Therapeutic targets for HIV-1 infection in the host proteome

BACKGROUND: Despite the success of HAART, patients often stop treatment due to the inception of side effects. Furthermore, viral resistance often develops, making one or more of the drugs ineffective. Identification of novel targets for therapy that may not develop resistance is sorely needed. Therefore, to identify cellular proteins that may be up-regulated in HIV infection and play a role in infection, we analyzed the effects of Tat on cellular gene expression during various phases of the cell cycle. RESULTS: SOM and k-means clustering analyses revealed a dramatic alteration in transcriptional activity at the G1/S checkpoint. Tat regulates the expression of a variety of gene ontologies, including DNA-binding proteins, receptors, and membrane proteins. Using siRNA to knock down expression of several gene targets, we show that an Oct1/2 binding protein, an HIV Rev binding protein, cyclin A, and PPGB, a cathepsin that binds NA, are important for viral replication following induction from latency and de novo infection of PBMCs. CONCLUSION: Based on exhaustive and stringent data analysis, we have compiled a list of gene products that may serve as potential therapeutic targets for the inhibition of HIV-1 replication. Several genes have been established as important for HIV-1 infection and replication, including Pou2AF1 (OBF-1), complement factor H related 3, CD4 receptor, ICAM-1, NA, and cyclin A1. There were also several genes whose role in relation to HIV-1 infection have not been established and may also be novel and efficacious therapeutic targets and thus necessitate further study. Importantly, targeting certain cellular protein kinases, receptors, membrane proteins, and/or cytokines/chemokines may result in adverse effects. If there is the presence of two or more proteins with similar functions, where only one protein is critical for HIV-1 transcription, and thus, targeted, we may decrease the chance of developing treatments with negative side effects

Modulation of GSK-3β activity in Venezuelan equine encephalitis virus infection

Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer\u27s. Hence inhibition of GSK-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC50 of ~0.5 µM and a CC50 of \u3e100 µM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3β inhibitors for modulating VEEV infection

Genetic Deletion of the Nociceptin/Orphanin FQ Receptor in the Rat Confers Resilience to the Development of Drug Addiction

The nociceptin (NOP) receptor is a G-protein-coupled receptor whose natural ligand is the nociceptin/orphanin FQ (N/OFQ) peptide. Evidence from pharmacological studies suggests that the N/OFQ system is implicated in the regulation of several addiction-related phenomena, such as drug intake, withdrawal and relapse. Here, to further explore the role of NOP system in addiction, we used NOP (-/-) rats to study the motivation for cocaine, heroin and alcohol self-administration in the absence of N/OFQ function. Conditioned place preference (CPP) and saccharin (0.2% w/v) self-administration were also investigated. Results showed that NOP (-/-) rats self-administer less cocaine (0.25, 0.125 or 0.5 mg/infusion) both under a Fixed Ratio 1 and a Progressive Ratio schedule of reinforcement compared to wild type (Wt) controls. Consistently, cocaine (10 mg/kg, i.p.) was able to induce CPP in Wt but not in NOP (-/-). When NOP (-/-) rats were tested for heroin (20 μg/infusion) and ethanol (10% v/v) self-administration, they showeda significantly lower drug intake compared to Wt. Conversely, saccharin self-administration was not affected by NOP deletion, excluding the possibility of nonspecific learning deficits or generalized disruption of reward mechanisms in NOP (-/-) rats. These findings were confirmed with pharmacological experiments using two selective NOP antagonists, SB-612111 and LY2817412. Both drugs attenuated alcohol self-administration in Wt rats but not in NOP (-/-) rats. In conclusion, our results demonstrate that genetic deletion of NOP receptors confers resilience to drug abuse and support a role for NOP receptor antagonism as a potential treatment option for drug addiction.Neuropsychopharmacology accepted article preview online, 26 August 2016. doi:10.1038/npp.2016.171

Small Molecules Targeted to a Non-Catalytic “RVxF” Binding Site of Protein Phosphatase-1 Inhibit HIV-1

HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q35VCF38, which resembles the PP1-binding “RVxF” motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the “RVxF”-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the “RVxF”-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners

Becoming and staying physically active in adolescents with cerebral palsy: protocol of a qualitative study of facilitators and barriers to physical activity

<p>Abstract</p> <p>Background</p> <p>Adolescents with cerebral palsy (CP) show a reduced physical activity (PA). Currently there are no interventions for adolescents with CP in this critical life phase that optimise and maintain the individuals' physical activity in the long term. To develop such a program it is important to fully understand the factors that influence physical activity behaviours in adolescents with CP. The aim of this study is to explore what makes it easy or hard for adolescents with CP to be and to become physically active.</p> <p>Methods/Design</p> <p>A qualitative research method is chosen to allow adolescents to voice their own opinion. Because we will investigate the lived experiences this study has a phenomenological approach. Thirty ambulatory and non-ambulatory adolescents (aged 10-18 years) with CP, classified as level I to IV on the Gross Motor Function Classification System and 30 parents of adolescents with CP will be invited to participate in one of the 6 focus groups or an individual interview. Therapists from all Children's Treatment Centres in Ontario, Canada, will be asked to fill in a survey. Focus groups will be audio- and videotaped and will approximately take 1.5 hours. The focus groups will be conducted by a facilitator and an assistant. In preparation of the focus groups, participants will fill in a demographic form with additional questions on physical activity. The information gathered from these questions and recent research on barriers and facilitators to physical activity will be used as a starting point for the content of the focus groups. Recordings of the focus groups will be transcribed and a content analysis approach will be used to code the transcripts. A preliminary summary of the coded data will be shared with the participants before themes will be refined.</p> <p>Discussion</p> <p>This study will help us gain insight and understanding of the participants' experiences and perspectives in PA, which can be of great importance when planning programs aimed at helping them to stay or to become physically active.</p

Reduced cAMP, Akt Activation and p65-c-Rel Dimerization: Mechanisms Involved in the Protective Effects of mGluR3 Agonists in Cultured Astrocytes

In recent decades, astrocytes have emerged as key pieces in the maintenance of normal functioning of the central nervous system. Any impairment in astroglial function can ultimately lead to generalized disturbance in the brain, thus pharmacological targets associated with prevention of astrocyte death are actually promising. Subtype 3 of metabotropic glutamate receptors (mGluR3) is present in astrocytes, its activation exerting neuroprotective roles. In fact, we have previously demonstrated that mGluR3 selective agonists prevent nitric oxide (NO)-induced astrocyte death. However, mechanisms responsible for that cytoprotective property are still subject to study. Although inhibition of adenylyl cyclase by mGluR3 activation was extensively reported, the involvement of reduced cAMP levels in the effects of mGluR3 agonists and the association between cAMP decrease and the downstream pathways activated by mGluR3 remain neglected. Thus, we studied intracellular signaling mediating anti-apoptotic actions of mGluR3 in cultured rat astrocytes exposed to NO. In the present work, we showed that the cytoprotective effect of mGluR3 agonists (LY379268 and LY404039) requires both the reduction of intracellular cAMP levels and activation of Akt, as assessed by MTT and TUNEL techniques. Moreover, dibutyryl-cAMP impairs Akt phosphorylation induced by LY404039, indicating a relationship between mGluR3-reduced cAMP levels and PI3K/Akt pathway activation. We also demonstrated, by co-immunoprecipitation followed by western-blot, that the mGluR3 agonists not only induce per se survival-linked interaction between members of the NF-κB family p65 and c-Rel, but also impede reduction of levels of p65-c-Rel dimers caused by NO, suggesting a possible anti-apoptotic role for p65-c-Rel. All together, these data suggest that mGluR3 agonists may regulate cAMP/Akt/p65-c-Rel pathway, which would contribute to the protective effect of mGluR3 against NO challenge in astrocytes. Our results widen the knowledge about mechanisms of action of mGluR3, potential targets for the treatment of neurodegenerative disorders where a pathophysiological role for NO has been established

p53 Activation following Rift Valley Fever Virus Infection Contributes to Cell Death and Viral Production

Rift Valley fever virus (RVFV) is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production

NF90 Binds the Dengue Virus RNA 3′ Terminus and is a Positive Regulator of Dengue Virus Replication

Background Viral RNA translation and replication are regulated by sequence and structural elements in the 5′ and 3′ untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5′ m7GpppG cap, and a conserved 3′-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3′ terminus. Methodology/Principal Findings Proteins eluted from a dengue 3′ SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny. Conclusions/Significance The results indicate that NF90 interacts with the 3′ SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.Ruth L. Kirschstein National Research Service Award (NIH-NRSA GM64985)UNCF-Merck Postdoctoral FellowshipNational Institute of Allergy and Infectious Diseases (U.S.)Ellison Medical Foundatio

Alteration of Blood–Brain Barrier Integrity by Retroviral Infection

The blood–brain barrier (BBB), which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1) Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans), both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies