A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5′ untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.RNA viruses are the cause of numerous human diseases. Because of their relatively simple genomes, successful infection by RNA viruses is intimately linked to host factors that can both contribute to, or antagonize the viral infection process (1–3). Infection by the hepatitis C virus (HCV)¹, a positive-sense RNA virus, can lead to liver cirrhosis and hepatocellular carcinoma. Approximately 2–3% of the world''s population is chronically infected with HCV, with more than 350,000 annual fatalities in recent years (4). As is typical for viruses, a large number of host factors have been reported to facilitate HCV infection including microRNA-122 (miR-122), CD81, claudin-1, cyclophilins, and lipoproteins, to name a few (5–9). These cellular factors interact with viral proteins or RNA, thus promoting HCV entry, genome translation, and replication.The 5′-untranslated region (5′-UTR) of the HCV RNA genome contains complex RNA structures that interact with cellular factors. These structures include the internal ribosomal entry site that regulates cap-independent translation of the viral RNA (10–11). The 5′-most stem-loop (SL) structure, namely SL1, has been reported to interact with miR-122 to increase the stability of the genomic RNA and facilitate HCV RNA replication in cells (12–13). However, host proteins that can bind to SL1 remain largely elusive because of a lack of proper tools. Previously, we have shown that functional protein microarrays, comprised of individually purified yeast proteins, are an ideal tool to identify proteins that directly interact with important RNA structures encoded by an RNA virus (14). Here, we took a similar approach using a human proteome microarray to identify human hnRNP K as a specific HCV SL1-binding protein that is required for efficient HCV RNA replication.     

QTL analysis of fruit components in the progeny of a Rennell Island Tall coconut (Cocos nucifera L.) individual

We investigated the genetic factors controlling fruit components in coconut by performing QTL analyses for fruit component weights and ratios in a segregating progeny of a Rennell Island Tall genotype. The underlying linkage map of this population was already established in a previous study, as well as QTL analyses for fruit production, which were used to complement our results. The addition of 53 new markers (mainly SSRs) led to minor amendments in the map. A total of 52 putative QTLs were identified for the 11 traits under study. Thirty-four of them were grouped in six small clusters, which probably correspond to single pleiotropic genes. Some additional QTLs located apart from these clusters also had relatively large effects on the individual traits. The QTLs for fruit component weight, endosperm humidity and fruit production were found at different locations in the genome, suggesting that efficient marker-assisted selection for yield can be achieved by selecting QTLs for the individual components. The detected QTLs descend from a genotype belonging to the “Pacific” coconut group. Based on the known molecular and phenotypic differences between “Pacific” and “Indo-Atlantic” coconuts, we suggest that a large fraction of coconut genetic diversity is still to be investigated by studying populations derived from crosses between these groups. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.     

Comparison of the effect of two excipients (karite nut butter and vaseline) on the efficacy of Cocos nucifera,Elaeis guineensis and Carapa procera oil-based repellents formulations against mosquitoes biting in Ivory Coast

Repellents in the form of dermal pomades are recommended as a protection against awakening and bedtime mosquito bites. If synthesis repellents are available, they are nevertheless not common and the prices remain out of reach for the communities concerned. The people therefore have to resort more and more to traditional concoctions, some of which have been shown to be effective. After demonstrating that oil-based formulations (lotions, creams, pomades) of Cocos nucifera (coconut), Elaeis guineensis (oil palm) and Carapa procera (gobi) were effective against mosquitoes, it became necessary to study the impact of the two excipients used in their manufacture, on the effectiveness of the repellents. Experiments were carried with Anopheles gambiae and Aedes aegypti under lobaratory conditions and any other mosquitoes collected under field conditions in Ivory Coast. The laboratory results indicate that the average protection times obtained with formulations with karite nut butter as excipient (54.8 +/- 37.0 mn and 74.6 +/- 26.4 mn respectively on An. gambiae and Ae. aegypti) are higher than those recorded with vaseline as excipient (respectively 42.7 +/- 30.0 mn and 60.8 +/- 33.9 mn). On the other hand, under field conditions, the biting rate percentage reduction obtained with the products with karite nut butter and vaseline excipient were similar (respectively 29.8% and 35.9% for all mosquitoes collected and 45.7% and 47.4% against An. gambiae). Nevertheless, the use of karite nut butter on repellent products should be encouraged because its sale price is very lower (10 time less) than the vaseline's.     

Participation of rab5, an early endosome protein, in hepatitis C virus RNA replication machinery

Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce "membranous web" formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.     

Mosquito (Diptera: Culicidae) Larval Ecology in Rubber Plantations and Rural Villages in Dabou (Côte d'Ivoire)

In Côte d'Ivoire, rubber cultivation has more than doubled since 2010. These mass agricultural areas require a large workforce with little information on how this environment might impact risk of mosquito-borne diseases. The objective of this study was to assess the larval ecology of mosquitoes in rubber areas of Dabou, Côte d'Ivoire. From January to June 2017, an entomological survey was conducted of mature (MP) and immature (IP) rubber plantations, as well as in villages surrounded by rubber plantations (SV) and remote from rubber plantations (RV). The number and type of potential and positive breeding sites were recorded, and mosquito larval densities and diversity were estimated. Seven genera divided into 31 species including major vector such as Anopheles gambiae s.l. and Aedes aegypti were identified. A total of 1,660 waterbodies were identified with a larvae positivity rate of 63.1%. A majority of waterbodies were identified in SV (N?=?875, 53.4% positivity rate), followed by MP (N?=?422, 81.8% positivity rate), IP (N?=?194, 72.2% positivity rate) and least in RV (N?=?169, 57.4% positivity rate). The most important breeding sites for disease vectors were leaf axils in IP (N?=?108, 77.1%), latex collection cups in MP (N?=?332, 96.2%) and the containers abandoned in the SV (N?=?242, 51.8%) as well as in the RV (N?=?59, 60.8%). All these results allow us to affirm that the cultivation of rubber trees has an impact on the larval ecology by increasing the number of available sites and favoring a high larval density and diversity.

     

Free-ranging pigs identified as a multi-reservoir of Trypanosoma brucei and Trypanosoma congolense in the Vavoua area,a historical sleeping sickness focus of Côte d’Ivoire

BackgroundThe existence of an animal reservoir of Trypanosoma brucei gambiense (T. b. gambiense), the agent of human African trypanosomiasis (HAT), may compromise the interruption of transmission targeted by World Health Organization. The aim of this study was to investigate the presence of trypanosomes in pigs and people in the Vavoua HAT historical focus where cases were still diagnosed in the early 2010’s.MethodsFor the human survey, we used the CATT, mini-anion exchange centrifugation technique and immune trypanolysis tests. For the animal survey, the buffy coat technique was also used as well as the PCR using Trypanosoma species specific, including the T. b. gambiense TgsGP detection using single round and nested PCRs, performed from animal blood samples and from strains isolated from subjects positive for parasitological investigations.ResultsNo HAT cases were detected among 345 people tested. A total of 167 pigs were investigated. Free-ranging pigs appeared significantly more infected than pigs in pen. Over 70% of free-ranging pigs were positive for CATT and parasitological investigations and 27–43% were positive to trypanolysis depending on the antigen used. T. brucei was the most prevalent species (57%) followed by T. congolense (24%). Blood sample extracted DNA of T. brucei positive subjects were negative to single round TgsGP PCR. However, 1/22 and 6/22 isolated strains were positive with single round and nested TgsGP PCRs, respectively.DiscussionFree-ranging pigs were identified as a multi-reservoir of T. brucei and/or T. congolense with mixed infections of different strains. This trypanosome diversity hinders the easy and direct detection of T. b. gambiense. We highlight the lack of tools to prove or exclude with certainty the presence of T. b. gambiense. This study once more highlights the need of technical improvements to explore the role of animals in the epidemiology of HAT.     

Conserved GXXXG- and S/T-like motifs in the transmembrane domains of NS4B protein are required for hepatitis C virus replication

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.     

In vitro induction of minitubers in yam (<Emphasis Type="Italic">Dioscorea cayenensis</Emphasis>- <Emphasis Type="Italic">D. rotundata</Emphasis> complex)

Two methods were used to produce yam minitubers from two different yam cultivars (cv. Krengle and cv. Kponan) using in vitro culture techniques. Method 1: Yam microtubers were first initiated in vitro and then transplanted to soil to generate plants from which minitubers were produced. Yam plants were obtained either by directly planting the microtubers to soil, or by inducing the germination of the microtubers using various chemical and physical treatments, before their transfer to soil. Method 2: Yam plantlets were first produced in vitro and then transplanted to soil for further development and tuber production. In both methods, the presence of jasmonic acid (JA) in the culture medium was found to be essential for yam tuberization, as well as for the germination of yam microtubers. In vitro production of yam microtubers was variety dependant. Compared to cv. Krengle, cv. Kponan responded better to microtuberization, and 2.5 μM JA was the optimum concentration resulting in 70 and 90% explants producing microtubers in the MS medium and the Tuberization medium (T-medium), respectively. Germination of the microtubers required treatment of JA at concentrations ranging from 1.0 to 2.5 μM. The overall length of the process to produce minitubers from microtubers took 32 weeks. In contrast, minitubers were obtained within 20 weeks when plantlets were directly transferred to soil. In this case, plantlets were first grown for 8 weeks on medium containing JA (0.1–1.0 μM) and 8% sucrose to initiate plant growth and rooting.     

An efficient mass propagation system for cassava (Manihot esculenta Crantz) based on nodal explants and axillary bud-derived meristems

Nodes from 3- to 5-week-old in vitro plants of different cassava cultivars were cultured for 2–3 days on solid Murashige and Skoog basal medium supplemented with cytokinin to induce the enlargement of axillary buds. Subculture of these buds on the same medium resulted in multiple shoot formation within 4–6 weeks. Of the four cytokinins tested (6-benzylaminopurine (BAP), thidiazuron (TDZ), zeatin, and kinetin), BAP induced shoot development most efficiently. The best results were obtained with cultivar TMS 30555, in which 63% of the explants each produced at least 25 shoots on medium with 10 mg/l BAP. In cultivars that did not produce shoots, the addition of the surfactant Pluronic F-68 (2% wt/vol) raised the percentage of explants forming at least 5 shoots from 0 to 20–60%. Axillary buds were also used to dissect meristems and test their ability to regenerate into shoots. Shoot formation from meristems of six different cultivars was observed after preculture on medium with 5 mg/l BAP followed by transfer to 10 mg/l BAP.Abbreviations MS Murashige and Skoog - BAP 6-Benzylaminopurine - TDZ Thidiazuron