Sandhya Boyapalle

Abrogation of Heat Shock Protein 70 Induction as a Strategy to Increase Antileukemia Activity of Heat Shock Protein 90 Inhibitor 17-Allylamino-Demethoxy Geldanamycin

17-Allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone association of heat shock protein 90 (hsp90) with the heat shock factor-1 (HSF-1), which induces the mRNA and protein levels of hsp70. Increased hsp70 levels inhibit death receptor and mitochondria-initiated signaling for apoptosis. Here, we show that ectopic overexpression of hsp70 in human acute myelogenous leukemia HL-60 cells (HL-60/hsp70) and high endogenous hsp70 levels in Bcr-Abl-expressing cultured CML-BC K562 cells confers resistance to 17-AAG-induced apoptosis. In HL-60/hsp70 cells, hsp70 was bound to Bax, inhibited 17-AAG-mediated Bax conformation change and mitochondrial localization, thereby inhibiting the mitochondria-initiated events of apoptosis. Treatment with 17-AAG attenuated the levels of phospho-AKT, AKT, and c-Raf but increased hsp70 levels to a similar extent in the control HL-60/Neo and HL-60/hsp70 cells. Pretreatment with 17-AAG, which induced hsp70, inhibited 1-beta-D-arabinofuranosylcytosine or etoposide-induced apoptosis in HL-60 cells. Stable transfection of a small interfering RNA (siRNA) to hsp70 completely abrogated the endogenous levels of hsp70 and blocked 17-AAG-mediated hsp70 induction, resulting in sensitizing K562/siRNA-hsp70 cells to 17-AAG-induced apoptosis. This was associated with decreased binding of Bax to hsp70 and increased 17-AAG-induced Bax conformation change. 17-AAG-mediated decline in the levels of AKT, c-Raf, and Bcr-Abl was similar in K562 and K562/siRNA-hsp70 cells. Cotreatment with KNK437, a benzylidine lactam inhibitor of hsp70 induction and thermotolerance, attenuated 17-AAG-mediated hsp70 induction and increased 17-AAG-induced apoptosis and loss of clonogenic survival of HL-60 cells. Collectively, these data indicate that induction of hsp70 attenuates the apoptotic effects of 17-AAG, and abrogation of hsp70 induction significantly enhances the antileukemia activity of 17-AAG.

Epidemiologic, Experimental, and Clinical Links between Respiratory Syncytial Virus Infection and Asthma

SUMMARY Virtually all children experience respiratory syncytial virus (RSV) infection at least once during the first 2 years of life, but only a few develop bronchiolitis and more severe disease requiring hospitalization, usually in the first 6 months of life. Children who recover from RSV-induced bronchiolitis are at increased risk for the development of recurrent wheeze and asthma in later childhood. Recent studies suggest that there is an association between RSV-induced bronchiolitis and asthma within the first decade of life but that this association is not significant after age 13. Despite the considerable progress made in our understanding of several aspects of respiratory viral infections, further work needs to be done to clarify the molecular mechanisms of early interactions between virus and host cell and the role of host gene products in the infection process. This review provides a critical appraisal of the literature in epidemiology and experimental research which links RSV infection to asthma. Studies to date demonstrate that there is a significant association between RSV infection and childhood asthma and that preventing severe primary RSV infections can decrease the risk of childhood asthma.

Comparison of Culture, Multiplex, and 5 Nuclease Polymerase Chain Reaction Assays for the Rapid Detection of Yersinia enterocolitica in Swine and Pork Products †

Bacteriological culture was compared with multiplex and fluorogenic (TaqMan) polymerase chain reaction (PCR) assays for the detection of attachment invasion locus (ail)-bearing Yersinia enterocolitica in market weight swine, chitterlings, and ground pork. The TaqMan assay detected 1 pg of purified Y. enterocolitica DNA, whereas conventional gel-based PCR detected I ng of the same. The presence of ail-bearing Y. enterocolitica was tested in pork and feces artificially inoculated with Y. enterocolitica strain NADC 5561. The sensitivity limits of culture, multiplex, and TaqMan PCR assays were 4 x 10(3), 4 x 10(2), and 0.4 CFU/g, respectively, for the artificially inoculated pork. The sensitivity limits were 4 x 10(2), 4 x 10(2), and 0.4 CFU/g, respectively, for feces after a 48-h enrichment in a Yersinia selective broth. By the culture method, Y. enterocolitica was not detected in any of the swine specimens (n = 2,403) examined. By contrast, it was detected in 48 (2%) of the swine samples screened using the multiplex PCR and in 656 (27.2%) of these samples using the TaqMan assay. Using the culture method, Y. enterocolitica was detected in 8% of chitterling samples (n = 350) and in none of the ground pork samples (n = 350). It was identified in 27% of the chitterling samples using multiplex PCR and in 79% of these samples using the TaqMan assay. Ten percent of the ground pork samples contained Y. enterocolitica, as determined by the multiplex PCR, and 38% based on the TaqMan assay. The results suggest that pork products harbor more ail-bearing Y. enterocolitica than selected organs of freshly slaughtered hogs and that the TaqMan assay is more sensitive than either the multiplex PCR or traditional culture methods.

Respiratory syncytial virus NS1 protein colocalizes with mitochondrial antiviral signaling protein MAVS following infection.

Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His6-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production.

A Small-Molecule E2F Inhibitor Blocks Growth in a Melanoma Culture Model

HLM006474 was identified using a computer-based virtual screen and the known crystal structure of the DNA-bound E2F4/DP2 heterodimer. Treatment of multiple cell lines with HLM006474 resulted in the loss of intracellular E2F4 DNA-binding activity as measured by electrophoretic mobility shift assay within hours. Overnight exposure to HLM006474 resulted in down-regulation of total E2F4 protein as well as known E2F targets. The effects of HLM006474 treatment on different cell lines varied but included a reduction in cell proliferation and an increase in apoptosis. HLM006474 induced apoptosis in a manner distinct from cisplatin and doxorubicin. E2F4-null mouse embryonic fibroblasts were less sensitive than wild-type counterparts to the apoptosis-inducing activity of the compound, revealing its biological specificity. A375 cells were extremely sensitive to the apoptosis-inducing activity of the compound in two-dimensional culture, and HLM006474 was a potent inhibitor of melanocytes proliferation and subsequent invasion in a three-dimensional tissue culture model system. Together, these results suggest that interference with E2F activity using small molecules may have clinical application in cancer therapy.

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection

Respiratory syncytial virus (RSV) infection accounts for approximately 64 million cases of respiratory disease and 200,000 deaths worldwide each year, yet no broadly effective prophylactic or treatment regimen is available. RSV deploys paired, self-associating, heptad repeat domains of its fusion protein, RSV-F, to form a fusogenic 6-helix bundle that enables the virus to penetrate the host cell membrane. Here, we developed hydrocarbon double-stapled RSV fusion peptides that exhibit stabilized α-helical structure and striking proteolytic resistance. Pretreatment with double-stapled RSV peptides that specifically bound to the RSV fusion bundle inhibited infection by both laboratory and clinical RSV isolates in cells and murine infection models. Intranasal delivery of a lead double-stapled RSV peptide effectively prevented viral infection of the nares. A chitosan-based nanoparticle preparation markedly enhanced pulmonary delivery, further preventing progression of RSV infection to the lung. Thus, our results provide a strategy for inhibiting RSV infection by mucosal and endotracheal delivery of double-stapled RSV fusion peptides.

A Baculovirus-Expressed Dicistrovirus That Is Infectious to Aphids

ABSTRACT Detailed investigation of virus replication is facilitated by the construction of a full-length infectious clone of the viral genome. To date, this has not been achieved for members of the family Dicistroviridae. Here we demonstrate the construction of a baculovirus that expresses a dicistrovirus that is infectious in its natural host. We inserted a full-length cDNA clone of the genomic RNA of the dicistrovirus Rhopalosiphum padi virus (RhPV) into a baculovirus expression vector. Virus particles containing RhPV RNA accumulated in the nuclei of baculovirus-infected Sf21 cells expressing the recombinant RhPV clone. These virus particles were infectious in R. padi, a ubiquitous aphid vector of major cereal viruses. The recombinant virus was transmitted efficiently between aphids, despite the presence of 119 and 210 vector-derived bases that were stably maintained at the 5′ and 3′ ends, respectively, of the RhPV genome. The maintenance of such a nonviral sequence was surprising considering that most RNA viruses tolerate few nonviral bases beyond their natural termini. The use of a baculovirus to express a small RNA virus opens avenues for investigating replication of dicistroviruses and may allow large-scale production of these viruses for use as biopesticides.

Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone

Availability of a cloned genome from which infectious RNA can be transcribed is essential for investigating RNA virus molecular mechanisms. To date, no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven expression of a cloned Rhopalosiphum padi virus (RhPV; Dicistroviridae) genome that was infectious to aphids, and we identified a cell line (GWSS-Z10) from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious. Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from transcript-infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool, and a more tractable system without interference from baculovirus infection, for investigating replication and pathogenesis of dicistroviruses.

Nanotechnology applications to HIV vaccines and microbicides

Human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) remains one of the most serious threats to global health. Today there are no HIV vaccines which can prevent HIV infection. All of the candidates being studied are in the experimental stage. Preventive vaccine candidates are being tested in HIV-negative people to see if they can prevent infection. With of the development of a safe and effective vaccine still likely to be years away, topical microbicide formulations that are applied vaginally and rectally are receiving greater interest as an effective alternative to slow down the global spread of HIV. Current microbicide trials that aim to prevent the sexual transmission of HIV are using gels, creams, rings, films and there is also work underway to explore other types of ‘delivery’ systems. There have been numerous reports on safety and lack of toxicity of the application of nanotechnology for targeted delivery and slow, sustained release of drugs, proteins, peptides or nucleic acids by any route to maximize effectiveness and minimize adverse effects. The application of nanotechnology for targeting drugs and macromolecules to specific tissues or cells is one of the most important areas in nanomedicine research. Thus far nanoparticles provide a strong platform to combine protein and DNA based vaccines/microbicides and will facilitate the production, preclinical evaluation and clinical testing in the future.

Respiratory syncytial virus (RSV) infection in elderly mice results in altered antiviral gene expression and enhanced pathology.

Elderly persons are more susceptible to RSV-induced pneumonia than young people, but the molecular mechanism underlying this susceptibility is not well understood. In this study, we used an aged mouse model of RSV-induced pneumonia to examine how aging alters the lung pathology, modulates antiviral gene expressions, and the production of inflammatory cytokines in response to RSV infection. Young (2–3 months) and aged (19–21 months) mice were intranasally infected with mucogenic or non-mucogenic RSV strains, lung histology was examined, and gene expression was analyzed. Upon infection with mucogenic strains of RSV, leukocyte infiltration in the airways was elevated and prolonged in aged mice compared to young mice. Minitab factorial analysis identified several antiviral genes that are influenced by age, infection, and a combination of both factors. The expression of five antiviral genes, including pro-inflammatory cytokines IL-1β and osteopontin (OPN), was altered by both age and infection, while age was associated with the expression of 15 antiviral genes. Both kinetics and magnitude of antiviral gene expression were diminished as a result of older age. In addition to delays in cytokine signaling and pattern recognition receptor induction, we found TLR7/8 signaling to be impaired in alveolar macrophages in aged mice. In vivo, induction of IL-1β and OPN were delayed but prolonged in aged mice upon RSV infection compared to young. In conclusion, this study demonstrates inherent differences in response to RSV infection in young vs. aged mice, accompanied by delayed antiviral gene induction and cytokine signaling.