Reversible disruption of XPO1-mediated nuclear export inhibits respiratory syncytial virus (RSV) replication

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract disease in infants, young children, the elderly and immunocompromised individuals. Therapy for RSV infections is limited to high risk infants and there are no safe and efficacious vaccines. Matrix (M) protein is a major RSV structural protein with a key role in virus assembly. Interestingly, M is localised to the nucleus early in infection and its export into the cytoplasm by the nuclear exporter, exportin-1 (XPO1) is essential for RSV assembly. We have shown previously that chemical inhibition of XPO1 function results in reduced RSV replication. In this study, we have investigated the anti-RSV efficacy of Selective Inhibitor of Nuclear Export (SINE) compounds, KPT-335 and KPT-185. Our data shows that therapeutic administration of the SINE compounds results in reduced RSV titre in human respiratory epithelial cell culture. Within 24 h of treatment, RSV replication and XPO1 expression was reduced, M protein was partially retained in the nucleus, and cell cycle progression was delayed. Notably, the effect of SINE compounds was reversible within 24 h after their removal. Our data show that reversible inhibition of XPO1 can disrupt RSV replication by affecting downstream pathways regulated by the nuclear exporter

Novel phylogenetic algorithm to monitor human tropism in Egyptian H5N1-HPAIV reveals evolution toward efficient human-to-human transmission

Years of endemic infections with highly pathogenic avian influenza (HPAI) A subtype H5N1 virus in poultry and high numbers of infections in humans provide ample opportunity in Egypt for H5N1-HPAIV to develop pandemic potential. In an effort to better understand the viral determinants that facilitate human infections of the Egyptian H5N1-HPAIVvirus, we developed a new phylogenetic algorithm based on a new distance measure derived from the informational spectrum method (ISM). This new approach, which describes functional aspects of the evolution of the hemagglutinin subunit 1 (HA1), revealed a growing group G2 of H5N1-HPAIV in Egypt after 2009 that acquired new informational spectrum (IS) properties suggestive of an increased human tropism and pandemic potential. While in 2006 all viruses in Egypt belonged to the G1 group, by 2011 these viruses were virtually replaced by G2 viruses. All of the G2 viruses displayed four characteristic mutations (D43N, S120(D,N), (S,L)129Δ and I151T), three of which were previously reported to increase binding to the human receptor. Already in 2006–2008 G2 viruses were significantly (p<0.02) more often found in humans than expected from their overall prevalence and this further increased in 2009–2011 (p<0.007). Our approach also identified viruses that acquired additional mutations that we predict to further enhance their human tropism. The extensive evolution of Egyptian H5N1-HPAIV towards a preferential human tropism underlines an urgent need to closely monitor these viruses with respect to molecular determinants of virulence

Cell autonomous regulation of herpes and influenza virus infection by the circadian clock.

Viruses are intracellular pathogens that hijack host cell machinery and resources to replicate. Rather than being constant, host physiology is rhythmic, undergoing circadian (∼24 h) oscillations in many virus-relevant pathways, but whether daily rhythms impact on viral replication is unknown. We find that the time of day of host infection regulates virus progression in live mice and individual cells. Furthermore, we demonstrate that herpes and influenza A virus infections are enhanced when host circadian rhythms are abolished by disrupting the key clock gene transcription factor Bmal1. Intracellular trafficking, biosynthetic processes, protein synthesis, and chromatin assembly all contribute to circadian regulation of virus infection. Moreover, herpesviruses differentially target components of the molecular circadian clockwork. Our work demonstrates that viruses exploit the clockwork for their own gain and that the clock represents a novel target for modulating viral replication that extends beyond any single family of these ubiquitous pathogens.A.B.R. acknowledges funding from the Wellcome Trust (083643/Z/07/Z, 100333/Z/12/Z and 100574/Z/12/Z), the European Research Council (ERC Starting Grant No. 281348, MetaCLOCK), the EMBO Young Investigators Programme, the Lister Institute of Preventative Medicine and the Medical Research Council (MRC_MC_UU_12012/5). A.D.N acknowledges funding from the People Programme (Marie Curie Actions) of the European Union Seventh Framework Programme (FP7/2007-2013; REA grant agreement 627630). We thank L. Ansel-Bollepalli for assistance with animal breeding, I. Robinson for assistance with pilot animal experiments, A. Snijders and H. Flynn (Francis Crick Institute Proteomics Core) for help with proteomics work, Cambridge NIHR BRC Cell Phenotyping Hub for flow cytometry assistance, A. Miyawaki (RIKEN Brain Science Institute, Japan) for Fucci2 lentiviral vectors, and H. Coleman, J. May and M. Jain for helpful discussions. We thank Prof J. Bass (Northwestern University, USA) for Bmal-/- mouse embryonic fibroblasts used in preliminary experiments, and N. Heaton and P. Palese (Icahn School of Medicine at Mount Sinai, USA) for PB2::Gaussia luciferase IAV (PR8 PB2::GLUC).This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.160189511

Characterization of the HIV-1 Restriction Factor Herc5

Herc5 is a potent HIV-1 restriction factor able to restrict HIV-1 by two mechanisms: 1) conjugation of ISG15 to gag polyprotein via E3 ligase activity, and 2) restricting the production of HIV-1 proteins by an unknown mechanism that is independent of its E3 ligase function. Herc5 mutations of the RCCl-like domain (RLD) and Spacer domains were generated to dissect this mechanism. Based on HIV-1 release assays, the RLD is important for preventing the production of viral proteins. Herc5 transfected cells show defects in cell cycle progression at the G2/M phase as well as abnormal nuclear morphology. C994A and ASpacer constructs did not impact these phenotypes. However, ARLD Herc5 constructs had normal cell cycle and nuclear morphology. Based on the results found, the RLD was found to be an important mediator of Herc5 cellular and antiviral activity

Cell cycle independent role of cyclin D3 in host restriction of influenza virus infection

To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G1 phase. M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se, and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into the S phase, failed to normalize virus production. Infection by IAV triggered redistribution of cyclin D3 from the nucleus to the cytoplasm followed by its proteasomal degradation. When over-expressed in HEK 293T cells cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.This work was supported by the Research Fund for the Control of Infectious Diseases (Grant RFCID 11101332) and the Area of Excellence Scheme of the University Grants Committee (Grant AoE/M-12/06) and partially supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. T11-705/14N)

Establishment and remodelling of the dendritic cell network in tissues

Conventional dendritic cells (cDCs) are leucocytes that act as sentinel cells, sensing the extracellular environment and initiating immune responses against infection and cancer. cDCs develop from a common progenitor in the bone marrow (BM) that travels via the blood in the form of a pre-cDC to seed tissues. How pre-cDCs colonise different organs, whether this is affected by infection and how BM production is matched to cDC demand in the periphery remains poorly understood. During my PhD I used a mouse model for multicolour fate mapping of cDC progenitors and found that many pre-cDCs and cDCs divide in tissues generating single cDC clones. Upon infection with influenza A virus, lung cDCs increase in number due to accelerated CCR2-dependent recruitment of pre-cDCs from the BM rather than local proliferation, diluting pre-existing clones. This recruitment generates new waves of cDCs in the lung that seem to be necessary for inducing antiviral immunity. Preliminary results using a reporter mouse for DC progenitors show that more cells localise close to BM sinusoids during infection, possibly to favour the rapid release of pre-cDCs into the blood circulation. Interestingly, cancer and vaccine adjuvants also mobilise BM cDC progenitors, demonstrating that this is probably a conserved mechanism by which the cDC network adapts to different challenges. In addition, pre-cDCs can directly sense pathogen-associated molecular patterns via toll-like receptors, which might be necessary for the progenitors to respond to infection or tissue damage. In sum, my results provide evidence for a tightly regulated cDC network that is often organised in clones. However, when a bigger arsenal of cDCs is required, the BM responds by pumping out more pre-cDCs, which is a new component of immunity to infection. More studies might reveal whether CCR2 also drive this phenomenon during cancer and the mechanism underlying pre-cDC exit from the BM

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate