Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells.
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The alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. To test this hypothesis, eastern equine encephalitis virus was passaged in BHK or mosquito cells, as well as in alternating (both) host cell passages. High and low multiplicities were used to examine the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with one-step growth curves and competition assays, and mutations were evaluated by nucleotide sequencing and RNA fingerprinting. All passages and assays were done at 32 degrees C to eliminate temperature as a selection factor. Viruses passaged in either cell type alone exhibited fitness declines in the bypassed cells, while high-multiplicity and clonal passages caused fitness declines in both types of cells. Bypassed cell fitness losses were mosquito and vertebrate specific and were not restricted to individual cell lines. Fitness increases occurred in the cell line used for single-host-adaptation passages and in both cells for alternately passaged viruses. Surprisingly, single-host-cell passage increased fitness in that cell type no more than alternating passages. However, single-host-cell adaptation resulted in more mutations than alternating cell passages. Mosquito cell adaptation invariably resulted in replacement of the stop codon in nsP3 with arginine or cysteine. In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positive Darwinian selection. Our results suggest that alternating host transmission cycles constrain the evolutionary rates of arboviruses but not their fitness for either host alone. (+info)
Structures of endogenous nonecotropic murine leukemia virus (MLV) long terminal repeats in wild mice: implication for evolution of MLVs.
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To develop a better understanding of the interaction between retroviruses and their hosts, we have investigated the polymorphism in endogenous murine leukemia proviruses (MLVs). We used genomic libraries of wild mouse DNAs and PCR to analyze genetic variation in the proviruses found in wild mouse species, including Mus musculus (M. m. castaneus, M. m. musculus, M. m. molossinus, and M. m. domesticus), Mus spretus, and Mus spicelegus, as well as some inbred laboratory strains. In this analysis, we detected several unique forms of sequence organization in the U3 regions of the long terminal repeats of these proviruses. The distribution of the proviruses with unique U3 structures demonstrated that xenotropic MLV-related proviruses were present only in M. musculus subspecies, while polytropic MLV-related proviruses were found in both M. musculus and M. spretus. Furthermore, one unique provirus from M. spicelegus was found to be equidistant from ecotropic provirus and nonecotropic provirus by phylogenetic analysis. This provirus, termed HEMV, was thus likely to be related to the common ancestor of these MLVs. Moreover, an ancestral type of polytropic MLV-related provirus was detected in M. spretus species. Despite their "ancestral" phylogenetic position, proviruses of these types are not widespread in mice, implying more-recent spread by infection rather than inheritance. These results imply that recent evolution of these proviruses involved alternating periods of replication as virus and residence in the germ line. (+info)
Combinatorial selection of high affinity RNA ligands to live African trypanosomes.
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African trypanosomiasis is a parasitic disease caused by a specific class of protozoan organisms. The best-studied representative of that group is Trypanosoma brucei which is transmitted by tsetse flies and multiplies in the blood of many mammals. Trypanosomes evade the immune system by altering their surface structure which is dominated by a layer of a variant surface glycoprotein (VSG). Although invariant surface proteins exist, they are inaccessible to the humoral immune response. Using a combinatorial selection method in conjunction with live trypanosomes as the binding target, we show that short RNA ligands (aptamers) for constant surface components can be isolated. We describe the selection of three classes of RNA aptamers that crosslink to a single 42 kDa protein located within the flagellar pocket of the parasite. The RNAs associate rapidly and with high affinity. They do not discriminate between two different trypanosome VSG variant strains and, furthermore, are able to bind to other trypanosome strains not used in the selection protocol. Thus, the aptamers have the potential to function as markers on the surface of the extracellular parasite and as such they might be modified to function as novel drugs against African trypanosomiasis. (+info)
A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex.
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Nuclear receptors play critical roles in the regulation of eukaryotic gene expression. We report the isolation and functional characterization of a novel transcriptional coactivator, termed steroid receptor RNA activator (SRA). SRA is selective for steroid hormone receptors and mediates transactivation via their amino-terminal activation function. We provide functional and mechanistic evidence that SRA acts as an RNA transcript; transfected SRA, unlike other steroid receptor coregulators, functions in the presence of cycloheximide, and SRA mutants containing multiple translational stop signals retain their ability to activate steroid receptor-dependent gene expression. Biochemical fractionation shows that SRA exists in distinct ribonucleoprotein complexes, one of which contains the nuclear receptor coactivator steroid receptor coactivator 1. We suggest that SRA may act to confer functional specificity upon multiprotein complexes recruited by liganded receptors during transcriptional activation. (+info)
Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants.
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Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson-Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 5' splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence. (+info)
The membrane-attached electron carrier cytochrome cy from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer.
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Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction. Among these species, Rhodobacter capsulatus has a periplasmic Cyt c2Rc and a membrane-bound bipartite Cyt cyRc. These electron carriers participate in both respiratory and photosynthetic electron-transfer chains. On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c2Rs. Recent work indicated that this species has a gene, cycYRs, that is highly homologous to cycYRc, and in the work presented here, functional properties of its gene product (Cyt cyRs) are defined. It was found that Cyt cyRs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R. capsulatus counterpart, Cyt cyRc. Chimeric constructs have shown that the photosynthetic incapability of Cyt cyRs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme. It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions. These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains? (+info)
The NDUFA1 gene product (MWFE protein) is essential for activity of complex I in mammalian mitochondria.
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The MWFE polypeptide of mammalian complex I (the proton-translocating NADH-quinone oxidoreductase) is 70 amino acids long, and it is predicted to be a membrane protein. The NDUFA1 gene encoding the MWFE polypeptide is located on the X chromosome. This polypeptide is 1 of approximately 28 "accessory proteins" identified in complex I, which is composed of 42 unlike subunits. It was considered accessory, because it is not one of the 14 polypeptides making up the core complex I; a homologous set of 14 polypeptides can make a fully functional proton-translocating NADH-quinone oxidoreductase in prokaryotes. One MWFE mutant has been identified and isolated from a collection of respiration-deficient Chinese hamster cell mutants. The CCL16-B2 mutant has suffered a deletion that would produce a truncated and abnormal MWFE protein. In these mutant cells, complex I activity is reduced severely (<10%). Complementation with hamster NDUFA1 cDNA restored the rotenone-sensitive complex I activity of these mutant cells to approximately 100% of the parent cell activity. Thus, it is established that the MWFE polypeptide is absolutely essential for an active complex I in mammals. (+info)
Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible.
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RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. We have now identified alternative exon 1 structures of human ADAR1 transcripts that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5'-rapid amplification of cDNA ends (RACE) cDNAs from human placenta established a linkage between exon 2 of ADAR1 and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrated that exon 1B-exon 2 transcripts were synthesized in the absence of IFN and were not significantly altered in amount by IFN treatment. By contrast, exon 1A-exon 2 transcripts were IFN inducible. Transient transfection analysis with reporter constructs led to the identification of two functional promoters, designated PC and PI. Exon 1B transcripts were initiated from the PC promoter whose activity in transient transfection reporter assays was not increased by IFN treatment. The 107-nt exon 1B mapped 14.5 kb upstream of exon 2. The 201-nt exon 1A that mapped 5.4 kb upstream of exon 2 was initiated from the interferon-inducible PI promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the ADAR1 gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed approximately 110-kDa ADAR1 protein (exon 1B) or the interferon-induced approximately 150-kDa ADAR1 protein (exon 1A). (+info)