Interferon Lambda Alleles Predict Innate Antiviral Immune Responses and Hepatitis C Virus Permissiveness

SummaryHepatitis C virus (HCV) infection can result in viral chronicity or clearance. Although host genetics and particularly genetic variation in the interferon lambda (IFNL) locus are associated with spontaneous HCV clearance and treatment success, the mechanisms guiding these clinical outcomes remain unknown. Using a laser capture microdissection-driven unbiased systems virology approach, we isolated and transcriptionally profiled HCV-infected and adjacent primary human hepatocytes (PHHs) approaching single-cell resolution. An innate antiviral immune signature dominated the transcriptional response but differed in magnitude and diversity between HCV-infected and adjacent cells. Molecular signatures associated with more effective antiviral control were determined by comparing donors with high and low infection frequencies. Cells from donors with clinically unfavorable IFNL genotypes were infected at a greater frequency and exhibited dampened antiviral and cell death responses. These data suggest that early virus-host interactions, particularly host genetics and induction of innate immunity, critically determine the outcome of HCV infection

Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells

Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well-characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV-infected cells. V protein-transduced HFLC supported enhanced (10 to 100-fold) levels of HCV infection relative to untransduced or control vector-transduced HFLC. Infection was assessed by measurement of virus-driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV-infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein-transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV-inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein-transduced cultures. Conclusion: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population.National Institutes of Health (U.S.) (NIH Roadmap for Medical Research Grant 1 R01 DK085713-01)Greenberg Institute for Medical ResearchStarr Foundatio

Multiple Breast Cancer Cell-Lines Derived from a Single Tumor Differ in Their Molecular Characteristics and Tumorigenic Potential

Background Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient’s breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT) treatment. Methods Five breast cancer cell lines were derived from a single patient’s primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER), CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC). In addition, a Fuorescent In Situ Hybridization (FISH) assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. Results We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. Conclusions All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to identify mechanisms of organ-specific metastasis of breast cancer

Tumor necrosis factor inhibits spread of hepatitis C virus among liver cells, independent from interferons

BACKGROUND & AIMS: Tumor necrosis factor (TNF) an inflammatory cytokine expressed by human fetal liver cells (HFLCs) following infection with cell culture-derived hepatitis C virus. TNF has been reported to increase entry of HCV pseudoparticles into hepatoma cells and inhibit signaling by interferon alpha (IFNA), but have no effect on replication of HCV RNA. We investigated the effects of TNF on HCV infection of and spread among Huh-7 hepatoma cells and primary HFLCs.  METHODS: Human hepatoma (Huh-7 and Huh-7.5) and primary HFLCs were incubated with TNF and/or recombinant IFNΑ2Α, IFNB, IFNL1, and IFNL2 before or during HCV infection. We used 2 fully infectious HCV chimeric viruses of genotype 2A in these studies: J6/JFH (Clone 2) and Jc1(p7-nsGluc2A) (Jc1G), which encodes a secreted luciferase reporter. We measured HCV replication, entry, spread, production, and release in hepatoma cells and HFLCs. RESULTS: TNF inhibited completion of the HCV infectious cycle in hepatoma cells and HFLC in a dose-dependent and time-dependent manner. This inhibition required TNF binding to its receptor. Inhibition was independent of IFNA, IFNB, IFNL1, IFNL2, or JAK signaling via STAT. TNF reduced production of infectious viral particles by Huh-7 and HFLC, and thereby reduced numbers of infected cells and size of foci. TNF had little effect on HCV replicons and increased entry of HCV pseudoparticles. When cells were incubated with TNF before infection, the subsequent anti-viral effects of IFNs were increased. CONCLUSION: In a cell culture system, we found TNF to have antiviral effects independently of, as well as in combination with, IFNs. TNF inhibits HCV infection despite increased HCV envelope glycoprotein-mediated infection of liver cells. These findings contradict those from other studies, which reported that TNF blocks signal transduction in response to IFNs. The destructive inflammatory effects of TNF must be considered along with its antiviral effects

Breast cancer cell lines express different levels of estrogen receptor and respond differently to estrogen and 4-OHT treatments.

<p>(A) bottom. Western blotting analyses of breast cancer cells for ER expression and top RT-qPCR for cMYC gene expression in RNA extracted from indicated cells treated with 10 nM of E2 for 24 h. (B) Cytotoxicity assay of MCF-7, (C) ARM-E and (D) ARM-H breast cancer cells treated for 3 days with different concentrations of 4-OHT. Left axis represents the relative percentage of live cells treated with 4-OHT compared to medium-treated cells. Similar results were obtained in three independent experiments. Data represents mean ± SD. Student’s t test was used to compare means of treated versus untreated samples with *p<0.05 considered statistically significant.</p