Plasmodium falciparum multidrug resistance protein 1 and artemisinin-based combination therapy in Africa.

Plasmodium falciparum response mechanisms to the major artemisinin-based combination therapies (ACTs) are largely unknown. Multidrug-resistance protein (MRP)-like adenosine triphosphate (ATP)-binding cassette transporters are known to be related to multidrug resistance in many organisms. Therefore, we hypothesized that sequence variation in pfmrp1 can contribute to decreased parasite sensitivity to ACT. Through sequencing of the pfmrp1 open reading frame for 103 geographically diverse P. falciparum infections, we identified 27 single-nucleotide polymorphisms (SNPs), of which 21 were nonsynonymous and 6 synonymous. Analyses of clinical efficacy trials with artesunate-amodiaquine and artemether-lumefantrine detected a specific selection of the globally prevalent I876V SNP in recurrent infections after artemether-lumefantrine treatment. Additional in silico studies suggested an influence of variation in amino acid 876 on the ATP hydrolysis cycle of pfMRP1 with potential impact on protein functionality. Our data suggest for the first time, to our knowledge, the involvement of pfMRP1 in P. falciparum in vivo response to ACT.

Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2).

BACKGROUND: Genotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported. METHODS: The original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis. RESULTS: The CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE. CONCLUSION: The improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens.

Influence of consecutive-day blood sampling on polymerase chain reaction-adjusted parasitological cure rates in an antimalarial-drug trial conducted in Tanzania.

We assessed the influence that consecutive-day blood sampling, compared with single-day blood sampling, had on polymerase chain reaction (PCR)-adjusted parasitological cure after stepwise genotyping of merozoite surface proteins 2 (msp2) and 1 (msp1) in 106 children in Tanzania who had uncomplicated falciparum malaria treated with either sulfadoxine-pyrimethamine or artemether-lumefantrine; 78 of these children developed recurrent parasitemia during the 42-day follow-up period. Initial msp2 genotyping identified 27 and 33 recrudescences by use of single- and consecutive-day sampling, respectively; in subsequent msp1 genotyping, 17 and 21 of these episodes, respectively, were still classified as recrudescences; these results indicate a similar sensitivity of the standard single-day PCR protocol--that is, 82% (27/33) and 81% (17/21), in both genotyping steps. Interpretation of PCR-adjusted results will significantly depend on methodology.

Binding of the La autoantigen to the hepatitis C virus 3' untranslated region protects the RNA from rapid degradation in vitro.

We have analysed hepatitis C virus (HCV) RNAs in an in vitro RNA degradation assay. We found that the 3' end of positive polarity HCV RNA is sensitive to cytosolic RNases whereas the 3' end of negative polarity HCV RNA is relatively stable. Interaction of the HCV 3' untranslated region with the cellular La protein prevented premature degradation of the HCV RNA. One may speculate that HCV RNAs interact with La protein in infected cells to prevent premature degradation of the viral RNAs.

Positive and negative effects on translation of the hepatitis C virus 3' untranslated region.

OBJECTIVES: To determine the role of the hepatitis C virus 3' untranslated region in viral mRNA translation in transfected cells and in cell extracts. STUDY DESIGN/METHODS: Noninfectious hepatitis C virus mini-genome RNAs with various deletions in the viral 3' untranslated region were transfected into cells or translated in vitro, and the translation efficiency was determined. RESULTS: We have found that the presence of the hepatitis C virus 3' untranslated region modestly increases mRNA translation. The positive effect correlated with the binding of a 45-kDa cytoplasmic factor to the hepatitis C virus 3' untranslated region. Furthermore, the U-rich sequence in the hepatitis C virus 3' untranslated region inhibits translation of capped and polyadenylated mRNAs as a result of the hybridization. CONCLUSIONS: The modest effect of the hepatitis C virus 3' untranslated region on translation suggests that it does not play a major role in mRNA translation. The inhibitory effect of the hepatitis C virus 3' untranslated region on translation of polyadenylated mRNAs supports the notion that translation of hepatitis C virus mRNAs occurs independently of a polyA tail.

Inhibition of translation by UAUUUAU and UAUUUUUAU motifs of the AU-rich RNA instability element in the HPV-1 late 3' untranslated region.

The human papillomavirus type 1 (HPV-1) late mRNAs contain a 57-nucleotide adenosine- and uridine-rich RNA instability element termed h1ARE in their late 3' untranslated regions. Here we show that five sequence motifs in the h1ARE (named I-V) affect the mRNA half-life in an additive manner. The minimal inhibitory sequence in motifs I and II was mapped to UAUUUAU, and the minimal inhibitory sequence in motifs III-V was mapped to UAUUUUUAU. We also provide evidence that the same motifs in the AU-RNA instability element inhibit mRNA translation, an effect that was entirely dependent on the presence of a poly(A) tail on the mRNA. Additional experiments demonstrated that the h1ARE interacted directly with the poly(A)-binding protein, suggesting that the h1ARE inhibits translation by interfering with the function of the poly(A)-binding protein.