is present on all the viral RNA species. Cotranslational capture of the full length genome ensures specificity. HIV-2 RNA dimerisation is thought to occur at the dimer initiation site (DIS) located in stem-loop 1 (SL-1), downstream of the main packaging determinant. However, the HIV-2 packaging signal also contains a palindromic sequence (pal) involved in dimerisation. In this study, we analysed the role of the HIV-2 packaging signal in genomic RNA dimerisation in vivo and its implication in viral replication.ResultsUsing a series of deletion and substitution mutants in SL-1 and the Psi region, we show that in fully infectious HIV-2, genomic RNA dimerisation is mediated by the palindrome pal. Mutation of the DIS had no effect on dimerisation or viral infectivity, while mutations in the packaging signal severely reduce both processes as well as RNA encapsidation. Electron micrographs of the Psi-deleted virions revealed a significant reduction in the proportion of mature particles and an increase in that of particles containing multiple cores.ConclusionIn addition to its role in RNA encapsidation, the HIV-2 packaging signal contains a palindromic sequence that is critical for genomic RNA dimerisation. Encapsidation of a dimeric genome seems required for the production of infectious mature particles, and provides a promising therapeutic target.

Evidence that the endosomal sorting complex required for transport-II (ESCRT-II) is required for efficient human immunodeficiency virus-1 (HIV-1) production

BackgroundEgress of a number of different virus species from infected cells depends on proteins of the endosomal sorting complexes required for transport (ESCRT) pathway. HIV has also hijacked this system to bud viruses outward from the cell surface. How ESCRT-I activates ESCRT-III in this process remains unclear with conflicting published evidence for the requirement of ESCRT-II which fulfils this role in other systems. We investigated the role of ESCRT-II using knockdown mediated by siRNA and shRNA, mutants which prevent ESCRT-I/ESCRT-II interaction and a CRISPR/Cas9 EAP45 knockout cell line.ResultsDepletion or elimination of ESCRT-II components from an HIV infected cell produces two distinct effects. The overall production of HIV-1 Gag is reduced leading to a diminished amount of intracellular virion protein. In addition depletion of ESCRT-II produces an effect similar to that seen when ESCRT-I and -III components are depleted, that of a delayed Gag p26 to p24 +p2 cleavage associated with a reduction in export of virion particles and a visible reduction in budding efficiency in virus producing cells. Mutants that interfere with ESCRT-I interacting with ESCRT-II similarly reduce virus export. The export defect is independent of the decrease in overall Gag production. Using a mutant virus which cannot use the ALIX mediated export pathway exacerbates the decrease in virus export seen when ESCRT-II is depleted. ESCRT-II knockdown does not lead to complete elimination of virus release suggesting that the late domain role of ESCRT-II is required for optimal efficiency of viral budding but that there are additional pathways that the virus can employ to facilitate this.ConclusionESCRT-II contributes to efficient HIV virion production and export by more than one pathway; both by a transcriptional or post transcriptional mechanism and also by facilitating efficient virus export from the cell through interactions with other ESCRT components.

Lentiviral-mediated overexpression of Bcl-xL protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis.

BACKGROUNDnEndothelial cells (EC) respond to mild injurious stimuli by upregulating anti-apoptotic gene expression to maintain endothelial integrity. EC dysfunction and apoptosis resulting from ischemia/reperfusion injury may contribute to chronic allograft rejection. We optimized conditions for lentiviral vector (LVV) transduction of rat aortic endothelial cells (RAEC) and investigated whether LVV delivery of the anti-apoptotic gene, Bcl-xL, protects RAEC from apoptotic death using in vitro models of hypoxia and ischemia/reperfusion injury.nnnMETHODSnLVV containing Bcl-xL were generated from a human immunodeficiency virus (HIV)-1 construct. EC were prepared from rat aorta. Hypoxia/reperfusion (H/R) or ischemia/reperfusion (I/R) injury was induced in vitro and apoptosis was assessed using caspase-3 activity, Annexin V/PI and TUNEL staining.nnnRESULTSnAfter in vitro induction of H/R or I/R injury, RAEC showed duration-dependent apoptosis. We confirmed the damaging effect of the reperfusion phase. Endogenous Bax expression increased with I/R injury, whereas endogenous Bcl-xL remained constant. RAEC transduced with LVV expressing Bcl-xL were protected from early apoptosis caused by I/R injury, correlating with reduced cytochrome c release into the cytosol.nnnCONCLUSIONSnOverexpressing Bcl-xL protects RAEC from I/R injury. This protective effect may be attributed to altering the balance of pro- and anti-apoptotic proteins, resulting in sequestration of the harmful Bax protein, and may open up new strategies for controlling chronic allograft rejection.

Replication of human immunodeficiency virus type 1 from entry to exit

It has been 25 years since the recognition of the disease acquired immunodeficiency syndrome (AIDS) in homosexual men in the United States. Much molecular progress in understanding the replication of the human immunodeficiency virus (HIV) has been advanced. Here, we review in a nonexhaustive manner our current knowledge of the replication of HIV-1 inside human cells from entry to exit of the virus.

Blockade of chemokine-induced signalling inhibits CCR5-dependent HIV infection in vitro without blocking gp120/CCR5 interaction.

BackgroundCellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.ResultsIn this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.ConclusionThese observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.

A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2′ hydroxyl reactivity (‘SHAPE’) that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. ‘XL-SHAPE’ was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.

Tri-partite complex for axonal transport drug delivery achieves pharmacological effect.

BackgroundTargeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System) neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior.ResultsWe developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle.ConclusionSpecific targeting of selected subpopulations of CNS neurons for drug delivery by axonal transport holds great promise. The data shown here provide a basic framework for the intraneural pharmacology of this tripartite complex. The pharmacologically efficacious drug delivery demonstrated here verify the fundamental feasibility of using axonal transport for targeted drug delivery.

Characterizing 3D RNA structure by single molecule FRET.

The importance of elucidating the three dimensional structures of RNA molecules is becoming increasingly clear. However, traditional protein structural techniques such as NMR and X-ray crystallography have several important drawbacks when probing long RNA molecules. Single molecule Förster resonance energy transfer (smFRET) has emerged as a useful alternative as it allows native sequences to be probed in physiological conditions and allows multiple conformations to be probed simultaneously. This review serves to describe the method of generating a three dimensional RNA structure from smFRET data from the biochemical probing of the secondary structure to the computational refinement of the final model.

Herpes simplex 1 encephalitis presenting as a brain haemorrhage with normal cerebrospinal fluid analysis: a case report

IntroductionHerpes simplex encephalitis is a potentially lethal infection that should be recognised as soon as possible. The combination of clinical history and examination, brain computed tomography or magnetic resonance imaging and lumbar puncture has been used to establish a diagnosis.Case presentationWe present a patient who had a suggestive history but a totally normal lumbar puncture and only evidence of intracerebral haemorrhage in the brain magnetic resonance imaging. Diagnosis was made by using the cerebrospinal fluid polymerase chain reaction for herpes simplex virus.ConclusionHerpes simplex encephalitis is being increasingly diagnosed with the availability of new diagnostic techniques. Herpes simplex encephalitis can present with the combination of haemorrhage and normal cerebrospinal fluid. Awareness of this common but, if left untreated, devastating condition should increase.