Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra-and intermolecular interactions required for NS4B structure/ function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.Hepatitis C virus (HCV) is an enveloped, positive-sense RNA virus responsible for 170 million cases of chronic infections worldwide. HCV is the only member of the genus Hepacivirus in the family Flaviviridae (51, 63), which includes other human pathogens, such as West Nile virus and dengue virus. Translation of the virus genome yields at least three structural proteins (core, E1, and E2), the highly hydrophobic p7 peptide, and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The NS proteins, including NS3 to NS5B, are sufficient to promote HCV replication in vitro (10,43). However, with the advent of the HCV cell culture system, many of the NS proteins (NS2, NS3, and NS5A) have been reported to play an active role in HCV assembly (5,46,50,69,73), further supporting the idea that the NS proteins in general have multiple functions in the HCV life cycle.NS3 is illustrative of multifunctionality. Its N-terminal serine protease activity is responsible for processing the NS proteins into their mature forms, whereas the C-terminal helicase activity may be required for the unwinding of HCV RNA (35,66). Similarly, NS4A is a cofactor of NS3 serine protease; it also assists NS3 in binding to host membranes (72) and facilitates the association of NS3 with the HCV replication complex (RC). NS5A may have multiple functions, including inhibition of the interferon response to virus infection and HCV RNA ...

. 85:6963-6976, 2011) have also reported NS4B's function in postreplication steps. Indeed, replacement of the NS4B C-terminal domain (CTD) in the HCV JFH1 (genotype 2a [G2a]) genome with sequences from Con1 (G1b) or H77 (G1a) had a negligible impact on JFH1 genome replication but attenuated virus production. Since NS4B interacts weakly with the HCV genome, we postulated that NS4B regulates the function of host or virus proteins directly involved in HCV production. In this study, we demonstrate that the integrity of the JFH1 NS4B CTD is crucial for efficient JFH1 genome encapsidation. Further, two adaptive mutations (NS4B N216S and NS5A C465S) were identified, and introduction of these mutations into the chimera rescued virus production to various levels, suggesting a genetic interaction between the NS4B and NS5A proteins. Interestingly, cells infected with chimeric viruses displayed a markedly decreased NS5A hyperphosphorylation state (NS5A p58) relative to JFH1, and the adaptive mutations differentially rescued NS5A p58 formation. However, immunofluorescence staining indicated that the decrease in NS5A p58 did not alter NS5A colocalization with the core around lipid droplets (LDs), the site of JFH1 assembly, suggesting that NS5A fails to facilitate the transfer of HCV RNA to the capsid protein on LDs. Alternatively, NS4B's function in HCV genome encapsidation may entail more than its regulation of the NS5A phosphorylation state. Hepatitis C virus (HCV) infects 2 to 3% of the world population, with ca. 160 to 170 million individuals chronically infected and more than 350,000 deaths annually due to complications from cirrhosis and hepatocellular carcinoma (1, 2). As a result of the error-prone nature of its polymerase (3), HCV is classified into at least 6 genotypes and more than 50 subtypes (4). HCV is an enveloped, positive-sense RNA virus with a 9.6-kb genome flanked by 5= and 3= noncoding regions (NCR) and a long open reading frame encoding one polyprotein ϳ3,011 amino acids (aa) in length. Processing of the polyprotein by host and viral proteases occurs co-or posttranslationally, giving rise to three structural proteins (the capsid protein core and the envelope glycoproteins E1 and E2), the viroporin protein p7, and six nonstructural (NS) proteins (NS2, -3, -4A, -4B, -5A, and -5B) (5). The p7 and NS2 proteins are involved in HCV assembly (6-8), while NS3 to NS5B are sufficient to promote virus genome replication in vitro (9, 10). Recently, many of the replicase proteins (NS3, NS4B, and NS5A) were also found to play an active role in HCV production (11-15), consistent with the interpretation that the NS proteins have multiple functions in the HCV life cycle.Recent studies suggest that NS5A physically links the HCV replication complex to the site of HCV assembly on lipid droplets (LDs) or the endoplasmic reticulum (ER) (6,16). This is possible in part because NS5A is a phosphoprotein that exists in two states, based on its migration distance after SDS-PAGE. Basal phosphorylation (NS5A p56) favors HCV genome re...

The use of low and no calorie sweeteners (LNCSs) has increased substantially the past several decades. Their high solubility in water, low absorption to soils, and reliable analytical methods facilitate their detection in wastewater and surface waters. Low and no calorie sweeteners are widely used in food and beverage products around the world, have been approved as food additives, and are considered safe for human consumption by the United States Food and Drug Administration (USFDA) and other regulatory authorities. Concerns have been raised, however, regarding their growing presence and potential aquatic toxicity. Recent studies have provided new empirical environmental monitoring, environmental fate, and ecotoxicity on acesulfame potassium (ACE‐K). Acesulfame potassium is an important high‐production LNCS, widely detected in the environment and generally reported to be environmentally persistent. Acesulfame‐potassium was selected for this environmental fate and effects review to determine its comparative risk to aquatic organisms. The biodegradation of ACE‐K is predicted to be low, based on available quantitative structure–activity relationship (QSAR) models, and this has been confirmed by several investigations, mostly published prior to 2014. More recently, there appears to be an interesting paradigm shift with several reports of the enhanced ability of wastewater treatment plants to biodegrade ACE‐K. Some studies report that ACE‐K can be photodegraded into potentially toxic breakdown products, whereas other data indicate that this may not be the case. A robust set of acute and chronic ecotoxicity studies in fish, invertebrates, and freshwater plants provided critical data on ACE‐K's aquatic toxicity. Acesulfame‐potassium concentrations in wastewater and surface water are generally in the lower parts per billion (ppb) range, whereas concentrations in sludge and groundwater are much lower (parts per trillion [ppt]). This preliminary environmental risk assessment establishes that ACE‐K has high margins of safety (MOSs) and presents a negligible risk to the aquatic environment based on a collation of extensive ACE‐K environmental monitoring, conservative predicted environmental concentration (PEC) and predicted no‐effect concentration (PNEC) estimates, and prudent probabilistic exposure modeling. Integr Environ Assess Manag 2020;16:421–437. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC)

The liver and the mammary gland have complementary metabolic roles during lactation. Substrates synthesized by the liver are released into the circulation and are taken up by the mammary gland for milk production. The aryl hydrocarbon receptor (AHR) has been identified as a lactation regulator in mice, and its activation has been associated with myriad morphological, molecular, and functional defects such as stunted gland development, decreased milk production, and changes in gene expression. In this study, we identified adverse metabolic changes in the lactation network (mammary, liver, and serum) associated with AHR activation using 1H nuclear magnetic resonance (NMR)-based metabolomics. Pregnant mice expressing Ahrd (low affinity) or Ahrb (high affinity) were fed diets containing beta naphthoflavone (BNF), a potent AHR agonist. Mammary, serum, and liver metabolomics analysis identified significant changes in lipid and TCA cycle intermediates in the Ahrb mice. We observed decreased amino acid and glucose levels in the mammary gland extracts of Ahrb mice fed BNF. The serum of BNF fed Ahrb mice had significant changes in LDL/VLDL (increased) and HDL, PC, and GPC (decreased). Quantitative PCR analysis revealed ∼50% reduction in the expression of key lactogenesis mammary genes including whey acid protein, α-lactalbumin, and β-casein. We also observed morphologic and developmental disruptions in the mammary gland that are consistent with previous reports. Our observations support that AHR activity contributes to metabolism regulation in the lactation network.

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.