David J. Rowlands

Mitochondrial transfer from bone-marrow–derived stromal cells to pulmonary alveoli protects against acute lung injury

Bone marrow–derived stromal cells (BMSCs) protect against acute lung injury (ALI). To determine the role of BMSC mitochondria in this protection, we airway-instilled mice first with lipopolysaccharide (LPS) and then with either mouse BMSCs (mBMSCs) or human BMSCs (hBMSCs). Live optical studies revealed that the mBMSCs formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the alveolar epithelia in these mice, releasing mitochondria-containing microvesicles that the epithelia engulfed. The presence of BMSC-derived mitochondria in the epithelia was evident optically, as well as by the presence of human mitochondrial DNA in mouse lungs instilled with hBMSCs. The mitochondrial transfer resulted in increased alveolar ATP concentrations. LPS-induced ALI, as indicated by alveolar leukocytosis and protein leak, inhibition of surfactant secretion and high mortality, was markedly abrogated by the instillation of wild-type mBMSCs but not of mutant, GJC-incompetent mBMSCs or mBMSCs with dysfunctional mitochondria. This is the first evidence, to our knowledge, that BMSCs protect against ALI by restituting alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer.

The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine

Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses [Harada, T. et al., (2000) J. Virol. 74, 9498–9506]. We show that p7 can be cross‐linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS‐tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza [Hay, A.J. et al. (1985) EMBO J. 4, 3021–3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485–489] and has recently been shown to be active in combination with current HCV therapies.

The Cell Attachment Site on Foot-and-Mouth Disease Virus Includes the Amino Acid Sequence RGD (Arginine-Glycine-Aspartic Acid)

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.

Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein

Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens1,2: the peptide sequence of amino acids 141–160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freunds adjuvant3–5. The immune response to these peptides is much lower than that to complete virus particles1 and the same sequence fused to the N terminus of β-galactosidase did not produce a more potent immunogen than synthetic peptide alone6. We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density. The immunogenicity of these structures approaches that of FMDV particles.

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation.

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.

A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71

Enterovirus 71 (EV71) is a major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. No vaccine or antiviral therapy is available. High-resolution structural analysis of the mature virus and natural empty particles shows that the mature virus is structurally similar to other enteroviruses. In contrast, the empty particles are markedly expanded and resemble elusive enterovirus-uncoating intermediates not previously characterized in atomic detail. Hydrophobic pockets in the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. These structures provide a model for enterovirus uncoating in which the VP1 GH loop acts as an adaptor-sensor for cellular receptor attachment, converting heterologous inputs to a generic uncoating mechanism, highlighting new opportunities for therapeutic intervention.

Substrate complexes of hepatitis C virus RNA polymerase (HC-J4): structural evidence for nucleotide import and de-novo initiation.

Several crystal structures of the hepatitis C virus NS5B protein (genotype-1b, strain J4) complexed with metal ions, single-stranded RNA or nucleoside-triphosphates have been determined. These complexes illustrate how conserved amino acid side-chains, together with essential structural features within the active site, control nucleotide binding and likely mediate de-novo initiation. The incoming nucleotide interacts with several basic residues from an extension on the NS5B fingers domain, a beta-hairpin from the NS5B thumb domain and the C-terminal arm. The modular, bi-partite fingers domain carries a long binding groove which guides the template towards the catalytic site. The apo-polymerase structure provides unprecedented insights into potential non-nucleoside inhibitor binding sites located between palm and thumb near motif E, which is unique to RNA polymerases and reverse transcriptases.

Intracellular Proton Conductance of the Hepatitis C Virus p7 Protein and Its Contribution to Infectious Virus Production

The hepatitis C virus (HCV) p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during an as yet uncharacterized maturation process.

Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus P7 protein in vitro

The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.

Immunological priming with synthetic peptides of foot-and-mouth disease virus.

A sub-immunizing dose of a synthetic peptide corresponding to the amino acids 141 to 160 region of protein VP1 from foot-and-mouth disease virus (FMDV), serotype O1, coupled to keyhole limpet haemocyanin (141-160KLH) has been shown to prime the immune system of guinea-pigs for an FMDV serotype-specific neutralizing antibody response to a second sub-immunizing dose of the same peptide. Optimal priming required an interval of 42 days between the priming dose and the booster dose. No priming was observed in the absence of adjuvant. The secondary response was not restricted by the carrier since animals primed with 141-160KLH could be boosted with uncoupled 141-160 or 141-160 coupled to tetanus toxoid. It has also been shown that uncoupled peptide 141-160 will prime for a neutralizing antibody response when it is incorporated into a relatively non-immunogenic carrier such as small unilamellar liposomes. These results indicate that the 141-160 peptide of FMDV, as well as containing an important neutralizing antibody site, can initiate its own T-helper cell response.