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.
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.
To identify cellular factors that interact with hepatitis C virus RNA, cellular extracts were subjected to UV cross-linking to radiolabeled RNAs corresponding to the hepatitis C virus 5' and 3' untranslated regions of positive and negative polarities. Our results demonstrate that the U-rich region of the hepatitis C virus 3' untranslated region of the positive RNA strand is a hot spot for cellular RNA binding proteins. Two of these proteins were identified as the ELAV-like HuR protein and hnRNP C. Interestingly, HuR and hnRNP C also interacted with the 3' end of the RNA representing the negative strand of the HCV genome. The binding of HuR and hnRNP C to the 3' ends of the HCV RNAs of both negative and positive polarities suggests that HuR and hnRNP C may be involved in the transcription of the HCV RNA genome. Alternatively, they act by protecting the HCV RNAs from premature degradation by binding to their 3' ends. However, we were unable to demonstrate an effect on HCV RNA stability by the HuR protein. These interactions may be necessary for the establishment of chronic active infections that may develop into cirrhosis or hepatocellular carcinoma.
We have analysed hepatitis C virus (HCV) RNAs in an in vitro RNA degradation assay. We found that the 3h end of positive polarity HCV RNA is sensitive to cytosolic RNases whereas the 3h end of negative polarity HCV RNA is relatively stable. Interaction of the HCV 3h 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.
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.
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 halflife 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.Human papillomaviruses (HPVs) 1 are a group of non-enveloped, double-stranded DNA tumor viruses with tropism for epithelial cells (1, 2). Expression of the late mRNAs is restricted to the terminally differentiated cells in the upper layers of the epithelium and at least four papillomaviruses (bovine papillomavirus type 1 (BPV-1), HPV-1, -16, and -31) have been shown to contain cis-acting inhibitory RNA elements located in the late 3Ј UTR (reviewed in Refs. 3-6). In addition, negative RNA elements have been identified in the HPV-16 L1 and L2 open reading frames (4,7,8).We have previously identified and characterized an inhibitory AU-rich element (ARE) located in the HPV-1 late 3Ј UTR region named h1ARE (4 -6, 9) ( Fig. 1). Using actinomycin D we showed that the presence of the h1ARE reduced the mRNA half-life (10). The minimal inhibitory sequence termed XB spans 57 nucleotides (nt) and contains 93% A and U. The element contains two AUUUA-and the three UUUUU-containing sequences (9, 10). Replacing two uridines (U) with cytidines (C) in each motif inactivated the h1ARE (10). The h1ARE interacts with cellular factors (11,12), which bind to the c-fos ARE (10). Two of the h1ARE binding factors interacted with the wild type h1ARE but not with a functionally inactive mutant of the h1ARE (10). These proteins were identified as HuR and hnRNP C (10, 13), and we later showed that binding of the HuR protein correlates with inhibitory activity of a panel of h1ARE mutants (13). While HuR binds to both AUUUA-and UUUUU-motifs (13), hnRNP C binds exclusively to the UUUUU-motifs (14). The role of hnRNP C in HPV-1 late gene expression is unclear. The HuR protein shuttles between the nucleus and the cytoplasm (15), and we observed that there was an inverse correlation between the levels of HuR in the cell cytoplasm and the inhibitory activity of the h1ARE (16), suggesting that the presence of high levels of HuR in the cytoplasm antagonizes the inhibitory effect of the h1ARE, whereas a primarily nuclear association of HuR is associated with inhibition of HPV-1 late gene expression. Interestingly, the HIV Rev and RRE, and the SRV-1 CTE can overcome the inhibition (9), suggesting that the h1ARE traps th...
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