Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Vector competence defines the ability of a vector to acquire, host, and transmit a pathogen. Understanding the molecular determinants of the mosquitos' competence to host dengue virus (DENV) holds promise to prevent its transmission. To this end, we employed RNA-seq to profile mRNA transcripts of the female Aedes aegypti mosquitos feeding on naïve vs viremic mouse. While most transcripts (12,634) did not change their abundances, 360 transcripts showed decreases. Biological pathway analysis revealed representatives of the decreased transcripts involved in the wnt signaling pathway and hippo signaling pathway. One thousand three hundred fourteen transcripts showed increases in abundance and participate in 21 biological pathways including amino acid metabolism, carbon metabolism, fatty acid metabolism, and oxidative phosphorylation. Inhibition of oxidative phosphorylation with antimycin A reduced oxidative phosphorylation activity and ATP concentration associated with reduced DENV replication in the Aedes aegypti cells. Antimycin A did not affect the amounts of the non-structural proteins 3 and 5, two major components of the replication complex. Ribavirin, an agent that reduces GTP concentration, recapitulated the effects of reduced ATP concentration on DENV replication. Knocking down one of the oxidative phosphorylation components, ATP synthase subunit ß, reduced DENV replication in the mosquitos. In summary, our results suggest that DENV enhances metabolic pathways in the female Aedes aegypti mosquitos to supply nutrients and energy for virus replication. ATP synthase subunit ß knockdown might be exploited to reduce the mosquitos' competence to host and transmit DENV. IMPORTANCE: Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

Unraveling blunt-end RNA binding and ATPase-driven translocation activities of the RIG-I family helicase LGP2.

The RIG-I family helicases, comprising RIG-I, MDA5 and LGP2, are cytoplasmic RNA sensors that trigger an antiviral immune response by specifically recognizing foreign RNAs. While LGP2 lacks the signaling domain necessary for immune activation, it plays a vital role in regulating the RIG-I/MDA5 signaling pathway. In this study, we investigate the mechanisms underlying this regulation by examining the oligomeric state, RNA binding specificity, and translocation activity of human LGP2 and the impact of ATPase activity. We show that LGP2, like RIG-I, prefers binding blunt-ended double-stranded (ds) RNAs over internal dsRNA regions or RNA overhangs and associates with blunt-ends faster than with overhangs. Unlike RIG-I, a 5'-triphosphate (5'ppp), Cap0, or Cap1 RNA-end does not influence LGP2's RNA binding affinity. LGP2 hydrolyzes ATP in the presence of RNA but at a 5-10 fold slower rate than RIG-I. Nevertheless, LGP2 uses its ATPase activity to translocate and displace biotin-streptavidin interactions. This activity is significantly hindered by a methylated RNA patch, particularly on the 3'-strand, suggesting a 3'-strand tracking mechanism like RIG-I. The preference of LGP2 for blunt-end RNA binding, its insensitivity to Cap0/Cap1 modification, and its translocation/protein displacement ability have substantial implications for how LGP2 regulates the RNA sensing process by MDA5/RIG-I.

Opioid agonist treatment reduces losses in quality of life and quality-adjusted life expectancy in heroin users: Evidence from real world data.

BACKGROUND: This study estimated the long-term changes of opioid agonist treatment (OAT) in quality of life (QOL) and quantified the quality-adjusted life years (QALY) from the loss of quality-adjusted life expectancy (QALE) in heroin users. METHODS: A total of 1283 heroin users stratified by OAT were linked to the National Mortality Registry for 8 years (2006-2014) to obtain survival functions, which were extrapolated to lifetime by applying a rolling extrapolation algorithm to survival ratio between the sub-cohorts and age- and sex-matched referents simulated from vital statistics of Taiwan. We performed cross-sectional measurement of EQ-5D on 349 participants, including those with a valid state of OAT or non-OAT plus newly recruited consecutive patients, during 2015-2017 for utility values, while the QOL of referents were abstracted from the 2009 National Health Interview Survey. The QALE was calculated by summing the products of the mean QOL and survival rate throughout life. The QALE difference between the cohort and corresponding referents was the loss-of-QALE. RESULTS: QOL of the OAT group was significantly better than that of the non-OAT group in every domain of the EQ-5D, which was quantified to be 0.23 for utility after controlling for other variables. After extrapolation to 70 years, the estimated QALE and loss-of-QALE were 17.8 and 18.2 QALY for OAT subjects, respectively, while those of the non-OAT group were 9.2 and 27.9 QALY. CONCLUSIONS: Receiving OAT could reduce QALE lost by 9.7 QALYs compared with non-OAT after accounting for QOL differences along time and different age and sex distributions.

Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus.

Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.

Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases.

Phosphorylation at serine 235 (S235) of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays a critical role in the viral life cycle. For medical and virological interests, we exploited the HEK293T kidney cells to test 3 candidate protein kinases on NS5A S235 phosphorylation. Inhibitors that inhibit casein kinase I α (CKIα), polo-like kinase I (PlKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIα was studied previously and PlKI had severe cytotoxicity, thus CaMKII was selected for validation in the Huh7.5.1 liver cells. In the HCV (J6/JFH1)-infected Huh7.5.1 cells, CaMKII inhibitor reduced NS5A S235 phosphorylation and HCV RNA levels without apparent cytotoxicity. RT-PCR analysis showed expression of CaMKII γ and δ isoforms in the Huh7.5.1 cells. Both CaMKII γ and δ directly phosphorylated NS5A S235 in vitro. CaMKII γ or δ single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKIα plus CaMKII (γ or δ) double knockdown reduced NS5A S235 phosphorylation and reduced HCV RNA levels; however, the HCV RNA levels were higher than those in the infected cells with CKIα single knockdown. We conclude that CKIα-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII γ and δ may have negative roles in the HCV life cycle.

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.

Estimation of life expectancy and the expected years of life lost among heroin users in the era of opioid substitution treatment (OST) in Taiwan.

BACKGROUND: Opioid substitution treatment (OST) has been implemented in Taiwan since 2006. We estimated the life expectancy (LE) and expected years of life lost (EYLL) in a cohort of heroin users stratified by OST for comparison. METHODS: A total of 1283 heroin users recruited from 2006 to 2008 were linked to the National Mortality Registry until the end of 2011. Among them, 983 received OST, while 300 did not. Kaplan-Meier estimation for survival was performed, and it was extrapolated to 50 years to obtain the LE using a semi-parametric method. We further estimated the EYLL for both cohorts by subtracting their life expectancies from the age- and sex-matched referents of the general population. Cause-specific standardized mortality ratios (SMRs) were calculated and compared with the national cohort to validate the representativeness of this sample. RESULTS: After extrapolation to 50 years of survival, the estimated average LE and EYLL were 27.4 and 10.6 for OST subjects, respectively, while those of the non-OST were 20.2 and 18.4 years. The all-cause mortality rates (per 1000 person-years) in the observational period for the OST and non-OST group were 15.5 and 23.9, respectively, representing a 7.5- and 10.2-fold SMR compared to the general population, indicating a high representativeness for our sample. But SMR of suicide mortality elevated 16.2 and 3.1 folds in OST and non-OST group, respectively. CONCLUSIONS: OST saves 7.8 EYLL more than non-OST after accounting for lead time bias. Effective suicide prevention programs could enhance its life-saving effect, especially among those co-morbid with depressive disorders.