Dengue virus NS1 protein interacts with the ribosomal protein RPL18: This interaction is required for viral translation and replication in Huh-7 cells

AbstractGiven dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle

Height and body-mass index trajectories of school-aged children and adolescents from 1985 to 2019 in 200 countries and territories: a pooled analysis of 2181 population-based studies with 65 million participants

Summary Background Comparable global data on health and nutrition of school-aged children and adolescents are scarce. We aimed to estimate age trajectories and time trends in mean height and mean body-mass index (BMI), which measures weight gain beyond what is expected from height gain, for school-aged children and adolescents. Methods For this pooled analysis, we used a database of cardiometabolic risk factors collated by the Non-Communicable Disease Risk Factor Collaboration. We applied a Bayesian hierarchical model to estimate trends from 1985 to 2019 in mean height and mean BMI in 1-year age groups for ages 5–19 years. The model allowed for non-linear changes over time in mean height and mean BMI and for non-linear changes with age of children and adolescents, including periods of rapid growth during adolescence. Findings We pooled data from 2181 population-based studies, with measurements of height and weight in 65 million participants in 200 countries and territories. In 2019, we estimated a difference of 20 cm or higher in mean height of 19-year-old adolescents between countries with the tallest populations (the Netherlands, Montenegro, Estonia, and Bosnia and Herzegovina for boys; and the Netherlands, Montenegro, Denmark, and Iceland for girls) and those with the shortest populations (Timor-Leste, Laos, Solomon Islands, and Papua New Guinea for boys; and Guatemala, Bangladesh, Nepal, and Timor-Leste for girls). In the same year, the difference between the highest mean BMI (in Pacific island countries, Kuwait, Bahrain, The Bahamas, Chile, the USA, and New Zealand for both boys and girls and in South Africa for girls) and lowest mean BMI (in India, Bangladesh, Timor-Leste, Ethiopia, and Chad for boys and girls; and in Japan and Romania for girls) was approximately 9–10 kg/m2. In some countries, children aged 5 years started with healthier height or BMI than the global median and, in some cases, as healthy as the best performing countries, but they became progressively less healthy compared with their comparators as they grew older by not growing as tall (eg, boys in Austria and Barbados, and girls in Belgium and Puerto Rico) or gaining too much weight for their height (eg, girls and boys in Kuwait, Bahrain, Fiji, Jamaica, and Mexico; and girls in South Africa and New Zealand). In other countries, growing children overtook the height of their comparators (eg, Latvia, Czech Republic, Morocco, and Iran) or curbed their weight gain (eg, Italy, France, and Croatia) in late childhood and adolescence. When changes in both height and BMI were considered, girls in South Korea, Vietnam, Saudi Arabia, Turkey, and some central Asian countries (eg, Armenia and Azerbaijan), and boys in central and western Europe (eg, Portugal, Denmark, Poland, and Montenegro) had the healthiest changes in anthropometric status over the past 3·5 decades because, compared with children and adolescents in other countries, they had a much larger gain in height than they did in BMI. The unhealthiest changes—gaining too little height, too much weight for their height compared with children in other countries, or both—occurred in many countries in sub-Saharan Africa, New Zealand, and the USA for boys and girls; in Malaysia and some Pacific island nations for boys; and in Mexico for girls. Interpretation The height and BMI trajectories over age and time of school-aged children and adolescents are highly variable across countries, which indicates heterogeneous nutritional quality and lifelong health advantages and risks

Heterogeneous contributions of change in population distribution of body mass index to change in obesity and underweight NCD Risk Factor Collaboration (NCD-RisC)

From 1985 to 2016, the prevalence of underweight decreased, and that of obesity and severe obesity increased, in most regions, with significant variation in the magnitude of these changes across regions. We investigated how much change in mean body mass index (BMI) explains changes in the prevalence of underweight, obesity, and severe obesity in different regions using data from 2896 population-based studies with 187 million participants. Changes in the prevalence of underweight and total obesity, and to a lesser extent severe obesity, are largely driven by shifts in the distribution of BMI, with smaller contributions from changes in the shape of the distribution. In East and Southeast Asia and sub-Saharan Africa, the underweight tail of the BMI distribution was left behind as the distribution shifted. There is a need for policies that address all forms of malnutrition by making healthy foods accessible and affordable, while restricting unhealthy foods through fiscal and regulatory restrictions

DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target

<div><p>Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication.</p></div

DENV infection down-regulates AMPK activity.

<p>In <b>A,</b> The AMPK activity, depicted as phosphorylation levels at Thr-172, was evaluated in Huh7 cells infected with DENV 2/4 (MOI 3) at 1, 12, and 24 hpi by ELISA, and NS3 viral protein levels <i>(A</i>, <i>lower panel)</i> were determined as infection test. AMPK activity was expressed as U/mL. *<i>p<0</i>.<i>05</i> compared to mock infected cells (0 hpi). Data are means ± standard error (S.E) of <i>n = 3</i> independent experiments realized by duplicate. <b>(B)</b> The levels of AMPK phosphorylated, AMPK total, and NS3 viral protein were analyzed by western blot in whole cell lysates obtained from Huh7 cells infected with DENV2 (MOI 0.1, 1 and 3) for 24 h. Graph represents the relative quantification of <i>p</i>AMPK respect to AMPK total protein. The <i>p</i>AMPK and total AMPK densitometry values were normalized with β-actin and pAMPK/AMPK ratios were calculated, Ratios are represented with respect to the indicated control. *<i>p<0</i>.<i>05</i> compared to mock infected cells. Data are means ± standard error (S.E) of <i>n = 4</i> independent experiments. <b>(C)</b> The AMPK activity and NS3 viral protein levels <i>(C</i>, <i>lower panel)</i> were determined in Mock or DENV 2/4 infected Huh7 cells treated with DMSO 0.5% (vehicle, VEH), 10 mM Metformin (MET, AMPK activator) or 10 μM Compound C (CC, AMPK inhibitor) for 24 h. <b><i>*</i></b> <i>p<0</i>.<i>05</i> compared to mock VEH-treated cells, ^{<b><i>ab</i></b>} <i>p<0</i>.<i>05</i> compared to mock MET-treated cells. Data are means ± standard error (S.E) of <i>n = 3</i> independent experiments realized by duplicate. (<b>D)</b> The levels of AMPK phosphorylated, AMPK total, and prM viral protein were analyzed by western blot in whole cell lysates obtained from Mock or DENV2 Huh7 infected cells (MOI 1 and 3) in the presence or absence of 10 mM metformin (MET) for 24h. Graph represents pAMPK/AMPK ratios normalized with respect to Mock infected cells with no MET treatment. pAMPK/AMPK ratios were obtained adjusting each protein with β-actin.</p

PP2A activity is not altered by DENV, but its inhibition by Okadaic acid has an antiviral effect.

<p>The PP2A activity was analyzed in Huh7 cells infected (MOI 3) with DENV 2/4 at 1, 12 and 24 hpi <b>(A)</b>, and in Mock or DENV 2/4 infected cells treated with DMSO 0.05% (vehicle) or 10 nM Okadaic acid (O. A) for 24h <b>(B)</b>. Activity is expressed as picomoles of phosphate (phosphates pmoles). From the same cell lysates, the levels of NS3 viral protein <i>(lower panels)</i> were determined by WB as infection test. <i>* p<0</i>.<i>05</i> compared to mock vehicle-treated cells. The antiviral effect of O. A (0, 1 and 10 nM) against DENV infection was evaluated in supernatants from Huh7 cells infected (MOI 3) with DENV2 <b>(C)</b> and DENV4 <b>(D)</b> by viral yield and NS1 secretion at 24 hpi. Viral yield is expressed as Foci Forming Units (FFU) / mL. NS1 secretion was normalized respect to infected non-treated cells and expressed as fold change vs 0 mM. <b>(E)</b> The number of viral genome copies of DENV 2/4 infected cells treated with O. A (0, 5, 10 nM) for 24h was examined by qRT-PCR, and expressed as Log of No. Copies. <i>* p<0</i>.<i>05</i> compared to non-treated cells. DMSO 0.05% was used as vehicle for all cases (0 nM). Data are means ± S.E of n = 3 independent experiments realized by duplicated.</p

DENV infection stimulates the intracellular cholesterol accumulation at replicative complexes through the activation of HMGCR.

<p>The distribution of intracellular cholesterol levels stained with filipin III complex <b><i>(blue)</i>,</b> and its co-localization with the viral protein NS4A <b><i>(green)</i></b> were evaluated by confocal microscopy in Huh7 cells non-infected, infected with DENV4 and treated with DMSO 0.5% (vehicle), 10 mM metformin or 50 μM lovastatin (HMGCR inhibitor) for 24 h. DENV4 infected cells are marked with <b>(+),</b> and non-infected cells marked with <b>(-)</b>. Nuclei were stained with propidium iodide <b><i>(red)</i></b>. Numbers inserted in images indicate the co-localization index between cholesterol and NS4A for that specific infected cell. Graph represents NS4A and cholesterol colocalization values as mean ± S.E of 50 infected cells analyzed from 3 independent experiments. Scale bar 10 μm. Images correspond to one representative experiment.</p

Intracellular distribution of HMGCR and NS4A viral protein during DENV infection.

<p><b>(A)</b>The distribution of the NS4A <b><i>(green)</i>,</b> a viral protein present at DENV-replication complexes, and the HMGCR <b><i>(red)</i>,</b> a cellular ER-resident protein, was evaluated by confocal microscopy in Huh7 cells infected (MOI 3) with DENV2 and DENV4 at 24 hpi. Nuclei were stained with Hoechst <b><i>(blue)</i></b>. Scale bar 10 μm. White dashed boxes are depicting the zoom area. <b>(B)</b> Histograms represent the fluorescence intensity for NS4A and HMGCR in determined area (white continuous line) demonstrating the correlation between two signals. In all infected cells, HMGCR colocalized with NS4A, however, the optical cut does not allow us to clearly observed this colocalization. <b>(C)</b>The table indicates HMGCR/NS4A colocalization values for region of interest (ROI, white dashed boxes) and colocalization per infected cell expressed as mean ± S.E. of 52 DENV2 infected cells and 47 DENV4 infected cells from three independent images.</p

Activated HMGCR is required for the formation of DENV-replication complexes and the maintenance of its architecture.

<p>The distribution of HMGCR and components of viral replication complexes (NS4A and E viral proteins) was evaluated by confocal microscopy in Huh7 cells infected with DENV2 (MOI 3) and treated with DMSO 0.5% (vehicle), 10 mM Metformin or 50 μM lovastatin (HMGCR inhibitor) for 24h. The integrity of replication complexes is depicted as the co-localization between NS4A and E proteins. In <b>A</b> is indicated the distribution of HMGCR <b>(red)</b>, NS4A <b>(light blue)</b>, and E protein <b>(green)</b> as well as the colocalization per infected cell of NS4A/HMGCR <b>(B)</b> and NS4A/E <b>(C)</b> represented by mean ± S.E of the colocalization of 60 infected cells per condition. <b>D</b> and <b>E</b> represent the mean fluorescence intensity of NS4A protein <b>(D)</b> and HMGCR <b>(E)</b> analyzed by flow cytometry. Graphs represent the mean fluorescence intensity ± S.E of three independent experiments, the histograms indicate the fluorescence intensity of a representative experiment.</p

HMGCR activity is increased during DENV infection through down-regulation of AMPK.

<p>In <b>A,</b> The enzymatic activity of HMGCR was evaluated in Huh7 cells infected with Mock or DENV 2/4 (serotype 2 or 4, MOI 3) at 24 hours post-infection (hpi). HMGCR activity was expressed as U/mg protein. *<i>p<0</i>.<i>05</i> compared to mock infected cells. From same cell lysates, levels of NS3 viral protein <i>(A</i>, <i>lower panel)</i> were determined by WB as infection test. <b>(B)</b> DENV4 infected Huh7 cells treated with DMSO 0.5% (vehicle, VEH), 10 mM metformin (MET, AMPK activator), 50 μM lovastatin or 10 nM Okadaic acid (O.A, PP2A inhibitor) for 24 hpi were assayed for HMGCR activity and NS3 viral protein levels <i>(B</i>, <i>lower panel)</i>. *<i>p<0</i>.<i>05</i> compared to mock infected cells, ^{<b><i>a</i></b>} <i>p<0</i>.<i>05</i> compared to DENV4 VEH-treated cells. Relative quantification of <i>NS3</i> levels (<i>numbers in italics</i>) was normalized to β-actin and represented with respect to the indicated control. (<b>C)</b> The levels of NS3, E (envelope), and prM viral proteins were analyzed by western blot in whole cell lysates obtained from Mock or DENV2 Huh7 infected cells (MOI 3) treated with DMSO 0.5% (vehicle, VEH), 10 mM metformin (MET, AMPK activator), 10 nM Okadaic acid (O.A, PP2A inhibitor), and 50 μM Lovastatin (LOV, HMGCR inhibitor) for 24 h. Graph represents the relative quantification each protein normalized to β-actin and represented with respect to the indicated control (VEH). All data are means ± standard error (S.E) of <i>n = 3</i> independent experiments.</p