Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Å resolution shows how the 995‐residue polypeptide forms a unique proteolytic chamber of more than 10 000 Å3 in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C‐terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N‐terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge‐bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.

Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher KM-values for polymerization but no change in KD-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency.

Argonaute (Ago) proteins interact with small regulatory RNAs such as microRNAs (miRNAs) and facilitate genesilencing processes. miRNAs guide Ago proteins to specific mRNAs leading to translational silencing or mRNA decay. In order to understand the mechanistic details of miRNA function, it is important to characterize Ago protein interactors. Although several proteomic studies have been performed, it is not clear how the Ago interactome changes on miRNA or mRNA binding. Here, we report the analysis of Ago protein interactions in miRNA-containing and miRNA-depleted cells. Using stable isotope labeling in cell culture in conjunction with Dicer knock out mouse embryonic fibroblasts, we identify proteins that interact with Ago2 in the presence or the absence of Dicer. In contrast to our current view, we find that Ago-mRNA interactions can also take place in the absence of miRNAs. Our proteomics approach provides a rich resource for further functional studies on the cellular roles of Ago proteins. Molecular & Cellular

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) andKaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (␥HV) infections. To completely explore the potential of the MHV-68 system for the investigation of ␥HV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of ␥HV miRNAs.Herpesviruses cause significant morbidity and mortality in the human population. The human gammaherpesviruses (␥HV) Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are associated with a number of tumors and lymphoproliferative disorders (37,40).The outcome of virus-host interactions is determined by many different factors. On the one hand, host immune responses play a pivotal role in the control of ␥HV infections and in pathogenesis. On the other hand, viral factors govern levels of infectivity, tropism, and immune evasion. Previous research on viral factors focused mainly on proteins encoded by viral genes. Recently, it was discovered that viruses, like the genomes of eukaryotic cells, also encode microRNAs (miRNAs). miRNAs are approximately 22-nucleotide noncoding RNAs generated from stem-loop precursors. Mature miRNAs interact directly with a member of the Argonaute (Ago) protein family to form the RNA-induced silencing complex (RISC), which silences gene expression posttranscriptionally by binding to the 3Ј untranslated regions (3Ј UTRs) of target mRNAs (reviewed in references 8, 18, and 31). It has been proposed that viral miRNAs participate in both lytic and latent infections and may be involved in virus-host interactions (25, 44). EBV was the first virus demonstrated to encode miRNAs (34). Shortly thereafter, other herpesviruses were found to encode miRNAs, for example, KSHV, murine gammaherpesvirus 68 (MHV-68), human cytomegalovirus, and rhesus monkey rhadinovirus (7,32,38,39). The functions of most virus-encoded miRNAs are still unknown, and for the majority of viruses, only a few miRNA targets have been identified so far (21, 47). For KSHV and EBV, a systematic analysis of viral miRNAmRNA interaction networks in latently infected cells has recently been performed by RISC immunop...