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1.  Autophagy Enhances Bacterial Clearance during P. aeruginosa Lung Infection 
PLoS ONE  2013;8(8):e72263.
Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.
PMCID: PMC3756076  PMID: 24015228
2.  Kaposi's Sarcoma-Associated Herpesvirus G-Protein-Coupled Receptor Prevents AU-Rich-Element-Mediated mRNA Decay 
Journal of Virology  2012;86(16):8859-8871.
During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these contain cis-acting AU-rich elements (AREs) in their 3′ untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells.
PMCID: PMC3421767  PMID: 22696654
3.  Viral subversion of autophagy impairs oncogene-induced senescence 
Autophagy  2012;8(7):1138-1140.
Many viruses have evolved elegant strategies to co-opt cellular autophagic responses to facilitate viral propagation and evasion of immune surveillance. Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a life-long persistent infection in its human host, and is etiologically linked to several cancers. KSHV gene products have been shown to modulate autophagy but their contribution to pathogenesis remains unclear. Our recent study demonstrated that KSHV subversion of autophagy promotes bypass of oncogene-induced senescence (OIS), an important host barrier to tumor initiation. These findings suggest that KSHV has evolved to subvert autophagy, at least in part, to establish an optimal niche for infection, concurrently dampening host antiviral defenses and allowing the ongoing proliferation of infected cells.
PMCID: PMC3429550  PMID: 22735194
KSHV; oncogene; DNA damage; autophagy; oncogene-induced senescence
5.  Hydrolyzable Tannins (Chebulagic Acid and Punicalagin) Target Viral Glycoprotein-Glycosaminoglycan Interactions To Inhibit Herpes Simplex Virus 1 Entry and Cell-to-Cell Spread▿ 
Journal of Virology  2011;85(9):4386-4398.
Herpes simplex virus 1 (HSV-1) is a common human pathogen that causes lifelong latent infection of sensory neurons. Non-nucleoside inhibitors that can limit HSV-1 recurrence are particularly useful in treating immunocompromised individuals or cases of emerging acyclovir-resistant strains of herpesvirus. We report that chebulagic acid (CHLA) and punicalagin (PUG), two hydrolyzable tannins isolated from the dried fruits of Terminalia chebula Retz. (Combretaceae), inhibit HSV-1 entry at noncytotoxic doses in A549 human lung cells. Experiments revealed that both tannins targeted and inactivated HSV-1 viral particles and could prevent binding, penetration, and cell-to-cell spread, as well as secondary infection. The antiviral effect from either of the tannins was not associated with induction of type I interferon-mediated responses, nor was pretreatment of the host cell protective against HSV-1. Their inhibitory activities targeted HSV-1 glycoproteins since both natural compounds were able to block polykaryocyte formation mediated by expression of recombinant viral glycoproteins involved in attachment and membrane fusion. Our results indicated that CHLA and PUG blocked interactions between cell surface glycosaminoglycans and HSV-1 glycoproteins. Furthermore, the antiviral activities from the two tannins were significantly diminished in mutant cell lines unable to produce heparan sulfate and chondroitin sulfate and could be rescued upon reconstitution of heparan sulfate biosynthesis. We suggest that the hydrolyzable tannins CHLA and PUG may be useful as competitors for glycosaminoglycans in the management of HSV-1 infections and that they may help reduce the risk for development of viral drug resistance during therapy with nucleoside analogues.
PMCID: PMC3126266  PMID: 21307190
6.  Phosphorylation and Function of the Kaposin B Direct Repeats of Kaposi's Sarcoma-Associated Herpesvirus 
Journal of Virology  2006;80(12):6165-6170.
Kaposi's sarcoma-associated herpesvirus encodes a protein, kaposin B, which is composed of multiple copies of 23-amino-acid direct repeats, termed DR2 and DR1. Kaposin B enhances the release of pathogenetically important proinflammatory cytokines by activating the p38 mitogen-activated protein kinase (MAPK)-MK2 kinase pathway and blocking cytokine mRNA decay. Here, we show that this mRNA stabilization function requires both the DR2 and DR1 elements of kaposin B; a monomeric form of the protein consisting of one copy of each repeat retains function. Furthermore, we show that p38 MAPK is capable of directly phosphorylating kaposin B in vitro and map the site of phosphorylation to a specific serine residue in DR1. Mutational ablation of this serine abolishes phosphorylation of the protein by p38 MAPK but does not affect kaposin B's ability to extend mRNA half-life.
PMCID: PMC1472581  PMID: 16731955

Results 1-7 (7)