BackgroundPolyketides are a diverse group of biotechnologically important secondary metabolites that are produced by multi domain enzymes called polyketide synthases (PKS).Methodology/Principal FindingsWe have estimated frequencies of type I PKS (PKS I) – a PKS subgroup – in natural environments by using Hidden-Markov-Models of eight domains to screen predicted proteins from six metagenomic shotgun data sets. As the complex PKS I have similarities to other multi-domain enzymes (like those for the fatty acid biosynthesis) we increased the reliability and resolution of the dataset by maximum-likelihood trees. The combined information of these trees was then used to discriminate true PKS I domains from evolutionary related but functionally different ones. We were able to identify numerous novel PKS I proteins, the highest density of which was found in Minnesota farm soil with 136 proteins out of 183,536 predicted genes. We also applied the protocol to UniRef database to improve the annotation of proteins with so far unknown function and identified some new instances of horizontal gene transfer.Conclusions/SignificanceThe screening approach proved powerful in identifying PKS I sequences in large sequence data sets and is applicable to many other protein families.

To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bel1 transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bel1 transactivation of the Kip2 gene are discussed.Studies on the replication and gene expression of different foamy viruses (FVs) have deepened our understanding of the molecular biology of these nonconventional and complex retroviruses (for reviews, see references 1, 4, 13, and 15). FVs have been developed into viral vectors for defined targeted gene delivery applications (22). Features favoring FV-based vectors are the lack of an overt disease association of naturally occurring FV infections and the low prevalence in humans, which has been primarily attributed to the rarity of interspecies transmission from FV-positive nonhuman primates to humans (7). Simian FVs have the capacity to establish a persistent and apparently apathogenic infection in humans (7).It has been reported that human FV (HFV) Env proteins are responsible for the characteristic cytopathic effects that result in formation of syncytia and cell lysis (1, 18). At present, it is unknown why FVs are apathogenic in the natural host whereas HFV-transgenic mice develop severe encephalopathy (1). The mechanisms responsible for induction and maintenance of HFV persistence are unclear. There is evidence, however, that cells that survive a lytic infection carry functionally inactivated FV genomes characterized by a deletion within the gene that encodes Bel1 (Tas), the transcriptional transactivator of FVs. This is achieved by reverse transcription of almost full-length genomic RNA spliced only at the intron of the accessory Bet protein of unknown function (13) that results in bel1-deleted proviruses (23). In contrast, some cell types allow a noncytopathic replication characterized by continuous production of genetically intact virus (30).A better understanding of the multiple mutual interactions of FVs with cellular genes ...

Expression of the human cyclin-dependent protein kinase inhibitor p57Kip2 gene was previously shown to be specifically and strongly activated by the retroviral trans-activator Bel1 of human foamy virus by means of expression profiling, Northern, and Western blot analysis. Here we report that Bel1-mediated trans-activation was conferred by a Bel1 response element (BRE) located in the second exon of p57Kip2 . The intragenic Kip2-BRE was capable of trans-activating the luciferase reporter gene upon cotransfection with Bel1. In electrophoretic mobility shift assays using 293T nuclear extracts or a purified glutathione S-transferase (GST)⅐Bel1 fusion protein, we identified the 55-nucleotide-long Kip2-BRE site that mainly consists of three direct repeats of 14-mers partially homologous to a functionally active BRE in the viral internal promoter. The specificity of the transactivator-DNA binding was shown by using mutated and shortened Kip2-BRE oligodeoxynucleotides in competition experiments with the authentic viral internal promoter and by Bel1-specific antibody that led to a supershift of the nuclear protein⅐Kip2-BRE and GST⅐Bel1⅐Kip2-BRE complex. The data indicate that Bel1 can directly bind to BRE sites. The cellular Kip2-BRE can be used to predict those human genes that are directly or indirectly activated by the Bel1 trans-activator.

To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bel1 transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bel1 transactivation of the Kip2 gene are discussed.