CK2 Inhibitors Increase the Sensitivity of HSV-1 to Interferon-β

Herpes simplex virus type 1 (HSV-1) requires the activities of cellular kinases for efficient replication. The host kinase, CK2, has been shown or is predicted to modify several HSV-1 proteins and has been proposed to affect one or more steps in the viral lifecycle. Furthermore, potential cellular and viral substrates of CK2 are involved in antiviral pathways and viral counter-defenses, respectively, suggesting that CK2 regulates these processes. Consequently, we tested whether pharmacological inhibitors of CK2 impaired HSV-1 replication, either alone or in combination with the cellular antiviral factor, interferon-β (IFN-β). Our results indicate that the use of CK2 inhibitors results in a minor reduction in HSV-1 replication but enhanced the inhibitory effect of IFN-β on replication. This effect was dependent on the HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), which impairs several host antiviral responses, including that produced by IFN-β. Inhibitors of CK2 did not, however, impede the ability of ICP0 to induce the degradation of two cellular targets: the promyelocyticleukemia protein (PML) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Notably, this effect was only apparent for HSV-1, as the CK2 inhibitors did not enhance the antiviral effect of IFN-β on either vesicular stomatitis virus or adenovirus type 5. Thus, our data suggest that the activity of CK2 is required for an early function during viral infection that assists the growth of HSV-1 in IFN-β-treated cells

hTERT Extends the Life of Human Fibroblasts without Compromising Type I Interferon Signaling

Primary cells are often used to study viral replication and host-virus interactions as their antiviral pathways have not been altered or inactivated; however, their use is restricted by their short lifespan. Conventional methods to extend the life of primary cultures typically utilize viral oncogenes. Many of these oncogenes, however, perturb or inactivate cellular antiviral pathways, including the interferon (IFN) response. It has been previously shown that expression of the telomerase reverse transcriptase (TERT) gene extends the life of certain cell types. The effect that TERT expression has on the innate antiviral response to RNA- and DNA-containing viruses has not been examined. In the current study, we introduced the human TERT (hTERT) gene into a primary human embryonic lung (HEL-299) cell strain, which is known to respond to the type I IFN, IFN-β. We show that the resulting HEL-TERT cell line is capable of replicating beyond 100 population doublings without exhibiting signs of senescence. Treatment with IFN-β resulted in the upregulation of four model IFN stimulated genes (ISGs) in HEL-299 and HEL-TERT cells. Both cell lines supported the replication of herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV) and impaired the replication of both viruses upon IFN-β pretreatment. Introduction of the viral oncoprotein, simian virus 40 (SV40) large T-antigen, which is frequently used to immortalize cells, largely negated this effect. Taken together, our data indicate that expression of hTERT does not alter type 1 IFN signaling and/or the growth of two viruses, making this cell line a useful reagent for studying viral replication and virus-cell interactions.This work was supported in part by grants from the National Institute of Allergy and Infectious Diseases (R01AI72357), the National Center for Research Resources (P20RR016475), and the National Institute of General Medical Sciences (P20GM103418) from the National Institutes of Health

Role of a cdk5-associated protein, p35, in herpes simplex virus type 1 replication in vivo

Previous studies have shown that herpes simplex virus type 1 (HSV-1) replication is inhibited by the cyclin-dependent kinase (cdk) inhibitor roscovitine. One roscovitine-sensitive cdk that functions in neurons is cdk5, which is activated in part by its binding partner, p35. Because HSV establishes latent infections in sensory neurons, we sought to determine the role p35 plays in HSV-1 replication in vivo. For these studies, wild-type (wt) and p35-/- mice were infected with HSV-1 using the mouse ocular model of HSV latency and reactivation. The current results indicate that p35 is an important determinant of viral replication in vivo

Serum Early Prostate Cancer Antigen (EPCA) Level and Its Association with Disease Progression in Prostate Cancer in a Chinese Population

BACKGROUND: Early prostate cancer antigen (EPCA) has been shown a prostate cancer (PCa)-associated nuclear matrix protein, however, its serum status and prognostic power in PCa are unknown. The goals of this study are to measure serum EPCA levels in a cohort of patients with PCa prior to the treatment, and to evaluate the clinical value of serum EPCA. METHODS: Pretreatment serum EPCA levels were determined with an ELISA in 77 patients with clinically localized PCa who underwent radical prostatectomy and 51 patients with locally advanced or metastatic disease who received primary androgen deprivation therapy, and were correlated with clinicopathological variables and disease progression. Serum EPCA levels were also examined in 40 healthy controls. RESULTS: Pretreatment mean serum EPCA levels were significantly higher in PCa patients than in controls (16.84 ± 7.60 ng/ml vs. 4.12 ± 2.05 ng/ml, P<0.001). Patients with locally advanced and metastatic PCa had significantly higher serum EPCA level than those with clinically localized PCa (22.93 ± 5.28 ng/ml and 29.41 ± 8.47 ng/ml vs. 15.17 ± 6.03 ng/ml, P = 0.014 and P<0.001, respectively). Significantly elevated EPCA level was also found in metastatic PCa compared with locally advanced disease (P < 0.001). Increased serum EPCA levels were significantly and positively correlated with Gleason score and clinical stage, but not with PSA levels and age. On multivariate analysis, pretreatment serum EPCA level held the most significantly predictive value for the biochemical recurrence and androgen-independent progression among pretreatment variables (HR = 4.860, P<0.001 and HR = 5.418, P<0.001, respectively). CONCLUSIONS: Serum EPCA level is markedly elevated in PCa. Pretreatment serum EPCA level correlates significantly with the poor prognosis, showing prediction potential for PCa progression

IGFBP-rP1, a potential molecule associated with colon cancer differentiation

<p>Abstract</p> <p>Background</p> <p>In our previous studies, we have demonstrated that insulin-like growth factor binding protein-related protein1 (IGFBP-rP1) played its potential tumor suppressor role in colon cancer cells through apoptosis and senescence induction. In this study, we will further uncover the role of IGFBP-rP1 in colon cancer differentiation and a possible mechanism by revealing responsible genes.</p> <p>Results</p> <p>In normal colon epithelium, immunohistochemistry staining detected a gradient IGFBP-rP1 expression along the axis of the crypt. IGFBP-rP1 strongly expressed in the differentiated cells at the surface of the colon epithelium, while weakly expressed at the crypt base. In colon cancer tissues, the expression of IGFBP-rP1 correlated positively with the differentiation status. IGFBP-rP1 strongly expressed in low grade colorectal carcinoma and weakly expressed in high grade colorectal carcinoma. In vitro, transfection of PcDNA3.1(IGFBP-rP1) into RKO, SW620 and CW2 cells induced a more pronounced anterior-posterior polarity morphology, accompanied by upregulation with alkaline phosphatase (AKP) activity. Upregulation of carcino-embryonic antigen (CEA) was also observed in SW620 and CW2 transfectants. The addition of IGFBP-rP1 protein into the medium could mimic most but not all effects of IGFBP-rP1 cDNA transfection. Seventy-eight reproducibly differentially expressed genes were detected in PcDNA3.1(IGFBP-rP1)-RKO transfectants, using Affymetrix 133 plus 2.0 expression chip platform. Directed Acyclic Graph (DAG) of the enriched GO categories demonstrated that differential expression of the enzyme regulator activity genes together with cytoskeleton and actin binding genes were significant. IGFBP-rP1 could upreguate Transgelin (TAGLN), downregulate SRY (sex determining region Y)-box 9(campomelic dysplasia, autosomal sex-reversal) (SOX9), insulin receptor substrate 1(IRS1), cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B), amphiregulin(schwannoma-derived growth factor) (AREG) and immediate early response 5-like(IER5L) in RKO, SW620 and CW2 colon cancer cells, verified by Real time Reverse Transcription Polymerase Chain Reaction (rtRT-PCR). During sodium butyrate-induced Caco2 cell differentiation, IGFBP-rP1 was upregulated and the expression showed significant correlation with the AKP activity. The downregulation of IRS1 and SOX9 were also induced by sodium butyrate.</p> <p>Conclusion</p> <p>IGFBP-rP1 was a potential key molecule associated with colon cancer differentiation. Downregulation of IRS1 and SOX9 may the possible key downstream genes involved in the process.</p

A Viral Ubiquitin Ligase Has Substrate Preferential SUMO Targeted Ubiquitin Ligase Activity that Counteracts Intrinsic Antiviral Defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection

Ectopic hTERT expression does not affect the ability of HEL cells to produce antiviral cytokines.

<p>HEL-299, HEL-TERT, and HEL-TERT-T cells were mock- or SeV-infected at 100 HAU/10⁶ cell. At 24 hpi, the media were transferred to naïve Vero cell monolayers. 6 h later, the Vero cells were infected with ∼200 PFU of VSV-eGFP per well. At 24 hpi with VSV-eGFP, the Vero cells were fixed and the number of fluorescent plaques counted. Data shown represents the average of two experiments performed in duplicate (± standard errors of the means).</p

Is 5 days of oral fluoroquinolone enough for acute uncomplicated pyelonephritis? The DTP randomized trial

International audienceThe treatment duration of acute uncomplicated pyelonephritis (AUP) is still under debate. As shortening treatment duration could be a means to reduce antimicrobial resistance, we aimed to establish whether 5 days of antibiotic treatment is non-inferior to 10 days in patients with AUP. We performed an open-label prospective randomized trial comparing 5 days to 10 days of fluoroquinolone treatment for AUP. The inclusion criteria were: female patients aged ≥18 years with clinical signs of urinary tract infection, fever >38 °C, and positive urinalysis. Patients were randomized to either 5 or 10 days of fluoroquinolone treatment. Outcome was cure at day 10 and day 30 after the end of treatment. One hundred patients were randomized and 12 were excluded after randomization. The mean ± standard deviation (SD) age was 31.8 ± 11 years old and the mean ± SD temperature was 38.6 ± 0.7 °C. The main bacterium involved was Escherichia coli (n = 86; 97.7%) and 3 (3.4%) patients had a positive blood culture. In the post-hoc analysis, clinical cure 10 days after the end of the treatment was 28/30 (93.3%) in the 5-day arm and 36/38 (94.7%) in the 10-day arm (p = 1.00). At day 30, the clinical cure rate was 23/23 (100%) in the 5-day arm and 20/20 (100%) in the 10-day arm (p = 1.00). The microbiological cure rate was 20/23 (87.0%) in the 5-day arm and 16/20 (80.0%) in the 10-day arm (p = 1.00). The efficacy of 5 days of fluoroquinolone treatment does not seem different from 10 days of treatment for AUP