Hepatitis C Virus Strain-Dependent Usage of Apolipoprotein E Modulates Assembly Efficiency and Specific Infectivity of Secreted Virions.

Hepatitis C virus (HCV) is extraordinarily diverse and uses entry factors in a strain-specific manner. Virus particles associate with lipoproteins, and apolipoprotein E (ApoE) is critical for HCV assembly and infectivity. However, whether ApoE dependency is common to all HCV genotypes remains unknown. Therefore, we compared the roles of ApoE utilizing 10 virus strains from genotypes 1 through 7. ApoA and ApoC also support HCV assembly, so they may contribute to virus production in a strain-dependent fashion. Transcriptome sequencing (RNA-seq) revealed abundant coexpression of ApoE, ApoB, ApoA1, ApoA2, ApoC1, ApoC2, and ApoC3 in primary hepatocytes and in Huh-7.5 cells. Virus production was examined in Huh-7.5 cells with and without ApoE expression and in 293T cells where individual apolipoproteins (ApoE1, -E2, -E3, -A1, -A2, -C1, and -C3) were provided in trans All strains were strictly ApoE dependent. However, ApoE involvement in virus production was strain and cell type specific, because some HCV strains poorly produced infectious virus in ApoE-expressing 293T cells and because ApoE knockout differentially affected virus production of HCV strains in Huh-7.5 cells. ApoE allelic isoforms (ApoE2, -E3, and -E4) complemented virus production of HCV strains to comparable degrees. All tested strains assembled infectious progeny with ApoE in preference to other exchangeable apolipoproteins (ApoA1, -A2, -C1, and -C3). The specific infectivity of HCV particles was similar for 293T- and Huh-7.5-derived particles for most strains; however, it differed by more than 100-fold in some viruses. Collectively, this study reveals strain-dependent and host cell-dependent use of ApoE during HCV assembly. These differences relate to the efficacy of virus production and also to the properties of released virus particles and therefore govern viral fitness at the level of assembly and cell entry.IMPORTANCE Chronic HCV infections are a major cause of liver disease. HCV is highly variable, and strain-specific determinants modulate the response to antiviral therapy, the natural course of infection, and cell entry factor usage. Here we explored whether host factor dependency of HCV in particle assembly is modulated by strain-dependent viral properties. We showed that all examined HCV strains, which represent all seven known genotypes, rely on ApoE expression for assembly of infectious progeny. However, the degree of ApoE dependence is modulated in a strain-specific and cell type-dependent manner. This indicates that HCV strains differ in their assembly properties and host factor usage during assembly of infectious progeny. Importantly, these differences relate not only to the efficiency of virus production and release but also to the infectiousness of virus particles. Thus, strain-dependent features of HCV modulate ApoE usage, with implications for virus fitness at the level of assembly and cell entry

The effectiveness of massage for reducing pregnant women's anxiety and depression : systematic review and meta-analysis

Objective: To critically appraise and synthesize the best available evidence on the effectiveness of massage to reduce antenatal women's anxiety and/ or depression. Design: Systematic review with meta-analysis Participants, interventions: Pregnant women over the age of 18 years who receive massage interventions. Measurements and findings: Eight studies were included in the review; seven were randomized controlled trials. Data were collected via pregnant women's self-reported ratings of anxiety or depression using validated tools. Meta-analysis of four studies revealed a moderate effect of massage therapy on women's depressive symptoms as measured by the Center for Epidemiologic Studies Depression Scale (CES-D) (MD = -5.95, 95%CI = -8.11 to -3.80, I2 = 0%) compared with usual care. A moderate effect of massage interventions on women's anxiety were also found based on five studies using various measures (SMD = -0.59, 95%CI = -1.06 to -0.12, I2 = 75%) when compared with usual care. However, none of the trials had a low risk of bias. Key conclusions: Non-pharmacologic treatments for mental health symptoms are an important option for women to use during pregnancy. As shown in meta-analysed data, massage therapy might be more effective in reducing pregnant women's anxiety and depression than usual care, although the current results may be prone to bias. Further high-quality research is required to fully evaluate the impact of massage therapy on pregnant women's mental health symptoms in the immediate and also longer term. Implications for practice: Massage therapy may be an acceptable and feasible approach for pregnant women to employ to reduce their anxiety and depressive symptoms. More research evidence examining the safety and effectiveness of massage is required before practice recommendations can be made

Ebola Virus Disease Is Characterized by Poor Activation and Reduced Levels of Circulating CD16+ Monocytes.

A number of previous studies have identified antigen-presenting cells (APCs) as key targets of Ebola virus (EBOV), but the role of APCs in human Ebola virus disease (EVD) is not known. We have evaluated the phenotype and kinetics of monocytes, neutrophils, and dendritic cells (DCs) in peripheral blood of patients for whom EVD was diagnosed by the European Mobile Laboratory in Guinea. Acute EVD was characterized by reduced levels of circulating nonclassical CD16(+) monocytes with a poor activation profile. In survivors, CD16(+) monocytes were activated during recovery, coincident with viral clearance, suggesting an important role of this cell subset in EVD pathophysiology

Maturation of secreted HCV particles by incorporation of secreted ApoE protects from antibodies by enhancing infectivity.

Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses.

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry

Liver-expressed <i>Cd302</i> and <i>Cr1l</i> limit hepatitis C virus cross-species transmission to mice

Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates

Schematic model and ribbon representation of wild type ApoC1 and mutant proteins.

<p><b>A:</b> Schematic depiction and <b>B:</b> predicted 3D structural models of wild-type ApoC1 (H1H2) and six mutants including swapped helices (H2H1), and proline insertions after given amino acid positions to disrupt formation of the first helix (H1p43H2 and H1p46H2) or the second helix (H1H2p67 and H1H2p71), or both helices (H1p43H2p71). Hydrophobic residues are colored grey while polar residues are colored cyan, green and yellow in the N-terminus segment, helix 1 and helix 2, respectively. The side-chain atoms of inserted proline residues are represented as red spheres of the corresponding van der Waals radius. The reader is looking at the polar sides of helices 1 and 2 (green and yellow, respectively) while their hydrophobic sides (colored gray) are on the opposite side, interacting with the membrane surface (represented by the background). SP = signal peptide</p

Switching the order of amphipathic alpha-helices in ApoC1 does not ablate its function in production of infectious HCV particles.

<p><b>A:</b> Secretion of wild-type ApoC1 (H1H2) and a mutant containing swapped amphipathic alpha-helices (H2H1) was quantified via HA-specific ELISA. Depicted are average values of 3 independent experiments with reciprocal dilutions of secreted proteins that reach an OD of 2-fold over background (cells expressing the empty vector). Significant changes compared to wild-type ApoC1 were calculated based on one-tailed unpaired t test with Welch’s correction. n.s. = non-significant (p > 0.05). <b>B:</b> and <b>C:</b> Depicted cell lines were transfected with HCV RNA (Jc1 wt) by electroporation and the expression of core protein, NS5A and ß-actin were determined by Western blotting. <b>C:</b> Absolute values of viral infectivity were determined by end point dilution assay. Plotted are individual results of three independent experiments with means presented as a dash. Data were analyzed by one-tailed unpaired t test with Welch’s correction (* = p < 0.05), n. d. = not detected.</p