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...

Large-scale production of recombinant spider silk proteins is a long-term goal for their industrial use. Therefore, we have recently developed a process for bacterial production. Due to a highly repetitive gene sequence of spider silks, the host strain E. coli BLR(DE3) was employed since it shows no homologue recombination. Although perfectly suited for production in full media, the BLR strain does not grow in cost-effective minimal media, indicating a previously not reported L: -isoleucine auxotrophy. We provide evidence that mutated threonine deaminase is likely responsible for the detected auxotrophy of BLR.

The human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), which are associated with a variety of diseases including tumors, produce various small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Like all herpesviruses, they show two stages in their life cycle: lytic replication and latency. During latency, hardly any viral proteins are expressed to avoid recognition by the immune system. Thus, sncRNAs might be exploited since they are less likely to be recognized. Specifically, it has been proposed that sncRNAs might contribute to the maintenance of latency. This has already been shown in vitro, but the respective evidence in vivo is very limited. A natural model system to explore this question in vivo is infection of mice with murine gammaherpesvirus 68 (MHV-68). We used this model to analyze a MHV-68 mutant lacking the expression of all miRNAs. In the absence of the miRNAs, we observed a higher viral genomic load during late latency in the spleens of mice. We propose that this is due to a disturbed regulation of the latent-to-lytic switch, altering the balance between latent and lytic infection. Hence, we provide for the first time evidence that gammaherpesvirus sncRNAs contribute to the maintenance of latency in vivo.

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