Three novel families of miniature inverted-repeat transposable elements are associated with genes of the yellow fever mosquito, Aedes aegypti

Three novel families of transposable elements, Wukong, Wujin, and Wuneng, are described in the yellow fever mosquito, Aedes aegypti. Their copy numbers range from 2,100 to 3,000 per haploid genome. There are high degrees of sequence similarity within each family, and many structural but not sequence similarities between families. The common structural characteristics include small size, no coding potential, terminal inverted repeats, potential to form a stable secondary structure, A+T richness, and putative 2- to 4-bp A+T-biased specific target sites. Evidence of previous mobility is presented for the Wukong elements. Elements of these three families are associated with 7 of 16 fully or partially sequenced Ae. aegypti genes. Characteristics of these mosquito elements indicate strong similarities to the miniature inverted-repeat transposable elements (MITEs) recently found to be associated with plant genes. MITE-like elements have also been reported in two species of Xenopus and in Homo sapiens. This characterization of multiple families of highly repetitive MITE-like elements in an invertebrate extends the range of these elements in eukaryotic genomes. A hypothesis is presented relating genome size and organization to the presence of highly reiterated MITE families. The association of MITE-like elements with Ae. aegypti genes shows the same bias toward noncoding regions as in plants. This association has potentially important implications for the evolution of gene regulation.

Aminoacylation identity switch of turnip yellow mosaic virus RNA from valine to methionine results in an infectious virus.

The turnip yellow mosaic virus genomic RNA terminates at its 3' end in a tRNA-like structure that is capable of specific valylation. By directed mutation, the aminoacylation specificity has been switched from valine to methionine, a novel specificity for viral tRNA-like structures. The switch to methionine specificity, assayed in vitro under physiological buffer conditions with wheat germ methionyl-tRNA synthetase, required mutation of the anticodon loop and the acceptor stem pseudoknot. The resultant methionylatable genomes are infectious and stable in plants, but genomes that lack strong methionine acceptance (as previously shown with regard to valine acceptance) replicate poorly. The results indicate that amplification of turnip yellow mosaic virus RNA requires aminoacylation, but that neither the natural (valine) specificity nor interaction specifically with valyl-tRNA synthetase is crucial.

Eight novel families of miniature inverted repeat transposable elements in the African malaria mosquito, Anopheles gambiae

Eight novel families of miniature inverted repeat transposable elements (MITEs) were discovered in the African malaria mosquito, Anopheles gambiae, by using new software designed to rapidly identify MITE-like sequences based on their structural characteristics. Divergent subfamilies have been found in two families. Past mobility was demonstrated by evidence of MITE insertions that resulted in the duplication of specific TA, TAA, or 8-bp targets. Some of these MITEs share the same target duplications and similar terminal sequences with MITEs and other DNA transposons in human and other organisms. MITEs in A. gambiae range from 40 to 1340 copies per genome, much less abundant than MITEs in the yellow fever mosquito, Aedes aegypti. Statistical analyses suggest that most A. gambiae MITEs are in highly AT-rich regions, many of which are closely associated with each other. The analyses of these novel MITEs underscored interesting questions regarding their diversity, origin, evolution, and relationships to the host genomes. The discovery of diverse families of MITEs in A. gambiae has important practical implications in light of current efforts to control malaria by replacing vector mosquitoes with genetically modified refractory mosquitoes. Finally, the systematic approach to rapidly identify novel MITEs should have broad applications for the analysis of the ever-growing sequence databases of a wide range of organisms.

Vitamin B12 and hepatitis C: Molecular biology and human pathology

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

A mechanism for the inhibition of fever by a virus.

Poxviruses encode proteins that block the activity of cytokines. Here we show that the study of such virulence factors can contribute to our understanding of not only virus pathogenesis but also the physiological role of cytokines. Fever is a nonspecific response to infection that contributes to host defense. Several cytokines induce an elevation of body temperature when injected into animals, but in naturally occurring fever it has been difficult to show that any cytokine has a critical role. We describe the first example of the suppression of fever by a virus and the molecular mechanism leading to it. Several vaccinia virus strains including smallpox vaccines express soluble interleukin 1 (IL-1) receptors, which bind IL-1 beta but not IL-1 alpha. These viruses prevent the febrile response in infected mice, whereas strains that naturally or through genetic engineering lack the receptor induce fever. Repair of the defective IL-1 beta inhibitor in the smallpox vaccine Copenhagen, a more virulent virus than the widely used vaccine strains Wyeth and Lister, suppresses fever and attenuates the disease. The vaccinia-induced fever was inhibited with antibodies to IL-1 beta. These findings provide strong evidence that IL-1 beta, and not other cytokines, is the major endogenous pyrogen in a poxvirus infection.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

A system for functional analysis of Ebola virus glycoprotein

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Effect of ATP Sulfurylase Overexpression in Bright Yellow 2 Tobacco Cells1 : Regulation of ATP Sulfurylase and SO42− Transport Activities

To determine if the ATP sulfurylase reaction is a regulatory step for the SO42−-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 35S promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO42− influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO42− (a toxic SO42− analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism.

Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication.

The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.

"Rattlesnake" structure of a filamentous plant RNA virus built of two capsid proteins.

Elongated particles of simple RNA viruses of plants are composed of an RNA molecule coated with numerous identical capsid protein subunits to form a regular helical structure, of which tobacco mosaic virus is the archetype. Filamentous particles of the closterovirus beet yellow virus (BYV) reportedly contain approximately 4000 identical 22-kDa (p22) capsid protein subunits. The BYV genome encodes a 24-kDa protein (p24) that is structurally related to the p22. We searched for the p24 in BYV particles by using immunoelectron microscopy with specific antibodies against the recombinant p24 protein and its N-terminal peptide. A 75-nm segment at one end of the 1370-nm filamentous viral particle was found to be consistently labeled with both types of antibodies, thus indicating that p24 is indeed the second capsid protein and that the closterovirus particle, unlike those of other plant viruses with helical symmetry, has a "rattlesnake" rather than uniform structure.