A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication

BACKGROUND: Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. RESULTS: Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. CONCLUSION: These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication

Binding of Equine Infectious Anemia Virus Rev to an Exon Splicing Enhancer Mediates Alternative Splicing and Nuclear Export of Viral mRNAs

In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs

Interaction Between Climatic, Environmental, and Demographic Factors on Cholera Outbreaks in Kenya

Background: Cholera remains an important public health concern in developing countries including Kenya where 11,769 cases and 274 deaths were reported in 2009 according to the World Health Organization (WHO). This ecological study investigates the impact of various climatic, environmental, and demographic variables on the spatial distribution of cholera cases in Kenya. Methods: District-level data was gathered from Kenya’s Division of Disease Surveillance and Response, the Meteorological Department, and the National Bureau of Statistics. The data included the entire population of Kenya from 1999 to 2009. Results: Multivariate analyses showed that districts had an increased risk of cholera outbreaks when a greater proportion of the population lived more than five kilometers from a health facility (RR: 1.025 per 1% increase; 95% CI: 1.010, 1.039), bordered a body of water (RR: 5.5; 95% CI: 2.472, 12.404), experienced increased rainfall from October to December (RR: 1.003 per 1 mm increase; 95% CI: 1.001, 1.005), and experienced decreased rainfall from April to June (RR: 0.996 per 1 mm increase; 95% CI: 0.992, 0.999). There was no detectable association between cholera and population density, poverty, availability of piped water, waste disposal methods, rainfall from January to March, or rainfall from July to September. Conclusion: Bordering a large body of water, lack of health facilities nearby, and changes in rainfall were significantly associated with an increased risk of cholera in Kenya

HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

BACKGROUND: Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1) differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2) control of HIV-1 alternative splicing, which is essential for optimal viral replication. RESULTS: Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins) and inhibitory factors (members of the hnRNP family). While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. CONCLUSION: While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative viral pre-mRNA splicing were observed. These changes correlated with changes in Tat expression and virus production and could play an important role in viral persistence in macrophages. This mechanism could provide a novel target for control of infection in this critical cell type, which would be necessary for eventual eradication of the virus from infected individuals

An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells

The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells

Introgression in the genus Campylobacter: generation and spread of mosaic alleles

Horizontal genetic exchange strongly influences the evolution of many bacteria, substantially contributing to difficulties in defining their position in taxonomic groups. In particular, how clusters of related bacterial genotypes – currently classified as microbiological species – evolve and are maintained remains controversial. The nature and magnitude of gene exchange between two closely related (approx. 15 % nucleotide divergence) microbiologically defined species, Campylobacter jejuni and Campylobacter coli, was investigated by the examination of mosaic alleles, those with some ancestry from each population. A total of 1738 alleles from 2953 seven-locus housekeeping gene sequence types (STs) were probabilistically assigned to each species group with the model-based clustering algorithm structure. Alleles with less than 75 % assignment probability to one of the populations were confirmed as mosaics using the structure linkage model. For each of these, the putative source of the recombinant region was determined and the allele was mapped onto a clonalframe genealogy derived from concatenated ST sequences. This enabled the direction and frequency of introgression between the two populations to be established, with 8.3 % of C. coli clade 1 alleles having acquired C. jejuni sequence, compared to 0.5 % for the reciprocal process. Once generated, mosaic genes spread within C. coli clade 1 by a combination of clonal expansion and lateral gene transfer, with some evidence of erosion of the mosaics by reacquisition of C. coli sequence. These observations confirm previous analyses of the exchange of complete housekeeping alleles and extend this work by describing the processes of horizontal gene transfer and subsequent spread within recipient species

Real-time PCR Demonstrates Ancylostoma duodenale Is a Key Factor in the Etiology of Severe Anemia and Iron Deficiency in Malawian Pre-school Children

Hookworm infections are a major cause of childhood anemia and iron deficiency. Two hookworm species exist of which Ancylostoma duodenale is the less common, yet causing more blood loss than Necator americanus. Although species differentiation and quantification are both of clinical importance, these are often not performed as the technique is complex and laborious using microscopy. Multiplex real-time PCR is a novel diagnostic tool which allows hookworm species differentiation and infection quantification. We applied this test in 830 stool samples of Malawian children with and without severe anemia. The prevalence of hookworm infections was high. A. duodenale was unexpectedly more prevalent than N. americanus. A. duodenale infections were associated with increased risk for severe anemia and iron deficiency, both of which increased with infection load. The study identifies a need for the quantitative screening of species-specific hookworm infections, which readily can be achieved by real-time-PCR. A. duodenale was independently associated with severe anemia and iron deficiency in our study population

Cost-effectiveness of intermittent preventive treatment of malaria in infants (IPTi) for averting anaemia in Gabon: a comparison between intention to treat and according to protocol analyses

ABSTRACT: BACKGROUND: In Gabon, the impact of intermittent preventive treatment of malaria in infants (IPTi) was not statistically significant on malaria reduction, but the impact on moderate anaemia was, with some differences between the intention to treat (ITT) and the according to protocol (ATP) trial analyses. Specifically, ATP was statistically significant, while ITT analysis was borderline. The main reason for the difference between ITT and ATP populations was migration. METHODS: This study estimates the cost-effectiveness of IPTi on the reduction of anaemia in Gabon, comparing results of the ITT and the ATP clinical trial analyses. Threshold analysis was conducted to identify when the intervention costs and protective efficacy of IPTi for the ATP cohort equalled the ITT cost-effectiveness ratio. RESULTS: Based on IPTi intervention costs, the cost per episode of moderate anaemia averted was US$12.88 (CI 95$11.30 (CI 95% 4.56, 26.66) using the ATP analysis. In order for the ATP results to equal the cost-effectiveness of ITT, total ATP intervention costs should rise from US$118.38 to US$134 or the protective efficacy should fall from 27% to 18.1%. The uncertainty surrounding the cost-effectiveness ratio using ITT trial results was higher than using the ATP results. CONCLUSIONS: Migration implies great challenges in the organization of health interventions that require repeat visits in Gabon. This was apparent in the study as the cost-effectiveness of IPTp-SP worsened when drop out from the prevention was taken into account. Despite such challenges, IPTi was both inexpensive and efficacious in averting cases of moderate anaemia in infant

Sm/Lsm Genes Provide a Glimpse into the Early Evolution of the Spliceosome

The spliceosome, a sophisticated molecular machine involved in the removal of intervening sequences from the coding sections of eukaryotic genes, appeared and subsequently evolved rapidly during the early stages of eukaryotic evolution. The last eukaryotic common ancestor (LECA) had both complex spliceosomal machinery and some spliceosomal introns, yet little is known about the early stages of evolution of the spliceosomal apparatus. The Sm/Lsm family of proteins has been suggested as one of the earliest components of the emerging spliceosome and hence provides a first in-depth glimpse into the evolving spliceosomal apparatus. An analysis of 335 Sm and Sm-like genes from 80 species across all three kingdoms of life reveals two significant observations. First, the eukaryotic Sm/Lsm family underwent two rapid waves of duplication with subsequent divergence resulting in 14 distinct genes. Each wave resulted in a more sophisticated spliceosome, reflecting a possible jump in the complexity of the evolving eukaryotic cell. Second, an unusually high degree of conservation in intron positions is observed within individual orthologous Sm/Lsm genes and between some of the Sm/Lsm paralogs. This suggests that functional spliceosomal introns existed before the emergence of the complete Sm/Lsm family of proteins; hence, spliceosomal machinery with considerably fewer components than today's spliceosome was already functional

Langmuir probe electronics upgrade on the tokamak a configuration variable

A detailed description of the Langmuir probe electronics upgrade for TCV (Tokamak a Configuration Variable) is presented. The number of amplifiers and corresponding electronics has been increased from 48 to 120 in order to simultaneously connect all of the 114 Langmuir probes currently mounted in the TCV divertor and main-wall tiles. Another set of 108 amplifiers is ready to be installed in order to connect 80 new probes, built in the frame of the TCV divertor upgrade. Technical details of the amplifier circuitry are discussed as well as improvements over the first generation of amplifiers developed at SPC (formerly CRPP) in 1993/1994 and over the second generation developed in 2012/2013. While the new amplifiers have been operated successfully for over a year, it was found that their silicon power transistors can be damaged during some off-normal plasma events. Possible solutions are discussed. (C) 2019 Author(s)