Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages.

Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure

Hepatitis C Virus Increases Occludin Expression via the Upregulation of Adipose Differentiation-Related Protein

The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread

Role of seipin in lipid droplet morphology and hepatitis C virus life cycle.

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell

Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages.

Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure

HCV increases occludin expression level <i>via</i> a mechanism dependent on ADRP.

<p>Huh-7 were transfected with Jc1 RNA (HCV) and occludin mRNA <b>(A)</b> and protein <b>(B-C)</b> expressions were determined 48 h post transfection by RT-qPCR and immunoblotting respectively. <b>(D)</b> Control or ADRP-silenced cells were transfected or not with Jc1 full length RNA (HCV). Forty eight hours post infection, mRNA occludin level was determined by RT-qPCR. <b>(E)</b> Correlation between ADRP and occludin mRNA expression level in HCV infected patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146000#pone.0146000.t001" target="_blank">Table 1</a>). Spearman’s r = 0.5236.</p

HCV upregulates ADRP expression in Huh-7 cells.

<p><b>(A)</b> Representative immunoblot of ADRP, core and β-actin in Huh-7 cells untransfected or transfected with Jc1 full length RNA (HCV). <b>(B)</b> Graph represents ADRP protein quantifications of at least three independent experiments. <b>(C)</b> ADRP mRNA levels were assessed by RT-qPCR.</p

Effect of ADRP overexpression on HCV particle production.

<p>GFP and ADRP transduced cells were infected by Jc1 viral particles. Intracellular proteins, RNA and particles were harvested 48 h post infection to assess the level of HCV core protein expression by immunoblot <b>(A,B)</b>, the relative number of intracellular HCV RNA copies by RT-qPCR <b>(C)</b> and the intracellular particle infectivity by infecting naive Huh-7.5 cells and calculating the TCID/50 <b>(D)</b>. In parallel, supernatants were collected to determine the relative number of extracellular HCV RNA copies by RT-qPCR <b>(E)</b> and the extracellular particle infectivity by infecting naive Huh-7.5 cells and calculating the TCID₅₀/ml <b>(F).</b></p