Characterization of the Agrobacterium octopine-cucumopine catabolic plasmid pAtAg67.

In addition to tumor-inducing agrobacteria, non-pathogenic strains are often isolated from crown gall tumors. Such non-pathogenic strains sometimes contain catabolic plasmids that allow them to take advantage of the opines produced in the tumors. Here we characterize for the first time an octopine catabolic plasmid, pAtAg67, which is derived from an Agrobacterium strain isolated from a grapevine tumor in Crete. By sequence analysis, we deduce that pAtAg67 enables its host to catabolize not only octopine, but also cucumopine and agrocinopine-like compounds. We found that a highly similar set of catabolic genes was present in the Ti plasmids of tumorigenic octopine-cucumopine grapevine strains such as pTiAg57. However, the catabolic genes in octopine-cucumopine Ti plasmids were interrupted by a T-DNA segment. As no T-DNA remnants, virulence genes or border repeats were found in pAtAg67, catabolic plasmid pAtAg67 does not appear to be a degenerate octopine-cucumopine Ti plasmid. In line, plasmid pAtAg67 was found to be compatible with incRh1 octopine Ti plasmids, but to be incompatible with the incRh2 agropine Ti plasmid pTiBo542, forming cointegrates by recombination in the homologous trb genes.

Complete genomic sequence and phylogenomics analysis of Agrobacterium strain AB2/73: a new Rhizobium species with a unique mega-Ti plasmid.

BACKGROUND: The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. RESULTS: We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. CONCLUSIONS: The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

EBV MicroRNA BART16 Suppresses Type I IFN Signaling.

Type I IFNs play critical roles in orchestrating the antiviral defense by inducing direct antiviral activities and shaping the adaptive immune response. Viruses have evolved numerous strategies to specifically interfere with IFN production or its downstream mediators, thereby allowing successful infection of the host to occur. The prototypic human gammaherpesvirus EBV, which is associated with infectious mononucleosis and malignant tumors, harbors many immune-evasion proteins that manipulate the adaptive and innate immune systems. In addition to proteins, the virus encodes >40 mature microRNAs for which the functions remain largely unknown. In this article, we identify EBV-encoded miR-BART16 as a novel viral immune-evasion factor that interferes with the type I IFN signaling pathway. miR-BART16 directly targets CREB-binding protein, a key transcriptional coactivator in IFN signaling, thereby inducing CREB-binding protein downregulation in EBV-transformed B cells and gastric carcinoma cells. miR-BART16 abrogates the production of IFN-stimulated genes in response to IFN-α stimulation and it inhibits the antiproliferative effect of IFN-α on latently infected BL cells. By obstructing the type I IFN-induced antiviral response, miR-BART16 provides a means to facilitate the establishment of latent EBV infection and enhance viral replication.

CRISPR/Cas9-Mediated Genome Editing of Herpesviruses Limits Productive and Latent Infections.

Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpes simplex virus (HSV) type 1 causes cold sores and herpes simplex keratitis, whereas HSV-2 is responsible for genital herpes. Human cytomegalovirus (HCMV) is the most common viral cause of congenital defects and is responsible for serious disease in immuno-compromised individuals. Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a broad range of malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and post-transplant lymphomas. Herpesviruses persist in their host for life by establishing a latent infection that is interrupted by periodic reactivation events during which replication occurs. Current antiviral drug treatments target the clinical manifestations of this productive stage, but they are ineffective at eliminating these viruses from the infected host. Here, we set out to combat both productive and latent herpesvirus infections by exploiting the CRISPR/Cas9 system to target viral genetic elements important for virus fitness. We show effective abrogation of HCMV and HSV-1 replication by targeting gRNAs to essential viral genes. Simultaneous targeting of HSV-1 with multiple gRNAs completely abolished the production of infectious particles from human cells. Using the same approach, EBV can be almost completely cleared from latently infected EBV-transformed human tumor cells. Our studies indicate that the CRISPR/Cas9 system can be effectively targeted to herpesvirus genomes as a potent prophylactic and therapeutic anti-viral strategy that may be used to impair viral replication and clear latent virus infection.

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors.

MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression through silencing of complementary target mRNAs. miRNAs are involved in many biological processes, including cell proliferation, differentiation, cell signaling and cellular defense responses to infection. Strategies that allow for strong and stable suppression of specific microRNA activity are needed to study miRNA functions and to develop therapeutic intervention strategies aimed at interfering with miRNA activity in vivo. One of these classes of miRNA inhibitors are Tough Decoys (TuD) RNAs, which comprise of an imperfect RNA hairpin structure that harbors two opposing miRNA binding sites. Upon developing TuDs targeting Epstein-Barr virus miRNAs, we observed a strong variation in inhibitory potential between different TuD RNAs targeting the same miRNA. We show that the composition of the 'bulge' sequence in the miRNA binding sites has a strong impact on the inhibitory potency of the TuD. Our data implies that miRNA inhibition correlates with the thermodynamic properties of the TuD and that design aimed at lowering the TuD opening energy increases TuD potency. Our study provides specific guidelines for the design and construction of potent decoy-based miRNA inhibitors, which may be used for future therapeutic intervention strategies.

Comprehensive profiling of functional Epstein-Barr virus miRNA expression in human cell lines.

BACKGROUND: Epstein-Barr virus (EBV) establishes lifelong infections in its human host. The virus is associated with a broad range of malignancies of lymphoid and epithelial origin, including Burkitt's lymphoma, post-transplant lymphoproliferative disease, nasopharyngeal carcinoma and gastric carcinoma. During the latent phase of its life cycle, EBV expresses more than 40 mature miRNAs that are highly abundant in tumor cells and may contribute to oncogenesis. Although multiple studies have assessed the relative expression profiles of EBV miRNAs in tumor cells, data linking these expression levels to functional target knockdown are mostly lacking. Therefore we set out to systematically assess the EBV miRNA expression levels in EBV(+) tumor cell lines, and correlate this to their functional silencing capacity in these cells. RESULTS: We provide comprehensive EBV miRNA expression profiles of the EBV(+) cell lines C666-1 (nasopharyngeal carcinoma), SNU-719 (gastric carcinoma), Jijoye (Burkitt's lymphoma), and AKBM (Burkitt's lymphoma) and of EBV(-) cells ectopically expressing the BART miRNA cluster. By deep sequencing the small RNA population and conducting miRNA-reporter experiments to assay miRNA potency, we were able to compare the expression profiles of the EBV miRNAs with their functional silencing efficacy. We observe a strong correlation between miRNA expression levels and functional miRNA activity. There is large variation in expression levels between EBV miRNAs in a given cell line, whereas the relative expression profiles are well maintained between cell lines. Furthermore, we show that miRNA arm selection bias is less pronounced for gamma-herpesvirus miRNAs than for human miRNAs. CONCLUSION: We provide an in depth assessment of the expression levels and silencing activity of all EBV miRNAs in B- and epithelial cell lines of different latency stages. Our data show a good correlation between relative EBV miRNA expression levels and silencing capacity, and suggest preferential processing of particular EBV miRNAs irrespective of cell-type. In addition to encoding the largest number of precursor miRNAs of all human herpesviruses, EBV expresses many miRNAs precursors that yield two functional miRNA strands, rather than one guide strand and a non-functional passenger strand. This reduced strand bias may increase the size of the EBV miRNA targetome.

Immune Evasion by Epstein-Barr Virus.

Epstein-Bar virus (EBV) is widespread within the human population with over 90% of adults being infected. In response to primary EBV infection, the host mounts an antiviral immune response comprising both innate and adaptive effector functions. Although the immune system can control EBV infection to a large extent, the virus is not cleared. Instead, EBV establishes a latent infection in B lymphocytes characterized by limited viral gene expression. For the production of new viral progeny, EBV reactivates from these latently infected cells. During the productive phase of infection, a repertoire of over 80 EBV gene products is expressed, presenting a vast number of viral antigens to the primed immune system. In particular the EBV-specific CD4+ and CD8+ memory T lymphocytes can respond within hours, potentially destroying the virus-producing cells before viral replication is completed and viral particles have been released. Preceding the adaptive immune response, potent innate immune mechanisms provide a first line of defense during primary and recurrent infections. In spite of this broad range of antiviral immune effector mechanisms, EBV persists for life and continues to replicate. Studies performed over the past decades have revealed a wide array of viral gene products interfering with both innate and adaptive immunity. These include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. The current review presents an overview of the evasion strategies that are employed by EBV to facilitate immune escape during latency and productive infection. These evasion mechanisms may also compromise the elimination of EBV-transformed cells, and thus contribute to malignancies associated with EBV infection.

The translocated virulence protein VirD5 causes DNA damage and mutation during Agrobacterium-mediated transformation of yeast.

The soil bacterium Agrobacterium tumefaciens is a preferred gene vector not only for plants but also for fungi. Agrobacterium delivers a small set of virulence proteins into host cells concomitantly with transferred DNA (T-DNA) to support the transformation process. Here, we find that expression of one of these proteins, called VirD5, in yeast host cells causes replication stress and DNA damage. This can result in both genomic rearrangements and local mutations, especially small deletions. Delivery of VirD5 during cocultivation with Agrobacterium led to mutations in the yeast genome that were unlinked to the integration of T-DNA. This load of mutations can be prevented by using a virD5 mutant for genome engineering, but this leads to a lower transformation frequency.

The genome sequence of hairy root Rhizobium rhizogenes strain LBA9402: Bioinformatics analysis suggests the presence of a new opine system in the agropine Ri plasmid.

We report here the complete genome sequence of the Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) strain LBA9402 (NCPPB1855rifR), a pathogenic strain causing hairy root disease. To assemble a complete genome, we obtained short reads from Illumina sequencing and long reads from Oxford Nanopore Technology sequencing. The genome consists of a 3,958,212 bp chromosome, a 2,005,144 bp chromid (secondary chromosome) and a 252,168 bp Ri plasmid (pRi1855), respectively. The primary chromosome was very similar to that of the avirulent biocontrol strain K84, but the chromid showed a 724 kbp deletion accompanied by a large 1.8 Mbp inversion revealing the dynamic nature of these secondary chromosomes. The sequence of the agropine Ri plasmid was compared to other types of Ri and Ti plasmids. Thus, we identified the genes responsible for agropine catabolism, but also a unique segment adjacent to the TL region that has the signature of a new opine catabolic gene cluster including the three genes that encode the three subunits of an opine dehydrogenase. Our sequence analysis also revealed a novel gene at the very right end of the TL-DNA, which is unique for the agropine Ri plasmid. The protein encoded by this gene was most related to the succinamopine synthases of chrysopine and agropine Ti plasmids and thus may be involved in the synthesis of the unknown opine that can be degraded by the adjacent catabolic cluster. The available sequence will facilitate the use of R. rhizogenes and especially LBA9402 in both the laboratory and for biotechnological purposes.