The transitional ER localization mechanism of Pichia pastoris Sec12

COPII vesicles assemble at ER subdomains called transitional ER (tER) sites, but the mechanism that generates tER sites is unknown. To study tER biogenesis, we analyzed the transmembrane protein Sec12, which initiates COPII vesicle formation. Sec12 is concentrated at discrete tER sites in the budding yeast Pichia pastoris. We find that P. pastoris Sec12 exchanges rapidly between tER sites and the general ER. The tER localization of Sec12 is saturable and is mediated by interaction of the Sec12 cytosolic domain with a partner component. This interaction apparently requires oligomerization of the Sec12 lumenal domain. Redistribution of P. pastoris Sec12 to the general ER does not perturb the localization of downstream tER components, suggesting that Sec12 and other COPII proteins associate with a tER scaffold. These results provide evidence that tER sites form by a network of dynamic associations at the cytosolic face of the ER.     

DEK binding to class II MHC Y-box sequences is gene- and allele-specific

Using electrophoretic mobility shift assays, we examined sequence-specific binding of DEK, a potential autoantigen in juvenile rheumatoid arthritis, to conserved Y-box regulatory sequences in class II MHC gene promoters. Nuclear extracts from several cell lines of different phenotypes contained sequence-specific binding activity recognizing DRA, DQA1*0101, and DQA1*0501 Y-box sequences. Participation of both DEK and NF-Y in the DQA1 Y-box binding complex was confirmed by 'supershifting' with anti-DEK and anti-NF-Y antibodies. Recombinant DEK also bound specifically to the DQA1*0101 Y box and to the polymorphic DQA1*0501 Y box, but not to the consensus DRA Y box. Measurement of the apparent dissociation constants demonstrated a two- to fivefold difference in DEK binding to the DQA1 Y-box sequence in comparison with other class II MHC Y-box sequences. Residues that are crucial for DEK binding to the DQA1*0101 Y box were identified by DNase I footprinting. The specific characteristics of DEK binding to these related sequences suggests a potential role for DEK in differential regulation of class II MHC expression, and thus in the pathogenesis of juvenile rheumatoid arthritis and other autoimmune diseases.     

Small dense nuclear bodies are the site of localization of herpes simplex virus 1 U(L)3 and U(L)4 proteins and of ICP22 only when the latter protein is present

The herpes simplex virus 1 U(L)3 and U(L)4 open reading frames are expressed late in infection and are not essential for viral replication in cultured cells in vitro. An earlier report showed that the U(L)4 protein colocalizes with the products of the alpha22/U(S)1.5 genes in small nuclear dense bodies. Here we report that the U(L)3 protein also colocalized in these small nuclear dense bodies and the localization of U(L)3 and U(L)4 proteins in these bodies required the presence of alpha22/U(S)1.5 genes. In cells infected with a mutant lacking intact alpha22/U(S)1.5 genes, U(L)3 was diffused throughout the nucleus even though the overall accumulation of the gamma2 U(L)3 protein was decreased. The results suggest that ICP22 acts both as a regulator of U(L)3 accumulation and as the structural component and anchor of these small dense nuclear bodies.     

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans

BackgroundApproximately 8% of the human genome consists of sequences of retroviral origin, a result of ancestral infections of the germ line over millions of years of evolution. The most recent of these infections is attributed to members of the human endogenous retrovirus type-K (HERV-K) (HML-2) family. We recently reported that a previously undetected, large group of HERV-K (HML-2) proviruses, which are descendants of the ancestral K111 infection, are spread throughout human centromeres.ResultsStudying the genomes of certain cell lines and the DNA of healthy individuals that seemingly lack K111, we discover new HERV-K (HML-2) members hidden in pericentromeres of several human chromosomes. All are related through a common ancestor, termed K222, which is a virus that infected the germ line approximately 25 million years ago. K222 exists as a single copy in the genomes of baboons and high order primates, but not New World monkeys, suggesting that progenitor K222 infected the primate germ line after the split between New and Old World monkeys. K222 exists in modern humans at multiple loci spread across the pericentromeres of nine chromosomes, indicating it was amplified during the evolution of modern humans.ConclusionsCopying of K222 may have occurred through recombination of the pericentromeres of different chromosomes during human evolution. Evidence of recombination between K111 and K222 suggests that these retroviral sequences have been templates for frequent cross-over events during the process of centromere recombination in humans.     

Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ～23.1 μM).     

A new in vivo termination function for DNA polymerase I of Escherichia coli K12

Escherichia coli deleted for the tus gene are viable. Thus there must be at least one other mechanism for terminating chromosome synthesis. The tus deletion strain yielded a small fraction of cells that overproduce DNA, as determined by flow cytometry after run-out chromosome replication in the presence of rifampicin and cephalexin. A plasmid, paraBAD tus+, prevented the excess DNA replication only when arabinose was added to the medium to induce the synthesis of the Tus protein. Transduction studies were done to test whether or not additional chromosomal deletions could enhance the excess chromosome replication in the tus deletion strain. A strain containing a second deletion in metE udp overproduced DNA at a high level during run-out replication. Further studies demonstrated that a spontaneous unknown mutation had occurred during the transduction. This mutation was mapped and sequenced. It is polA(G544D). Transduction of polA(G544D) alone into the tus deletion strain produced the high DNA overproduction phenotype. The polA(G544D) and six other polA alleles were then tested in wild-type and in tus deletion backgrounds. The two alleles with low levels of 5'-->3' exonuclease (exo) overproduced DNA while those with either high or normal exo overproduce much less DNA in run-out assays in the wild-type background. In contrast, all seven mutant polA alleles caused the high DNA overproduction phenotype in a tus deletion background. To explain these results we propose a new in vivo function for wild-type DNA polymerase I in chromosome termination at site(s) not yet identified.     

Ultrastructural Analysis of ICP34.5? Herpes Simplex Virus 1 Replication in Mouse Brain Cells In Vivo

Replication-competent forms of herpes simplex virus 1 (HSV-1) defective in the viral neurovirulence factor infected cell protein 34.5 (ICP34.5) are under investigation for use in the therapeutic treatment of cancer. In mouse models, intratumoral injection of ICP34.5-defective oncolytic HSVs (oHSVs) has resulted in the infection and lysis of tumor cells, an associated decrease in tumor size, and increased survival times. The ability of these oHSVs to infect and lyse cells is frequently characterized as exclusive to or selective for tumor cells. However, the extent to which ICP34.5-deficient HSV-1 replicates in and may be neurotoxic to normal brain cell types in vivo is poorly understood. Here we report that HSV-1 defective in ICP34.5 expression is capable of establishing a productive infection in at least one normal mouse brain cell type. We show that γ34.5 deletion viruses replicate productively in and induce cellular damage in infected ependymal cells. Further evaluation of the effects of oHSVs on normal brain cells in animal models is needed to enhance our understanding of the risks associated with the use of current and future oHSVs in the brains of clinical trial subjects and to provide information that can be used to create improved oHSVs for future use.Several types of replication-competent neuroattenuated herpes simplex viruses (HSVs) are currently being evaluated in clinical cancer trials for safety and therapeutic activity (32), as well as for vaccine development (20). A critical safety concern associated with the clinical use of these oncolytic HSVs (oHSVs) is their ability to enter, replicate in, and spread to a wide range of cell types in different regions of the nervous system. One potential complication resulting from invasion of the central nervous system by HSV is herpes simplex encephalitis (HSE), an infection that causes lifelong neurological damage or death. A limited number of genes have been demonstrated to contribute to the virus''s ability to trigger HSE. The viral gene γ34.5 encodes the neurovirulence protein infected cell protein 34.5 (ICP34.5) (29). Viruses lacking the γ34.5 gene (e.g., R3616 and 1716) were found to be 5 logs less neurovirulent than wild-type strains of HSV-1 (4, 19, 36), as quantified by the intracranial LD₅₀, i.e., the lethal dose in 50% of mice inoculated intracerebroventricularly with the virus. The basis for this neuroattenuation was initially reported to be the inability of the γ34.5 deletion viruses to infect or replicate in brain cells (4). Subsequent immunohistochemical studies on infected brain tissue of intracerebroventricularly inoculated mice suggested that γ34.5 deletion viruses retained the ability to infect a wide range of brain cell types and to replicate in and, by day 7, destroy ependymal cells (ECs) (16, 21).To create a more neuroattenuated and thus safer virus, the virus G207 was constructed from the γ34.5 deletion virus R3616 by insertional mutagenesis of the U_L39 gene (25). The U_L39 gene encodes the large subunit of the viral ribonucleotide reductase (vRR) (29). Cellular ribonucleotide reductase is a DNA synthetic enzyme which is of low abundance in quiescent cells but is critical for the synthesis of DNA precursors and is thus abundant in mitotically active cells such as cancer cells. Based on the phenotype of viruses mutated in the vRR alone (13), this double-deletion virus lacking both ICP34.5 and vRR expression is predicted to restrict viral replication to cancer cells expressing cellular RR at levels sufficient to support viral replication (25). In preclinical studies with mice, inoculation with G207 via the intracerebroventricular route failed to destroy the EC layer at 5 days postinoculation (34). These studies supported the concept that a double-deletion virus may be safer in clinical trials than a virus lacking only ICP34.5 expression.To test the hypothesis that productive replication of γ34.5 deletion viruses is restricted to cancer cells, we developed sensitive methods to examine the ability of γ34.5 deletion viruses, with either intact or mutated vRR, to replicate productively in vivo and to complete the multistep process of virion assembly and egress.Common to most models of HSV virion assembly and egress is the observation that capsid proteins translated in the cytoplasm are imported to the nucleus, where a capsid shell is assembled and viral DNA is subsequently packaged. Capsids containing viral DNA are distinguished by an electron-dense (dark) center, whereas capsids lacking viral DNA contain a core protein visible by electron microscopy (EM) often as an inner concentric circle. In subsequent steps, DNA-filled capsids acquire an envelope by budding through the inner nuclear membrane into the perinuclear space. Capsids observed between the inner and outer nuclear membranes have an envelope and tegument and resemble mature extracellular virions (10).Consensus is lacking on the specific sequence of subsequent stages of viral egress, and multiple pathways may exist (3, 18, 24, 30). In the subsequent step of the envelopment-deenvelopment-reenvelopment model (18, 30), enveloped capsids in the perinuclear space lose their envelope by fusion with the outer nuclear membrane as the capsids enter the cytoplasm. In this model, progeny viruses are thus present in the cytoplasm as naked capsids. Cytoplasmic naked capsids acquire their mature envelope as they bud into a cytoplasmic organelle (e.g., a Golgi body).According to an alternative model, enveloped capsids move within the perinuclear space into the endoplasmic reticulum (ER), which is continuous with the perinuclear space (33). From this space, enveloped capsids, individually or in groups, bud off within a vesicle membrane characteristic of the outer nuclear membrane/ER. Within these vesicles, enveloped virions are transported through the cytoplasm. In a final step common to both models, the cytoplasmic vesicle releases mature enveloped virions into the extracellular space by fusing with the cell membrane.ECs are an ideal cell type for these studies due to their distinct morphology and location (described below) and their reported function as neural stem cells (15). We reasoned that since mitotic activity is the reported basis for the productive replication and selectivity of γ34.5 deletion viruses in cancer cells (9, 34), and ECs may be mitotically active, if any normal brain cell type were to support productive replication of γ34.5 deletion viruses, ECs would be the most likely candidate.ECs line the cerebral ventricles, acting as a semipermeable barrier between the brain parenchyma and the cerebrospinal fluid (CSF) in the ventricles (7, 12). Their location thus makes them easily exposed to the virus via intraventricular injections. Their location, combined with their morphologically distinct cuboid shape with kinocilia and microvilli that protrude into the CSF, allows them to be easily excised and recognized under both light microscopy and EM.Here we report the results of a side-by-side comparative study evaluating whether a double-deletion virus similar to G207 and a virus lacking only ICP34.5 expression differ from each other and from a wild-type virus in the ability to infect and replicate productively in ECs of the mouse brain in vivo. The results of these studies are consistent with results of other studies in that they demonstrate that viruses similar to those used in clinical trials (e.g., G207, HSV1716) have a greatly attenuated ability to replicate compared to that of a wild-type virus. However, our data also show very clearly that γ34.5 deletion viruses do replicate productively in infected mouse brain ECs in vivo. These studies suggest that (i) ECs can serve as an exquisitely sensitive model for future evaluations of the ability of oHSVs to replicate productively in normal mouse brain cells and (ii) the potential exists for double-deletion oHSVs to damage normal brain cells. Thus, further comparative studies are warranted to determine whether this risk is sufficiently high to restrict the administration of ICP34.5 deletion viruses in or near the cerebral ventricles in clinical studies.