Andrew M. L. Lever

Sepsis: definition, epidemiology, and diagnosis

On 29 March 2005 a 41 year old journalist died of sepsis six days after a minor surgical procedure; she had consulted eight doctors over the intervening Easter bank holiday weekend. Whereas the national press focused on the political question relating to the provision of out of hours medical services in the United Kingdom, the coroner pointed out that “non-recognition of the seriousness of her condition contributed [to her death].” With an estimated annual mortality of between 30 and 50 deaths per 100 000 population,1 2 this condition ranks in the top 10 causes of death,3 affects all ages, and occurs in the community, in long term care facilities, and among patients admitted to hospital under the care of any, and every, medical specialty. #### Summary points Systemic illness caused by microbial invasion of normally sterile parts of the body is referred to as “sepsis.” This is a term that specifically serves to differentiate an illness of microbial origin from an identical clinical syndrome that can arise in several non-microbial conditions, of which pancreatitis is the archetype. The similarity in clinical picture is explained by the pathophysiological role …

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET.

The Gag polyprotein is the major structural protein of human immunodeficiency virus‐1 (HIV‐1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals –Ψ) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Ψ+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Ψ– RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Ψ does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Ψ+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV‐1 Ψ thus acts as a subcellular localisation signal as well as a high‐affinity‐binding site for Gag.

HIV Gag polyprotein: processing and early viral particle assembly

Over the past several decades, extensive research into the Gag polyprotein, the main structural protein of HIV-1 and all other retroviruses, has changed the way that we describe Gags role within viral lifecycles. Initially thought of as a simple scaffold protein forming the viral core, Gag has demonstrated the ability to specifically recognize genomic RNA and both viral and host proteins as it traffics to the cell membrane. There, Gag forms higher ordered structures required for the correct assembly, budding, and maturation of new infectious particles.

Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo

Abstract. Vectors based on lentiviruses such as human immunodeficiency virus (HIV) type-1 have many advantages for gene therapy, including the ability to infect non-dividing cells, long-term transgene expression and the absence of induction of an inflammatory/immune response. This study was initiated to determine whether lentiviruses would efficiently transfer genes to both neonatal and adult cardiac cells in culture and, by direct injection, to the heart in vivo. A three-plasmid expression system, including a packaging defective helper construct, a plasmid coding for a heterologous (VSV-G) envelope protein and a vector construct harboring reporter genes –E-GFP (enhanced green fluorescent protein) and puro (puromycin-resistance protein) was used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells. We demonstrated efficient gene transfer into neonatal and adult cardiac myocytes in vitro and identified conditions in which virtually 100 % of cultured neonatal and 70 % of adult cardiac myocytes express the reporter gene. Transduction of adult cardiac myocytes with high titre lentiviral vectors did not affect the cell number, morphology or viability compared to untransduced cells. We delivered HIV-1-based vectors to the intact heart by direct injection. Hearts transduced with pseudotyped HIV-1 vectors showed levels of transgene expression comparable to that achieved by adenovirus vectors. This study demonstrates for the first time that lentivirus-based vectors can successfully transduce adult cardiomyocytes both in vitro and in vivo, and opens up the prospect of lentivirus-based vectors becoming an important gene delivery system in the cardiovascular field.

The Major Human Immunodeficiency Virus Type 2 (HIV-2) Packaging Signal Is Present on All HIV-2 RNA Species: Cotranslational RNA Encapsidation and Limitation of Gag Protein Confer Specificity

ABSTRACT Deletion of a region of the human immunodeficiency virus type 2 (HIV-2) 5′ leader RNA reduces genomic RNA encapsidation to about 5% that of wild-type virus with no defect in viral protein production but severely limits virus spread in Jurkat T cells, indicating that this region contains a major cis-acting encapsidation signal, or psi (Ψ). Being upstream of the major splice donor, it is present on all viral transcripts. We have shown that HIV-2 selects its genomic RNA for encapsidation cotranslationally, rendering wild-type HIV-2 unable to encapsidate vector RNAs in trans . Virus with Ψ deleted, however, encapsidates an HIV-2 vector, demonstrating competition for Gag protein. HIV-2 overcomes the lack of packaging signal location specificity by two novel mechanisms, cotranslational packaging and competition for limiting Gag polyprotein.

Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms, and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

ABSTRACT Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.

HIV‐1 RNA Packaging

Publisher Summary This chapter focuses on the concept of human immunodeficiency virus type 1 (HIV‐1) RNA packaging. RNA encapsidation by retroviruses is a remarkable process by which the virus negotiates the trafficking of a minority species of mRNA through a particular cellular pathway to become its genome. During this, in the case of HIV, it may first be translated before being selected by the viral Gag protein, highly specifically, from the cellular background pool of mRNAs. These processes involve the recognition of RNA secondary and tertiary structures and an RNA–RNA intermolecular interaction to package a diploid dimeric RNA genome. RNA transport and encapsidation involves cellular chaper-one proteins of which, as yet, few are identified. The structural detail of the interaction between the viral RNA and the Gag protein is described in the chapter. This process requires flexibility and conformational change in the RNA and reflects the fact that transport from transcriptional site to virion likely involves the genomic RNA adopting a number of different structures to display the relevant stage‐specific cis‐acting signals. The specificity of this process and the virus‐specific nature of RNA export from a cell make these processes attractive therapeutic targets.

Comparison of Viral Genomic RNA Sorting Mechanisms in Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Moloney Murine Leukemia Virus

ABSTRACT Genomic RNA sorting between translation and packaging was examined for human immunodeficiency virus type 1 (HIV-1) and HIV-2 using actinomycin D and leptomycin B treatment. Both viruses behaved differently from a simple retrovirus under actinomycin D treatment. With leptomycin B, the lack of apparent functional separation between translation and packaging functions in lentiviruses was confirmed. HIV-2 RNA levels were more stable, but reverse transcriptase production declined similarly to HIV-1.

Smad-Dependent and Smad-Independent Induction of Id1 by Prostacyclin Analogues Inhibits Proliferation of Pulmonary Artery Smooth Muscle Cells In Vitro and In Vivo

Rationale: Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH). Mutations lead to reduced Smad1/5-driven expression of inhibitor of DNA binding protein 1 (Id1) and loss of the growth suppressive effects of BMPs. The impact of existing PAH therapies on BMP signaling is lacking. Objective: Because prostacyclin analogues are effective treatments for clinical PAH, we hypothesized that these agents enhance Smad1/Id1 signaling. Methods and Results: Iloprost alone induced Id1 expression in human pulmonary artery smooth muscle cells (PASMCs), an effect that was independent of Smad1/5 activation but dependent on a cAMP-responsive element in the Id1 promoter. In addition, iloprost and treprostinil enhanced BMP-induced phosphorylation of Smad1/5 and Id1 expression in a cAMP-dependent manner. The mechanism involved suppression of inhibitory Smad, Smad6. Furthermore, iloprost rescued the deficit in Smad1/5 phosphorylation and Id gene expression in PASMCs harboring mutations in BMPR-II and restored growth suppression to BMP4 in mutant PASMCs. We confirmed a critical role for Id1 in PASMC proliferation. Reduced expression of Id1 was observed in concentric intimal lesions of heritable PAH cases. In the monocrotaline rat model of PAH, associated with reduced BMPR-II expression, we confirmed that treprostinil inhibited smooth muscle cell proliferation and prevented progression of PAH while enhancing Smad1/5 phosphorylation and Id1 gene expression. Conclusions: Prostacyclin analogues enhance Id1 expression in vitro and in vivo and restore deficient BMP signaling in BMPR-II mutant PASMCs.

Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation.

ABSTRACT The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5′ untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5′ UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.