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ABSTRACT: Viruses can modify conditions inside cells to make them more favorable for replication and progeny virus production. One way of doing this is through manipulation of the cell cycle, a process that describes the ordered growth and division of cells. Analysis of model cell lines, such as A549 cells and primary airway epithelial cells, infected with human respiratory syncytial virus (HRSV) has shown alteration of the cell cycle during infection, although the signaling events were not clearly understood. In this study, targeted transcriptomic analysis of HRSV-infected primary airway epithelial cells revealed alterations in the abundances of many mRNAs encoding cell cycle-regulatory molecules, including decreases in the D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]). These alterations were reflected in changes in protein abundance and/or relocalization in HRSV-infected cells; taken together, they were predicted to result in G(0)/G(1) phase arrest. In contrast, there was no change in the abundances of D-type cyclins in A549 cells infected with HRSV. However, the abundance of the G(1)/S phase progression inhibitor p21(WAF1/CIP1) was increased over that in mock-treated cells, and this, again, was predicted to result in G(0)/G(1) phase arrest. The G(0)/G(1) phase arrest in both HRSV-infected primary cells and A549 cells was confirmed using dual-label flow cytometry that accurately measured the different stages of the cell cycle. Comparison of progeny virus production in primary and A549 cells enriched in G(0)/G(1) using a specific CDK4/6 kinase inhibitor with asynchronously replicating cells indicated that this phase of the cell cycle was more efficient for virus production.
Journal of Virology 07/2011; 85(19):10300-9. · 5.40 Impact Factor
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Sheena M Cruickshank,
Matthew L Deschoolmeester,
Marcus Svensson, Gareth Howell,
Aikaterini Bazakou,
Larisa Logunova,
Matthew C Little,
Nicholas English,
Matthias Mack,
Richard K Grencis,
Kathryn J Else,
Simon R Carding
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ABSTRACT: The large intestine is a major site of infection and disease, yet little is known about how immunity is initiated within this site and the role of dendritic cells (DCs) in this process. We used the well-established model of Trichuris muris infection to investigate the innate response of colonic DCs in mice that are inherently resistant or susceptible to infection. One day postinfection, there was a significant increase in the number of immature colonic DCs in resistant but not susceptible mice. This increase was sustained at day 7 postinfection in resistant mice when the majority of the DCs were mature. There was no increase in DC numbers in susceptible mice until day 13 postinfection. In resistant mice, most colonic DCs were located in or adjacent to the epithelium postinfection. There were also marked differences in the expression of colonic epithelial chemokines in resistant mice and susceptible mice. Resistant mice had significantly increased levels of epithelium-derived CCL2, CCL3, CCL5, and CCL20 compared with susceptible mice. Furthermore, administering neutralizing CCL5 and CCL20 Abs to resistant mice prevented DC recruitment. This study provides clear evidence of differences in the kinetics of DC responses in hosts inherently resistant and susceptible to infection. DC responses in the colon correlate with resistance to infection. Differences in the production of DC chemotactic chemokines by colonic epithelial cells in response to infection in resistant vs susceptible mice may explain the different kinetics of the DC response.
The Journal of Immunology 04/2009; 182(5):3055-62. · 5.79 Impact Factor
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ABSTRACT: Localisation of both viral and cellular proteins to the nucleolus is determined by a variety of factors including nucleolar localisation signals (NoLSs), but how these signals operate is not clearly understood. The nucleolar trafficking of wild type viral proteins and chimeric proteins, which contain altered NoLSs, were compared to investigate the role of NoLSs in dynamic nucleolar trafficking. Three viral proteins from diverse viruses were selected which localised to the nucleolus; the coronavirus infectious bronchitis virus nucleocapsid (N) protein, the herpesvirus saimiri ORF57 protein and the HIV-1 Rev protein. The chimeric proteins were N protein and ORF57 protein which had their own NoLS replaced with those from ORF57 and Rev proteins, respectively. By analysing the sub-cellular localisation and trafficking of these viral proteins and their chimeras within and between nucleoli using confocal microscopy and photo-bleaching we show that NoLSs are responsible for different nucleolar localisations and trafficking rates.
Virology 10/2008; 380(2):191-202. · 3.35 Impact Factor
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ABSTRACT: The nucleocapsid (N) protein of infectious bronchitis virus (IBV) localizes to the cytoplasm and nucleolus and contains an eight-amino-acid nucleolar retention motif. In this study, a leucine-rich nuclear export signal (NES) (291-LQLDGLHL-298) present in the C-terminal region of the IBV N protein was analyzed by using alanine substitution and deletion mutagenesis to investigate the relative contributions that leucine residues make to nuclear export and where these residues are located on the structure of the IBV N protein. The analysis indicated that Leu296 and Leu298 are required for efficient nuclear export of the protein. Structural information indicated that both of these amino acids are available for interaction with protein complexes involved in this process. However, export of N protein from the nucleus/nucleolus was not inhibited by leptomycin B treatment, indicating that N protein nuclear export is independent of the CRM1-mediated export pathway.
Journal of Virology 05/2007; 81(8):4298-304. · 5.40 Impact Factor
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Jane E Dalton,
Sheena M Cruickshank,
Charlotte E Egan,
Rainy Mears,
Darren J Newton,
Elizabeth M Andrew,
Beth Lawrence, Gareth Howell,
Kathryn J Else,
Marc-Jan Gubbels,
Boris Striepen,
Judith E Smith,
Stanley J White,
Simon R Carding
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ABSTRACT: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection.
Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity.
In the absence of gammadelta+ iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in gammadelta+ iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of gammadelta T-cell receptor (TCR-Vgamma7+) iIELs that, after reconstituting gammadelta iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection.
These findings identify a novel role for gammadelta+ iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.
Gastroenterology 09/2006; 131(3):818-29. · 11.68 Impact Factor
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ABSTRACT: The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.
Journal of General Virology 01/2006; 86(Pt 12):3303-10. · 3.36 Impact Factor
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ABSTRACT: Gammadelta T cells have a direct role in resolving the host immune response to infection by eliminating populations of activated macrophages. Macrophage reactivity resides within the Vgamma1/Vdelta6.3 subset of gammadelta T cells, which have the ability to kill activated macrophages following infection with Listeria monocytogenes (Lm). However, it is not known how gammadelta T cell macrophage cytocidal activity is regulated, or what effector mechanisms gammadelta T cells use to kill activated macrophages. Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta-/- mice were incubated with splenocytes from wild-type and Fas ligand (FasL)-deficient mice (gld), the ability of Vgamma1 T cells to bind macrophages was shown to be dependent upon Fas-FasL interactions. Combinations of anti-TCR and FasL Abs completely abolished binding to and killing of activated macrophages by Vgamma1 T cells. In addition, confocal microscopy showed that Fas and the TCR colocalized on Vgamma1 T cells at points of contact with macrophages. Collectively, these studies identify an accessory or coreceptor-like function for Fas-FasL that is essential for the interaction of Vgamma1 T cells with activated macrophages and their elimination during the resolution stage of pathogen-induced immune responses.
The Journal of Immunology 10/2004; 173(6):3660-7. · 5.79 Impact Factor
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ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.
Virology.
