Host Cell Nucleolin Is Required To Maintain the Architecture of Human Cytomegalovirus Replication Compartments

Drastic reorganization of the nucleus is a hallmark of herpesvirus replication. This reorganization includes the formation of viral replication compartments, the subnuclear structures in which the viral DNA genome is replicated. The architecture of replication compartments is poorly understood. However, recent work with human cytomegalovirus (HCMV) showed that the viral DNA polymerase subunit UL44 concentrates and viral DNA synthesis occurs at the periphery of these compartments. Any cellular factors involved in replication compartment architecture are largely unknown. Previously, we found that nucleolin, a major protein component of nucleoli, associates with HCMV UL44 in infected cells and is required for efficient viral DNA synthesis. Here, we show that nucleolin binds to purified UL44. Confocal immunofluorescence analysis demonstrated colocalization of nucleolin with UL44 at the periphery of replication compartments. Pharmacological inhibition of viral DNA synthesis prevented the formation of replication compartments but did not abrogate association of UL44 and nucleolin. Thus, association of UL44 and nucleolin is unlikely to be a nonspecific effect related to development of replication compartments. No detectable colocalization of 5-ethynyl-2′-deoxyuridine (EdU)-labeled viral DNA with nucleolin was observed, suggesting that nucleolin is not directly involved in viral DNA synthesis. Small interfering RNA (siRNA)-mediated knockdown of nucleolin caused improper localization of UL44 and a defect in EdU incorporation into viral DNA. We propose a model in which nucleolin anchors UL44 at the periphery of replication compartments to maintain their architecture and promote viral DNA synthesis

Maintaining a sense of direction during long-range communication on DNA

Many biological processes rely on the interaction of proteins with multiple DNA sites separated by thousands of base pairs. These long-range communication events can be driven by both the thermal motions of proteins and DNA, and directional protein motions that are rectified by ATP hydrolysis. The present review describes conflicting experiments that have sought to explain how the ATP-dependent Type III restriction–modification enzymes can cut DNA with two sites in an inverted repeat, but not DNA with two sites in direct repeat. We suggest that an ATPase activity may not automatically indicate a DNA translocase, but can alternatively indicate a molecular switch that triggers communication by thermally driven DNA sliding. The generality of this mechanism to other ATP-dependent communication processes such as mismatch repair is also discussed

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (? 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ? 2.8-3.6 ?(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources

Multi-strain Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus.

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains

Lipoprotein N-Acylation in Staphylococcus aureus Is Catalyzed by a Two-Component Acyl Transferase System

Although it has long been known that S. aureus forms triacylated Lpps, a lack of homologs to known N-acylation genes found in Gram-negative bacteria has until now precluded identification of the genes responsible for this Lpp modification. Here, we demonstrate N-terminal Lpp acylation and chemotype conversion to the tri-acylated state is directed by a unique acyl transferase system encoded by two noncontiguous staphylococci genes (lnsAB). Since triacylated Lpps stimulate TLR2 more weakly than their diacylated counterparts, Lpp N-acylation is an important TLR2 immunoevasion factor for determining tolerance or nontolerance in niches such as in the skin microbiota. The discovery of the LnsAB system expands the known diversity of Lpp biosynthesis pathways and acyl transfer biochemistry in bacteria, advances our understanding of Lpp structural heterogeneity, and helps differentiate commensal and noncommensal microbiota.Bacterial lipoproteins (Lpps) are a class of membrane-associated proteins universally distributed among all bacteria. A characteristic N-terminal cysteine residue that is variably acylated anchors C-terminal globular domains to the extracellular surface, where they serve numerous roles, including in the capture and transport of essential nutrients. Lpps are also ligands for the Toll-like receptor 2 (TLR2) family, a key component of the innate immune system tasked with bacterial recognition. While Lpp function is conserved in all prokaryotes, structural heterogeneity in the N-terminal acylation state is widespread among Firmicutes and can differ between otherwise closely related species. In this study, we identify a novel two-gene system that directs the synthesis of N-acylated Lpps in the commensal and opportunistic pathogen subset of staphylococci. The two genes, which we have named the lipoprotein N-acylation transferase system (Lns), bear no resemblance to previously characterized N-terminal Lpp tailoring enzymes. LnsA (SAOUHSC_00822) is an NlpC/P60 superfamily enzyme, whereas LnsB (SAOHSC_02761) has remote homology to the CAAX protease and bacteriocin-processing enzyme (CPBP) family. Both LnsA and LnsB are together necessary and alone sufficient for N-acylation in Staphylococcus aureus and convert the Lpp chemotype from diacyl to triacyl when heterologously expressed in Listeria monocytogenes. Acquisition of lnsAB decreases TLR2-mediated detection of S. aureus by nearly 10-fold and shifts the activated TLR2 complex from TLR2/6 to TLR2/1. LnsAB thus has a dual role in attenuating TLR2 signaling in addition to a broader role in bacterial cell envelope physiology

Neuronal miR-9 promotes HSV-1 epigenetic silencing and latency by repressing Oct-1 and Onecut family genes

Abstract Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation

Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion

Many DNA-interacting proteins diffuse on DNA to perform their biochemical functions. Processivity factors diffuse on DNA to permit unimpeded elongation by their associated DNA polymerases, but little is known regarding their rates and mechanisms of diffusion. The processivity factor of herpes simplex virus DNA polymerase, UL42, unlike “sliding clamp” processivity factors that normally form rings around DNA, binds DNA directly and tightly as a monomer, but can still diffuse on DNA. To investigate the mechanism of UL42 diffusion on DNA, we examined the effects of salt concentration on diffusion coefficient. Ensemble studies, employing electrophoretic mobility shift assays on relatively short DNAs, showed that off-rates of UL42 from DNA depended on DNA length at higher but not lower salt concentrations, consistent with the diffusion coefficient being salt-dependent. Direct assays of the motion of single fluorescently labeled UL42 molecules along DNA revealed increased diffusion at higher salt concentrations. Remarkably, the diffusion coefficients observed in these assays were ≈104-fold higher than those calculated from ensemble experiments. Discrepancies between the single-molecule and ensemble results were resolved by the observation, in single-molecule experiments, that UL42 releases relatively slowly from the ends of DNA in a salt-dependent manner. The results indicate that UL42 “hops” rather than “slides,” i.e., it microscopically dissociates from and reassociates with DNA as it diffuses rather than remaining so intimately associated with DNA that cation condensation on the phosphate backbone does not affect its motion. These findings may be relevant to mechanisms of other processivity factors and DNA-binding proteins

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1*

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-ΔC) was solved in an oligomeric state. The molecular structure of BMRF1-ΔC shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-ΔC, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-ΔC exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-ΔC associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-ΔC probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-ΔC is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively