Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist

Newcastle disease virus (NDV) edits its P gene by inserting one or two G residues at the conserved editing site (UUUUUCCC, genome sense) and transcribes the P mRNA (unedited), the V mRNA (with a +1 frameshift), and the W mRNA (with a +2 frameshift). All three proteins are amino coterminal but vary at their carboxyl terminus in length and amino acid composition. Little is known about the role of the V and W proteins in NDV replication and pathogenesis. We have constructed and recovered two recombinant viruses in which the expression of the V or both the V and W proteins has been abolished. Compared to the parental virus, the mutant viruses showed impaired growth in cell cultures, except in Vero cells. However, transient expression of the carboxyl-terminal portion of the V protein enhanced the growth of the mutant viruses. In embryonated chicken eggs, the parental virus grew to high titers in embryos of different gestational ages, whereas the mutant viruses showed an age-dependent phenomenon, growing to lower titer in more-developed embryos. An interferon (IFN) sensitivity assay showed that the parental virus was more resistant to the antiviral effect of IFN than the mutant viruses. Moreover, infection with the parental virus resulted in STAT1 protein degradation, but not with the mutant viruses. These findings indicate that the V protein of NDV possesses the ability to inhibit alpha IFN and that the IFN inhibitory function lies in the carboxyl-terminal domain. Pathogenicity studies showed that the V protein of NDV significantly contributes to the virus virulence.     

Lycopene and the LXRα agonist T0901317 synergistically inhibit the proliferation of androgen-independent prostate cancer cells via the PPARγ-LXRα-ABCA1 pathway

In our previous study, we demonstrated that lycopene can inhibit the proliferation of androgen-dependent prostate LNCaP cancer cells through the activation of the peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor alpha (LXRα)-ATP-binding cassette transporter 1 (ABCA1) pathway. However, it is still unclear whether lycopene possesses similar effects in androgen-independent prostate cancer cells DU145 and PC-3. As lycopene inhibited the proliferation of both cell types to a similar extent, we chose DU145 cells for most of the subsequent studies. We show that lycopene significantly increased protein and mRNA expression of PPARγ, LXRα and ABCA1 and cholesterol efflux (i.e., decreased cellular cholesterol and increased cholesterol in culture medium). Lycopene (10 μM) in the presence of a specific antagonist of PPARγ (GW9662) or of LXRα (GGPP) restored the proliferation of DU145 cells and significantly suppressed lycopene-induced protein and mRNA expression of PPARγ and LXRα and cholesterol efflux. Liver X receptor α knockdown by siRNA against LXRα significantly promoted the proliferation of DU145 cells, whereas si-LXRα knockdown followed by incubation with lycopene (10 μM) restored the proliferation to the control level. Furthermore, lycopene in combination with the LXRα agonist T0901317 exhibited synergistic effects on cell proliferation and protein expression of PPARγ, LXRα and ABCA1. These results demonstrate that lycopene can inhibit DU145 cell proliferation via PPARγ-LXRα-ABCA1 pathway and that lycopene and T0901317 exhibit synergistic effects.     

Inhibition of transcription of the beta interferon gene by the human herpesvirus 6 immediate-early 1 protein

Human herpesviruses (HHV) are stealth pathogens possessing several decoy or immune system evasion mechanisms favoring their persistence within the infected host. Of these viruses, HHV-6 is among the most successful human parasites, establishing lifelong infections in nearly 100% of individuals around the world. To better understand this host-pathogen relationship, we determined whether HHV-6 could interfere with the development of the innate antiviral response by affecting interferon (IFN) biosynthesis. Using inducible cell lines and transient transfection assays, we have identified the immediate-early 1 (IE1) protein as a potent inhibitor of IFN-beta gene expression. IE1 proteins from both HHV-6 variants were capable of suppressing IFN-beta gene induction. IE1 prevents IFN-beta gene expression triggered by Sendai virus infection, double-stranded RNA (dsRNA) and dsDNA transfection, or the ectopic expression of IFN-beta gene activators such as retinoic inducible gene I protein, mitochondrial antiviral signaling protein, TBK-1, IkappaB kinase epsilon (IKKepsilon), and IFN regulatory factor 3 (IRF3). While the stability of IFN-beta mRNA is not affected, IE1-expressing cells have reduced levels of dimerized IRF3 and nucleus-translocated IRF3 in response to activation by TBK-1 or IKKepsilon. Using nuclear extracts and gel shift experiments, we could demonstrate that in the presence of IE1, IRF3 does not bind efficiently to the IFN-beta promoter sequence. Overall, these results indicate that the IE1 protein of HHV-6, one of the first viral proteins synthesized upon viral entry, is a potent suppressor of IFN-beta gene induction and likely contributes to favor the establishment of and successful infection of cells with this virus.     

Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2′-5′-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.     

Enhanced steatosis by nuclear receptor ligands: A study in cultured human hepatocytes and hepatoma cells with a characterized nuclear receptor expression profile

Steatosis is the first step in the development of non-alcoholic fatty liver disease (NAFLD). However, the mechanisms involved in its pathogenesis are not fully understood. Many nuclear receptors (NRs) involved in energy homeostasis and biotransformation constitute a network connecting fatty acids, cholesterol and xenobiotic metabolisms; therefore, multiple NRs and their ligands may play a prominent role in liver fat metabolism and accumulation. In this study we have attempted to gain insight into the relevance of the NR superfamily in NAFLD by investigating the steatogenic potential of 76 different NR ligands in fatty acid overloaded human hepatocytes and hepatoma cells. Moreover, we have determined the mRNA expression level of 24 NRs to correlate the steatogenic potential of the ligands with the expression of their associated NRs in the cultured cells. Our results demonstrate that 18% of the examined NR ligands enhanced lipid accumulation in human hepatocytes and/or hepatoma cells. Among them, ligands of PPARγ (e.g., thiazolidinediones), LXR (paxilline and 24(S),25-epoxycholesterol), PXR (hyperforin), CAR (3α,5α-androstenol), ERα (tamoxifen), FXR (Z-guggulsterone), VDR (25-hydroxyvitamin D3) and particular retinoids and farnesoids showed a significant pro-steatotic effect. The mRNA level of most of the NRs examined was well preserved in human hepatocytes, but HepG2 showed a deranged profile, where many of the receptors had a marginal or negligible level of expression in comparison with the human liver. By comparing the steatogenic effect of NR ligands with the NR expression levels, we conclude that LXR, PXR, RAR and PPARγ ligands likely induce fat accumulation by a NR-dependent mechanism. Indeed, over-expression of PXR in HepG2 cells enhanced the steatogenic effect of hyperforin and rifampicin. However, the accumulation of fat induced by other ligands did not correlate with the expression of their associated NR. Our results also suggest that human hepatocytes cultured with free fatty acids offer a highly valuable in vitro system to investigate the pathogenesis and therapeutics of the human fatty liver.     

Rhesus cytomegalovirus particles prevent activation of interferon regulatory factor 3

One of the most important innate host defense mechanisms against viral infection is the induction of interferon (IFN)-stimulated genes (ISGs). Immediately upon entry, viruses activate interferon-regulatory factor 3 (IRF3), as well as nuclear factor kappaB (NF-kappaB), which transactivate a subset of ISGs, proinflammatory genes, as well as IFN genes. Most large DNA viruses exhibit countermeasures against induction of this response. However, whereas human cytomegalovirus (HCMV) inhibits IFN-dependent induction of ISGs, IFN-independent induction of ISGs is observed both in the presence and, even moreso, in the absence of viral gene expression. Rhesus CMV (RhCMV) is an emerging animal model for HCMV sharing important similarities in primary structure, epidemiology, and pathogenesis. To determine whether RhCMV would similarly induce ISGs, we performed DNA microarray and quantitative PCR analysis of ISG expression in rhesus fibroblasts infected with RhCMV or HCMV. In contrast to HCMV, however, RhCMV did not induce expression of ISGs or proinflammatory genes at any time after infection. Moreover, dimerization and nuclear accumulation of IRF3, readily observed in HCMV-infected cells, was absent from RhCMV-infected cells, whereas neither virus seemed to activate NFkappaB. RhCMV also blocked IRF3 activation by live or UV-inactivated HCMV, suggesting that RhCMV inhibits viral IRF3 activation and the resultant ISG induction with extraordinary efficiency. Since infection during inhibition of protein expression by cycloheximide or inactivation of viral gene expression by UV treatment did not trigger IRF3 activation or ISG expression by RhCMV, we conclude that RhCMV virions contain a novel inhibitor of IFN-independent viral induction of ISG expression by IRF3.     

α-Lipoic acid ameliorates foam cell formation via liver X receptor α-dependent upregulation of ATP-binding cassette transporters A1 and G1

α-Lipoic acid (α-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether α-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with α-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, α-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, α-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by α-LA depended on liver X receptor α (LXRα), as evidenced by an increase in the nuclear levels of LXRα and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXRα expression with small interfering RNA (siRNA). Consistently, α-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXRα siRNA treatment. In conclusion, LXRα-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of α-LA on foam cell formation.