Aminoglycosides are potent antibacterials, but therapy is compromised by substantial toxicity causing, in particular, irreversible hearing loss. Aminoglycoside ototoxicity occurs both in a sporadic dose-dependent and in a genetically predisposed fashion. We recently have developed a mechanistic concept that postulates a key role for the mitochondrial ribosome (mitoribosome) in aminoglycoside ototoxicity. We now report on the surprising finding that apramycin, a structurally unique aminoglycoside licensed for veterinary use, shows little activity toward eukaryotic ribosomes, including hybrid ribosomes which were genetically engineered to carry the mitoribosomal aminoglycoside-susceptibility A1555G allele. In ex vivo cultures of cochlear explants and in the in vivo guinea pig model of chronic ototoxicity, apramycin causes only little hair cell damage and hearing loss but it is a potent antibacterial with good activity against a range of clinical pathogens, including multidrug-resistant Mycobacterium tuberculosis. These data provide proof of concept that antibacterial activity can be dissected from aminoglycoside ototoxicity. Together with 3D structures of apramycin-ribosome complexes at 3.5-Å resolution, our results provide a conceptual framework for further development of less toxic aminoglycosides by hypothesis-driven chemical synthesis.antibiotic | translation | misreading | deafness | mycobacteria

Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, a novel semisynthetic series of spectinomycin analogs was generated with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross-resistance with existing tuberculosis therapeutics, activity against MDR/XDR-tuberculosis, and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target based tuberculosis drug discovery.

Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.RNA silencing, also referred to as RNA interference and posttranscriptional gene silencing, is an evolutionarily conserved mechanism that protects cells against invasive nucleic acids, such as viruses, transposons, and transgenes (19). RNA silencing is triggered by double-stranded RNA (dsRNA), effects sequence-specific degradation of cognate viral or endogenous RNA, and, at least in plants, causes de novo methylation of homologous DNA (33). In plants, silencing is increasingly viewed as an adaptive immune system targeting pathogenic RNA and DNA (28, 52). To counteract this defense system, viruses have evolved suppressor proteins (4, 6, 37) that interfere with different steps of the RNA silencing pathway (11), thus allowing efficient viral replication in a single cell and systemic spread of the infection. For example, the coat protein of Turnip crinkle virus blocks generation of small interfering RNAs (siRNAs) (38), derived from dsRNA processing by the RNase III-like enzyme Dicer at an early initiation step of silencing. p19 of tombusviruses binds siRNAs (27, 51), thereby inhibiting a downstream step involving cleavage of cognate RNA by an siRNA-guided, RNA-induced silencing complex. Movement protein P25 of Potato virus X prevents systemic spread of silencing through the vascular system (54). Potyvirus protein HC-Pro might interfere with both the initiation and spread of silencing, although the mechanism of HC-Pro action is still a matter of debate (reference 32 and references therein). Interestingly, HC-Pro and other viral suppressors not only are able to suppress RNA silencing but also can interfere with a relate...

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan β-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and βC1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and βC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.

Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virusVigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.The family Geminiviridae comprises small circular singlestranded DNA viruses that cause severe diseases in major crop plants worldwide. On the basis of genome organization, host range, and type of insect vector, the family is divided into four genera: Mastrevirus, Curtovirus, Topocuvirus, and Begomovirus (42). Members of the largest genus, Begomovirus (10), infect primarily dicotyledonous plants and are transmitted by the whitefly Bemisia tabaci. Many begomoviruses have a bipartite genome, with a DNA-A component encoding all the protein functions necessary for virus replication in a single cell while the DNA-B component provides movement functions required for systemic spread.Transcription regulation in begomoviruses has been extensively studied in both transgenic plants (13,19,23,40,48,60) and protoplast systems (8,9,13,16,17,24,46,47,49,51,52,53,60). Transcription start and poly(A) sites on both DNA-A and -B have been partially or completely mapped for African cassava mosaic virus (ACMV) (54), Tomato golden mosaic virus (TGMV) (19,36,45,50), Abutilon mosaic virus (AbMV) (14), and the monopartite Tomato leaf curl virus (TLCV) (34). However, major gaps in our understanding of the structural organization of begomovirus promoters and transcription units, and of the regulation of transcription by viral proteins, remain. Bidirectional promoters have been identified in the intergenic regions (IGR) of DNA-A and DNA-B, which share a common region (CR) of about 160 to 200 bp. The CR includes all cisacting elements required for DNA replication and a core promoter driving leftward transcription of the AC1 gene encoding the replication-associated protein (Rep) (reviewed in reference 20). AC1 also functions as a negativ...

SummaryDrug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug-resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug-resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug-resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A-site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem-loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug-resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug-resistant tuberculosis disease.

Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.RNA silencing is a posttranscriptional, RNA-guided, gene regulatory mechanism that operates through RNA-mediated sequence-specific interactions in the cytoplasm of eukaryotes, including plants (5,47,57).RNA silencing is generally induced by double-stranded RNA (dsRNA), which can originate from various sources, such as transgenes, viral replication intermediates, or experimentally introduced dsRNA sequences. Central to the silencing process are dicers or "dicer-like" enzymes that cleave dsRNA into small double-stranded fragments, called small interfering RNAs (siRNAs). Single-stranded siRNAs are then incorporated into multicomponent RNA-induced silencing complexes (RISC), which contain an "argonaute" (AGO) family protein (in plants usually AGO1) (3) and inactivate homologous RNA through endonucleolytic cleavage. In addition to siRNAs, which are usually derived from foreign elements such as transgenes and viruses, other small RNA (sRNA) species are encoded by specific noncoding RNA genes. Among these, micro-RNAs (miRNAs) have predominant roles during plant development (28) and are processed from miRNA precursors encoded by miRNA genes. Similarly to siRNAs, miRNAs are incorporated into AGO-containing RISC complexes to guide the recognition of target RNAs. In plants, miRNA-RISC complexes usually cause target RNA cleavage, whereas in most mammalian cases miRNA-RISC inhibits translation of target mRNA (37). Plant siRNAs and miRNAs (commonly referred to as sRNAs) ...