A comprehensive mapping of interactions among Epstein-Barr virus (EBV) proteins and interactions of EBV proteins with human proteins should provide specific hypotheses and a broad perspective on EBV strategies for replication and persistence. Interactions of EBV proteins with each other and with human proteins were assessed by using a stringent high-throughput yeast two-hybrid system. Overall, 43 interactions between EBV proteins and 173 interactions between EBV and human proteins were identified. EBV-EBV and EBV-human protein interaction, or ''interactome'' maps provided a framework for hypotheses of protein function. For example, LF2, an EBV protein of unknown function interacted with the EBV immediate early R transactivator (Rta) and was found to inhibit Rta transactivation. From a broader perspective, EBV genes can be divided into two evolutionary classes, ''core'' genes, which are conserved across all herpesviruses and subfamily specific, or ''noncore'' genes. Our EBV-EBV interactome map is enriched for interactions among proteins in the same evolutionary class. Furthermore, human proteins targeted by EBV proteins were enriched for highly connected or ''hub'' proteins and for proteins with relatively short paths to all other proteins in the human interactome network. Targeting of hubs might be an efficient mechanism for EBV reorganization of cellular processes.herpesvirus ͉ interactome ͉ replication ͉ yeast two hybrid

Human TRIM5␣ (TRIM5␣ hu ) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIV mac ). Alteration of arginine 332 in the TRIM5␣ hu B30.2 domain to proline, the residue found in rhesus monkey TRIM5␣, has been shown to create a potent restricting factor for both HIV-1 and SIV mac. Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5␣ hu. The increase in restricting activity correlated with an increase in the ability of TRIM5␣ hu mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5␣ hu R332P binding and allowed escape from restriction. The ability of TRIM5␣ hu to restrict SIV mac could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5␣ hu B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5␣ hu might potentiate the innate resistance of human cells to HIV-1 infection.Primates express dominant restriction factors that block retrovirus infection soon after entry but prior to reverse transcription (1, 2, 5). Genetic studies of virus variants and restriction factor competition studies indicate that the viral capsid is the determinant of susceptibility to restriction (3, 3b, 6, 13, 14). Most early restriction in primates is mediated by TRIM5␣ (7,10,16,23,26). TRIM5␣ is a member of the tripartite motif family of proteins and contains RING, B-box 2, and coiled-coil (RBCC) domains (17). TRIM5␣ also contains a C-terminal B30.2/SPRY domain, which is required for retroviral restriction (23). Deletion of the B30.2 domain disrupts the ability of the TRIM5␣ protein to bind viral capsid complexes (20,24). Differences in the B30.2 domains of TRIM5␣ proteins from distinct primate species account for patterns of retrovirus restriction. For example, the rhesus monkey TRIM5␣ (TRIM5␣ rh ) potently blocks human immunodeficiency virus type 1 (HIV-1), which is only weakly inhibited by human TRIM5␣ (TRIM5␣ hu ) (23). Neither TRIM5␣ rh nor TRIM5␣ hu efficiently restricts simian immunodeficiency virus (SIV mac ) (23). Four variable regions (v1 to v4) are found in the B30.2 domains of TRIM5␣ proteins from different primates (19,21). Differences in the v1 regions of TRIM5␣ rh and TRIM5␣ hu account for the differences in anti-HIV-1 potency of these TRIM5␣ variants (15,19,24,27). Alteration of arginine 332 in the v1 region of TRIM5␣ hu to the proline residue found in TRIM5␣ rh results in a protein that can potently restrict HIV-1 and, surprisingly, SIV mac infection (24, 27). Here we investigate the specific v1 sequences in TRIM5␣ hu required for efficient antiviral activity against HIV-1 and SIV mac and provide a mechanistic explanation for the observed enhancement of restriction that results from changes in this region. MATERIALS AND METHODS Plasmids a...

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MotivationWe present an overview of the Kappa platform, an integrated suite of analysis and visualization techniques for building and interactively exploring rule-based models. The main components of the platform are the Kappa Simulator, the Kappa Static Analyzer and the Kappa Story Extractor. In addition to these components, we describe the Kappa User Interface, which includes a range of interactive visualization tools for rule-based models needed to make sense of the complexity of biological systems. We argue that, in this approach, modeling is akin to programming and can likewise benefit from an integrated development environment. Our platform is a step in this direction.ResultsWe discuss details about the computation and rendering of static, dynamic, and causal views of a model, which include the contact map (CM), snaphots at different resolutions, the dynamic influence network (DIN) and causal compression. We provide use cases illustrating how these concepts generate insight. Specifically, we show how the CM and snapshots provide information about systems capable of polymerization, such as Wnt signaling. A well-understood model of the KaiABC oscillator, translated into Kappa from the literature, is deployed to demonstrate the DIN and its use in understanding systems dynamics. Finally, we discuss how pathways might be discovered or recovered from a rule-based model by means of causal compression, as exemplified for early events in EGF signaling.Availability and implementationThe Kappa platform is available via the project website at kappalanguage.org. All components of the platform are open source and freely available through the authors’ code repositories.

In primary human infection, Epstein-Barr virus (EBV) replicates in the oropharyngeal epithelium (60) and establishes latency III infection in B lymphocytes (48,62,67). During latency III infection, the EBV Cp or Wp EBNA promoters drive expression of six nuclear antigen proteins (EBNA2, EBNALP, EBNA3A, EBNA3B, EBNA3C, and EBNA1) from a single alternatively spliced transcript (37, 54). The latency III primary EBNA transcripts include many open reading frames (ORFs) expressed in EBV replication and are the likely source of the 3 BHRF1 micro-RNAs (miRNAs), which are encoded in an intron of most EBNA RNAs (11,50). In latency III infection, EBV also expresses three integral membrane protein (LMP1, LMP2A, and LMP2B)-encoding mRNAs, two small RNAs (EBER1 and -2), BamHI A rightward transcripts (BARTs) (7,15,22,37,54,56,58), and 24 BART miRNAs (11,29,50). Latency III EBV gene expression causes continuous cell proliferation, which results in vitro in lymphoblastoid cell lines (LCLs) and in vivo in lymphoproliferative diseases (37,54). Only BART miRNAs are detected in latency I-or IIinfected cells, in which EBNA1 is the only EBNA expressed, from a promoter downstream of BHRF1. However, latency III-associated proteins are also detected with EBV replication in epithelial cells in vivo (68) or late in EBV replication in latency I-infected Burkitt's lymphoma (BL) cells (72).miRNAs are small non-protein-coding 20-to 25-nucleotide (nt) single-strand RNAs, which negatively control protein expression, by inhibiting translation or cleaving of mRNA (2, 6). Most miRNAs are processed in the cell nucleus from RNA polymerase II capped and polyadenylated RNAs by the RNase III enzyme Drosha to release 70-nt RNA hairpin pre-miRNA (6, 10, 39, 40). Pre-miRNAs are exported to the cytoplasm by exportin 5 (44, 70). In the cytoplasm, pre-miRNA can be cleaved by the RNase III enzyme Dicer (33) in association with TRBP (17) to generate 22-nt mature miRNAs (21). Mature miRNAs can be incorporated into RNA-induced silencing complexes (RISC) and can direct RISC to complementary mRNA targets (6). The targets of the EBV miRNAs are not known, although miR-BART2 may cleave EBV DNA polymerase (BALF5) mRNA (11,26,50).The experiments reported here investigate EBV miR-BHRF1-1, -2, and -3, which are encoded within introns of EBNA transcripts and are expressed in latency III-infected lymphoblasts, but not in latency I-infected BL or latency IIassociated nasopharyngeal carcinoma (NPC) cells (Fig. 1A) (11, 50). miR-BHRF1-1, -2, and -3 are likely to be Droshacleaved products of EBNA introns. BHRF1 is an antiapoptotic Bcl-2 homologue, which is expressed early in EBV replication (31). Although RNAs that initiate upstream of the BHRF1 promoter and include the BHRF1 ORF are detected in latency III-infected lymphoblasts (3, 49, 52, 58), BHRF1 monoclonal antibody (MAb) rarely detects BHRF1 protein until early in EBV replication, when BHRF1 abundantly accumulates (49). miR-BHRF1-1 overlaps with the BHRF1 mRNA transcriptional start site and is therefore not encoded in BHR...

RNA helicase A (RHA) has been shown to promote HIV-1 replication at both the translation and reverse transcription stages. A prerequisite step for reverse transcription involves the annealing of tRNA 3 Lys , the primer for reverse transcription, to HIV-1 RNA. tRNA 3 Lys annealing is a multistep process that is initially facilitated by Gag prior to viral protein processing. Herein, we report that RHA promotes this annealing through increasing both the quantity of tRNA 3 Lys annealed by Gag and the ability of tRNA 3 Lys to prime the initiation of reverse transcription. This improved annealing is the result of an altered viral RNA conformation produced by the coordinate action of Gag and RHA. Since RHA has been reported to promote the translation of unspliced viral RNA to Gag protein, our observations suggest that the conformational change in viral RNA induced by RHA and newly produced Gag may help facilitate the switch in viral RNA from a translational mode to one facilitating tRNA 3 Lys annealing.

The purpose of this study was to examine the occurrence of fosfomycin-resistant Escherichia coli from chickens and to characterize the plasmids carrying fosA3. A total of 661 E. coli isolates of chicken origin collected from 2009 to 2011 were screened for plasmid-mediated fosfomycin resistance determinants by PCR. Plasmids were characterized using PCR-based replicon typing, plasmid multilocus sequence typing, and restriction fragment length polymorphisms. Associated addiction systems and resistance genes were identified by PCR. PCR-mapping was used for analysis of the genetic context of fosA3. Fosfomycin resistance was detected in 58 isolates that also carried the fosA3 gene. Fifty-seven, 17, and 52 FosA3-producers also harbored blaCTX−M, rmtB, and floR genes, respectively. Most of the 58 fosA3-carrying isolates were clonally unrelated, and all fosA3 genes were located on plasmids belonged to F33:A-:B- (n = 18), IncN-F33:A-:B- (n = 7), IncHI2/ST3 (n = 10), IncI1/ST71 (n = 3), IncI1/ST108 (n = 3), and others. The genetic structures, IS26-ISEcp1-blaCTX−M−55-orf477-blaTEM-1-IS26-fosA3-1758bp-IS26 and ISEcp1-blaCTX−M−65-IS903-iroN-IS26-fosA3-536bp-IS26 were located on highly similar F33:A-:B- plasmids. In addition, blaCTX−M−14-fosA3-IS26 was frequently present on similar IncHI2/ST3 plasmids. IncFII plasmids had a significantly higher frequency of addiction systems (mean 3.5) than other plasmids. Our results showed a surprisingly high prevalence of fosA3 gene in E. coli isolates recovered from chicken in China. The spread of fosA3 can be attributed to horizontal dissemination of several epidemic plasmids, especially F33:A-:B- plasmids. Since coselection by other antimicrobials is the major driving force for the diffusion of the fosA3 gene, a strict antibiotic use policy is urgently needed in China.

RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.