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bioRxiv2017

A helicase-containing module defines a family of pCD630-like plasmids in Clostridium difficile

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile. We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile.

microbiology↗

bioRxiv2017

A Multimodal Strategy Used By A Large c-di-GMP Network

The Pseudomonas fluorescens genome encodes for 50+ proteins involved in -di-GMP signaling. Here, we demonstrate that when tested across 188 nutrients, these enzymes and effectors appear capable of impacting biofilm formation. Transcriptional analysis of network members across [~]50 nutrient conditions indicates that altered gene expression can explain a subset, but not all, of biofilm-formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing [~]2000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy, using combinations of ligand-mediated signals, protein-protein interaction and/or transcriptional regulation to further fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria.\n\nAbstract ImportanceCyclic diguanylate (c-di-GMP) is a key signalling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break or bind this dinucleotide. Thus, a major open question in the field is how signalling specificity is conferred in this context with a soluble signalling molecule. Here, we take a systems approach, using mutational analysis, transcriptional studies and bacterial two-hybrid analysis to interrogate this network. We find that the network typically combines two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) to generate an observed output.

microbiology↗

bioRxiv2017

The Proteus mirabilis rffG gene is essential for swarm-associated cell elongation through an Rcs-mediated pathway

Individual cells of the bacterium Proteus mirabilis can elongate up to 40-fold on surfaces before engaging in a cooperative surface-based motility termed swarming. How cells regulate this dramatic morphological remodeling remains an open question. In this paper, we move forward the understanding of this regulation by demonstrating that P. mirabilis requires the gene rffG for swarmer cell elongation and subsequent swarm motility. The rffG gene encodes a protein homologous to the dTDP-glucose 4,6 dehydratase protein of Escherichia coli, which contributes to Enterobacterial Common Antigen biosynthesis. Here we characterize the rffG gene in P. mirabilis, demonstrating that it is required for the production of large lipopolysaccharide-linked moieties necessary for wild-type cell envelope integrity. We show that absence of the rffG gene induces several stress-responsive pathways including those controlled by the transcriptional regulators RpoS, CaiF, and RcsB. We further show that in rffG-deficient cells, suppression of the Rcs phosphorelay, via loss of RcsB, is sufficient to induce cell elongation and swarm motility. However, loss of RcsB does not rescue cell envelope integrity defects and instead results in abnormally shaped cells, including cells producing more than two poles. We conclude that a RcsB-mediated response acts to suppress emergence of shape defects in cell envelope-compromised cells, suggesting an additional role for RcsB in maintaining cell morphology under stress conditions. We further propose that the composition of the cell envelope acts as a checkpoint before cells initiate swarmer cell elongation and motility.\n\nImportance statementP. mirabilis swarm motility has been implicated in pathogenesis. We have found that cells deploy multiple uncharacterized strategies to handle cell envelope stress beyond the Rcs phosphorelay when attempting to engage in swarm motility. While RcsB is known to directly inhibit the master transcriptional regulator for swarming, we have shown an additional role for RcsB in protecting cell morphology. These data support a growing appreciation that the Rcs phosphorelay is a multi-functional regulator of cell morphology in addition to its role in microbial stress responses. These data also strengthen the paradigm that outer membrane composition is a crucial checkpoint for modulating entry into swarm motility. Furthermore, the rffG-dependent moieties provide a novel, attractive target for potential antimicrobials.

microbiology↗

bioRxiv2017

Cell surface vimentin is involved in matrix stiffness-dependent infection of endothelial cells by Listeria monocytogenes

Extracellular matrix (ECM) stiffness is one of many mechanical forces acting on mammalian adherent cells that influence cellular function. We have addressed the open question of how ECM stiffness might alter the susceptibility of host cells to infection by bacterial pathogens. We manufactured hydrogels of varying physiologically-relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm) and found that adhesion of Lm onto host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1, and demonstrated that bacterial infection of these host cells is decreased when surface vimentin is perturbed. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of host cells to bacterial infection.

microbiology↗

bioRxiv2017

Immunological tolerance, pregnancy and pre-eclampsia: the roles of semen microbes and the father

Although it is widely recognised as involving two stages (poor placentation followed by oxidative stress/inflammation), the precise originating causes of pre-eclampsia (PE) remain elusive. We have previously brought together some of the considerable evidence that a (dormant) microbial component is commonly a significant part of its aetiology. However, apart from recognising, consistent with this view, that the many inflammatory markers of PE are also increased in infection, we had little to say about immunity, whether innate or adaptive. In addition, we focussed on the gut, oral and female urinary tract microbiomes as the main sources of the infection. We here marshall further evidence for an infectious component in PE, focussing on the immunological tolerance characteristic of pregnancy, and the well-established fact that increased exposure to the fathers semen assists this immunological tolerance. As well as these benefits, however, semen is not sterile, microbial tolerance mechanisms may exist, and we also review the evidence that semen may be responsible for inoculating the developing conceptus with microbes, not all of which are benign. It is suggested that when they are not, this may be a significant cause of preeclampsia. A variety of epidemiological and other evidence is entirely consistent with this, not least correlations between semen infection, infertility and PE. Our view also leads to a series of other, testable predictions. Overall, we argue for a significant paternal role in the development of PE through microbial infection of the mother via insemination. O_QD\"In one of the last articles which he wrote, the late Professor F J Browne (1958) expressed the opinion that all the essential facts about pregnancy toxaemia are now available and that all that is required to solve the problem is to fit them together in the right order, like the pieces of a jigsaw puzzle\" [1]\n\nC_QD O_QD\"It appears astonishing how little attention has been given in reproductive medicine to the maternal immune system over the last few decades.\" [2]\n\nC_QD

microbiology↗

bioRxiv2017

Auranofin inhibits virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence in P. aeruginosa. We demonstrate that auranofin acts by targeting Vfr, which in turn leads to inhibition of quorum sensing (QS) and Type IV pili (TFP). Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.

microbiology↗

bioRxiv2017

Cell surface associated LapA of Pseudomonas fluorescens is anchored inside its Type-1 Secretion TolC-like Pore

The type-1 secretion system (T1SS) of gram-negative bacteria enables a one-step translocation strategy known to move functionally diverse proteins from the cytoplasm into the extracellular environment without a periplasmic intermediate. LapA of Pseudomonas fluorescens Pf0-1 is a giant type-1 secreted (T1S) adhesin that facilitates biofilm formation only when displayed at the cell surface. A LapA-targeting periplasmic protease, LapG, connects intracellular cyclic diguanylate (c-di-GMP) levels with cell surface-associated LapA by cleaving and absolving LapA from the cell surface under conditions unsuitable for biofilm formation. Here, we demonstrate that LapA contains a novel N-terminal element, called the retention module (RM), which prohibits classical one-step T1S of LapA. We provide evidence that the RM of LapA tethers LapA at the cell surface through its outer membrane TolC-like pore, LapE, where LapA is accessible to the periplasmic protease LapG. We also demonstrate that this unusual retention strategy is likely conserved among LapA-like proteins and represents a new subclass of T1SS ABC transporters exclusively involved in transporting LapA-like adhesins.\n\nSignificance StatementBacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is often key for initiating contact with a preferred substrate to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence these adhesins are secreted by a new subclass of T1SS ABC transporters.

microbiology↗

bioRxiv2017

Phosphate starvation induces replacement of phospholipids with the betaine lipid diacylglycerol-N,N,N-trimethylhomoserine in the human fungal pathogen Candida albicans

We have previously demonstrated that phosphate starvation induces replacement of phosphatidylcholine with the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) in fungi. In Neurospora crassa, the BTA1 gene encodes the betaine lipid synthase, which is necessary and sufficient for DGTS synthesis. BTA1 expression and DGTS accumulation are part of the fungal phosphorus (Pi) deprivation (PHO) regulon, mediated by the NUC-1/Pho4p transcription factor. We now demonstrate that the human pathogen Candida albicans encodes a BTA1 ortholog (CaBTA1), which is activated during Pi scarcity. The CaBTA1 gene is also induced under certain biofilm-promoting conditions independent of Pi starvation. RNA-seq and qRT-PCR showed a significant increase in CaBTA1 expression in response to Pi limitation. Thin-layer chromatography and LC-ESI-MS/MS confirmed the replacement of PC with DGTS in wild-type under low Pi and showed the absence of DGTS in the bta1{Delta}{Delta} mutant.\n\nPi limitation in the gut of critically ill patients also triggers the switching of C. albicans into an invasive filamentous form. To assess the role of BTA1 and DGTS in the pathogenicity of C. albicans in vitro, we compared the growth and morphology of bta1{Delta}{Delta} and wild type in hyphaeinducing media and observed defects in biofilm formation and invasive growth in the bta1{Delta}{Delta} mutant. This observation is complemented by RNA-seq data demonstrating that Pi starvation in planktonic C. albicans cells induces the expression of virulence-associated cell surface proteins. Taken together, these results show novel functional interactions between lipid metabolism and remodeling, biofilm formation, and the phosphate starvation response of C. albicans.

microbiology↗

bioRxiv2017

Probabilistic data integration identifies reliable gametocyte-specific proteins and transcripts in malaria parasites

Plasmodium gametocytes are the sexual forms of the malaria parasite essential for transmission to mosquitoes. To better understand how gametocytes differ from asexual blood-stage parasites, we performed a systematic analysis of available omics data for P. falciparum and other Plasmodium species. 18 transcriptomic and proteomic data sets were evaluated for the presence of curated \"gold standards\" of 41 gametocyte-specific versus 46 non-gametocyte genes and integrated using Bayesian probabilities, resulting in gametocyte-specificity scores for all P. falciparum genes.\n\nTo illustrate the utility of the gametocyte score, we explored newly predicted gametocyte-specific genes as potential biomarkers of gametocyte carriage and exposure. We analyzed the humoral immune response in field samples against 30 novel gametocyte-specific antigens and found five antigens to be differentially recognized by gametocyte carriers as compared to malaria-infected individuals without detectable gametocytes. We also validated the gametocyte-specificity of 15 identified gametocyte transcripts on culture material and samples from naturally infected individuals, resulting in eight transcripts that were >1000-fold higher expressed in gametocytes compared to asexual parasites and whose transcript abundance allowed gametocyte detection in naturally infected individuals. Our integrated genome-wide gametocyte-specificity scores provide a comprehensive resource to identify targets and monitor P. falciparum gametocytemia.

microbiology↗

bioRxiv2017

Repurposed kinase inhibitors and β-lactams as a novel therapy for antibiotic resistant bacteria

Antibiotic resistant bacteria are an increasing global problem, and pathogenic actinomycetes and firmicutes are particularly challenging obstacles. These pathogens share several eukaryotic-like kinases that present antibiotic development opportunities. We used computational modelling to identify human kinase inhibitors that could be repurposed towards bacteria as part of a novel combination therapy. The computational model suggested a family of inhibitors, the imidazopyridine aminofurazans (IPAs), bind PknB with high affinity. We found that these inhibitors biochemically inhibit PknB, with potency roughly following the predicted models. A novel x-ray structure confirmed that the inhibitors bind as predicted and made favorable protein contacts with the target. These inhibitors also have antimicrobial activity towards Mycobacteria and Nocardia, and normally ineffective {beta}-lactams can potentiate IPAs to more efficiently inhibit growth of these pathogens. Collectively, our data show that in silico modeling can be used as a tool to discover promising drug leads, and the inhibitors we discovered can synergize with clinically relevant antibiotics to restore their efficacy against bacteria with limited treatment options.

microbiology↗

bioRxiv2017

High Dynamic Range Bacterial Cell Detection in a Novel "Lab-In-A-Comb" for High-Throughput Antibiotic Susceptibility Testing of Clinical Urine Samples

Antibiotic resistance in urinary tract infection is a major global challenge, and improved cost-effective and rapid antibiotic susceptibility tests (AST) are urgently needed to inform correct antibiotic selection. Although microfluidic technology can miniaturise AST, the high dynamic range of pathogen density found in clinical urine samples makes direct testing of clinical samples - rather than testing colonies from overnight agar plates - extremely challenging. We evaluated for the first time how pathogen concentration in urine affects microfluidic AST using a novel microplate-compatible high-throughput microfluidic AST system termed \"Lab-on-a-Comb\". When tested with clinical E. coli isolates at standardised density, these devices gave identical antibiotic susceptibility profiles to standard disc diffusion and microtitre plate tests. Bacterial detection directly in synthetic urine spiked with clinical E. coli UTI isolates was possible over a very large dynamic range of starting cell densities, from 103 - 108 CFU/mL which covers the range of pathogen cell densities found in patient urine. The lowest cell density where cell growth was reproducibly detected optically was 9.6x102 CFU/mL, corresponding to one single CFU detected in a 1 L microcapillary-an unprecedented level of sensitivity. Cell growth kinetics followed a simple Monod model with fast growth limited by the substrate availability and an estimated doubling time of 24.5 min, indicating optimal E. coli growth conditions within these microfluidic devices. There was a trade-off between sensitivity and speed of detection, with 105 CFU/mL detection possible within 2h, but 6h incubation required at 103 CFU/mL.

microbiology↗

bioRxiv2017

High-throughput fitness profiling of Zika virus E protein reveals different roles for N-linked glycosylation during infection of mammalian and mosquito cells

Zika virus (ZIKV) infection causes Guillain-Barre syndrome and severe birth defects. ZIKV envelope (E) protein is the major viral protein involved in cell receptor binding and entry and therefore considered one of the major determinants in ZIKV pathogenesis. Here, we report a gene-wide mapping of functional residues of ZIKV E protein using a mutant library with changes covering every nucleotide position. By comparing the replication fitness of every viral mutant between mosquito and human cells, we identified that mutations affecting N-linked glycosylation at N154 position display the most divergence. Through characterizing individual mutants, we show that, while ablation of N-linked glycosylation selectively benefits ZIKV infection of mosquito cells by enhancing cell entry, it either had little impact on ZIKV infection on certain human cells or decreased infection through entry factor DC-SIGN. In conclusion, we define the roles of individual residues of ZIKV envelope protein, which contribute to ZIKV replication fitness in human and mosquito cells.\n\nHighlightsO_LIGene-wide mapping of functional residues of E protein in human and mosquito cells.\nC_LIO_LIMutations affecting N-linked glycosylation display the most dramatic difference.\nC_LIO_LIN-linked glycosylation decreases ZIKV entry into mosquito cells.\nC_LIO_LIN-linked glycosylation is important for DC-SIGN mediated infection of human cells.\nC_LI

microbiology↗

bioRxiv2017

Umbilical Cord Blood Diagnostics for Early Onset Sepsis in Premature Infants: Detection of Bacterial DNA and Systemic Inflammatory Response

Statement of financial supportThis work has been supported by a Thrasher Research Fund Early Career Award (#12573), National Institutes of Health (K23 HL093302 [KKM], R01GM108494 [PCS])), the Gerber Foundation, the Hartwall Foundation, and the Northwestern Memorial Foundation (Friends of Prentice Grants Initiative).\n\nDisclosure statementThe authors of this manuscript have no conflicts of interest to disclose.\n\nCategory of StudyTranslational

microbiology↗

bioRxiv2017

Nasty Prophages and the Dynamics of Antibiotic-Tolerant Persister Cells

Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and the activation of toxin-antitoxin (TA) modules. This model found support in the field, but also generated controversy as part of recent heated debates on the validity of significant parts of the literature. In this study, we therefore used our previous work as a model to critically examine common experimental procedures in order to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variation of culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage {varphi}80, a notorious laboratory contaminant, has apparently caused several phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon, but do not anymore support a role of TA modules in E. coli persister formation. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.\n\nImportanceThe recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies have identified multiple genetic pathways involved in persister formation, but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely different results based on ill-controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis of future studies on the formation and eradication of antibiotic-tolerant persisters.

microbiology↗

bioRxiv2017

Protein-protein interactions with fructose-1-kinase alter function of the central Escherichia coli transcription regulator, Cra

In E. coli, the master transcription regulator Cra regulates >100 genes in central metabolism by binding upstream DNA operator sequences. Genes encoding glycolytic enzymes are repressed, whereas those for gluconeogenesis and the citric acid cycle are activated. Cra-DNA binding is allosterically diminished by binding to either fructose-1-phosphate (F-1-P, generated upon fructose import) or fructose-1,6-bisphosphate (F-1,6-BP). F-1,6-BP is generated from F-1-P by the enzyme fructose-1-kinase (FruK) or from other sugars and is a key intermediate in glycolysis. Here, we report that Cra directly interacts with FruK to form a tight protein-protein complex. Further, growth assays with a fruK knockout strain show that FruK has a broader role in metabolism than its known role in fructose catabolism. Biochemical experiments show that F-1,6-BP binding enhances either the Cra/FruK interaction and/or CRA binding to DNA and that FruK can catalyze the reverse reaction of F-1,6-BP to F-1-P. Results were used to propose a model in which the Cra-FruK complex enhances activation of gluconeogenic genes. Finally, since FruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients.

microbiology↗

bioRxiv2017

Comprehensive skin microbiome analysis reveals the uniqueness of human-associated microbial communities among the class Mammalia

Skin is the largest organ of the body and represents the primary physical barrier between mammals and their external environment. The objective of this research was to generate a skin microbiota baseline for members of the class Mammalia, testing the effects of host species, geographic location, body region, and biological sex. The back, torso, and inner thigh regions of 177 non-human mammals were collected to include representatives from 38 species and 10 mammalian orders. Animals were collected from local farms, zoos, households, and the wild. All samples were amplified using the V3-V4 16S rRNA gene region and sequenced using a MiSeq (Illumina). For reference, previously published skin microbiome data from 20 human participants, sampled using an identical protocol to the non-human mammals, were included in the analysis. Human skin was significantly less diverse than all other mammalian orders and the factor most strongly associated with community variation for all samples was whether the host was a human. Within non-human samples, host taxonomic order was the most significant factor influencing the skin community, followed by the geographic location of the habitat. By comparing the congruence between known host phylogeny and microbial community dendrograms, we observed that Artiodactyla (even-toed ungulates) and Perissodactyla (odd-toed ungulates) had significant congruence, providing first evidence of phylosymbiosis between skin communities and their hosts.\n\nSignificanceSkin forms a critical protective barrier between a mammal and its external environment. Baseline data on the mammalian skin microbiome is crucial for making informed decisions related to veterinary research and biodiversity conservation strategies, in addition to providing insight into mammalian evolutionary history. To our knowledge, this study represents the largest mammalian skin microbiota project to date. These findings demonstrate that human skin is distinct, not only from other Primates, but from all 10 mammalian orders sampled. Using phylosymbiosis analysis, we provide the first evidence that co-evolution may be occurring between skin communities and their mammalian hosts, which warrants more in-depth future studies of the relationships between mammals and their skin microbiota.

microbiology↗

bioRxiv2017

N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi’s sarcoma-associated herpesvirus infection

Methylation at the N6 position of adenosine (m6A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m6A deposition and proteins involved in the m6A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m6A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposis sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m6A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m6A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m6A levels across much of the host transcriptome. However, we found that depletion of the m6A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m6A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m6A modified. In contrast, although the ORF50 mRNA was also m6A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m6A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types.\n\nAuthor SummaryIn addition to its roles in regulating cellular RNA fate, methylation at the N6 position of adenosine (m6A) of mRNA has recently emerged as a mechanism for regulating viral infection. While it has been known for over 40 years that the mRNA of nuclear replicating DNA viruses contain m6A, only recently have studies began to examine the distribution of this modification across viral transcripts, as well as characterize its functional impact upon viral lifecycles. Here, we apply m6A-sequencing to map the location of m6A modifications throughout the transcriptome of the oncogenic human DNA virus Kaposis sarcoma-associated herpesvirus (KSHV). We show that the m6A machinery functions in a cell type specific manner to either promote or inhibit KSHV gene expression. Thus, the KSHV lifecycle is impacted by the m6A pathway, but the functional outcome may depend on cell lineage specific differences in m6A-based regulation.

microbiology↗

bioRxiv2017

IFITM proteins inhibit HIV-1 protein synthesis

Interferon induced transmembrane proteins (IFITMs) inhibit the cellular entry of a broad range of viruses, but it has been suspected that for HIV-1 IFITMs may also inhibit a post-integration replicative step. We show that IFITM expression reduces HIV-1 viral protein synthesis by preferentially excluding viral mRNA transcripts from translation and thereby restricts viral production. Codon-optimization of proviral DNA rescues viral translation, implying that IFITM-mediated restriction requires recognition of viral RNA elements. In addition, we find that expression of the viral accessory protein Nef can help overcome the IFITM-mediated inhibition of virus production. Our studies identify a novel role for IFITMs in inhibiting HIV replication at the level of translation, but show that the effects can be overcome by the lentiviral protein Nef.

microbiology↗