Mihnea Bostina

Poliovirus RNA Is Released from the Capsid near a Twofold Symmetry Axis

ABSTRACT After recognizing and binding to its host cell, poliovirus (like other nonenveloped viruses) faces the challenge of translocating its genome across a cellular membrane and into the cytoplasm. To avoid entanglement with the capsid, the RNA must exit via a single site on the virion surface. However, the mechanism by which a single site is selected (from among 60 equivalents) is unknown; and until now, even its location on the virion surface has been controversial. To help to elucidate the mechanism of infection, we have used single-particle cryo-electron microscopy and tomography to reconstruct conformationally altered intermediates that are formed by the poliovirion at various stages of the poliovirus infection process. Recently, we reported icosahedrally symmetric structures for two forms of the end-state 80S empty capsid particle. Surprisingly, RNA was frequently visible near the capsid; and in a subset of the virions, RNA was seen on both the inside and outside of the capsid, caught in the act of exiting. To visualize RNA exiting, we have now determined asymmetric reconstructions from that subset, using both single-particle cryo-electron microscopy and cryo-electron tomographic methods, producing independent reconstructions at ∼50-Å resolution. Contrary to predictions in the literature, the footprint of RNA on the capsid surface is located close to a viral 2-fold axis, covering a slot-shaped area of reduced density that is present in both of the symmetrized 80S reconstructions and which extends by about 20 Å away from the 2-fold axis toward each neighboring 5-fold axis.

Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy.

ABSTRACT Poliovirus infection requires that the particle undergo a series of conformational transitions that lead to cell entry and genome release. In an effort to understand the conformational changes associated with the release of the RNA genome, we have used cryo-electron microscopy to characterize the structure of the 80S “empty” particles of poliovirus that are thought to represent the final product of the cell entry pathway. Using two-dimensional classification methods, we show that preparations of 80S particles contain at least two structures, which might represent snapshots from a continuous series of conformers. Using three-dimensional reconstruction methods, we have solved the structure of two distinct forms at subnanometric resolution, and we have built and refined pseudoatomic models into the reconstructions. The reconstructions and the derived models demonstrate that the two structural forms are both slightly expanded, resulting in partial disruption of interprotomer interfaces near their particle 2-fold axes, which may represent the site where RNA is released. The models demonstrate that each of the two 80S structures has undergone a unique set of movements of the capsid proteins, associated with rearrangement of flexible loops and amino-terminal extensions that participate in contacts between protomers, between pentamers, and with the viral RNA.

Altered Proliferation and Differentiation Properties of Primary Mammary Epithelial Cells from BRCA1 Mutation Carriers

Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MEC) with altered proliferation and differentiation properties. Using a three-dimensional culture technique to grow MECs ex vivo, we found that the ability to form colonies, an indication of clonality, was restricted to the aldehyde dehydrogenase 1-positive fraction in MECs but not in HCC1937 BRCA1-mutant cancer cells. Primary MECs from BRCA1 mutation carriers (n = 9) had a 28% greater ability for clonal growth compared with normal controls (n = 6; P = 0.006), and their colonies were significantly larger. Colonies in controls and BRCA1 mutation carriers stained positive for BRCA1 by immunohistochemistry, and 79% of the examined single colonies from BRCA1 carriers retained heterozygosity for BRCA1 (ROH). Colonies from BRCA1 mutation carriers frequently showed high epidermal growth factor receptor (EGFR) expression (71% EGFR positive versus 44% in controls) and were negative for estrogen receptor (ERalpha; 32% ER negative, 44% mixed, 24% ER positive versus 90% ER positive in controls). Expression of CK14 and p63 were not significantly different. Microarray studies revealed that colonies from BRCA1-mutant PMECs anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is up-regulated. We conclude that BRCA1 haploinsufficiency leads to an increased ability for clonal growth and proliferation in the PMECs of BRCA1 mutation carriers, possibly as a result of EGFR pathway activation. These altered growth and differentiation properties may render BRCA1-mutant PMECs vulnerable to transformation and predispose to the development of ER-negative, EGFR-positive breast cancers.

RNA Transfer from Poliovirus 135S Particles across Membranes Is Mediated by Long Umbilical Connectors

ABSTRACT During infection, the binding of poliovirus to its cell surface receptor at 37°C triggers an expansion of the virus in which internal polypeptides that bind to membranes are externalized. Subsequently, in a poorly understood process, the viral RNA genome is transferred directly across an endosomal membrane, and into the host cell cytoplasm, to initiate infection. Here, cryoelectron tomography demonstrates the results of 37°C warming of a poliovirus-receptor-liposome model complex that was produced using Ni-nitrilotriacetic acid lipids and His-tagged receptor ectodomains. In total, 651 subtomographic volumes were aligned, classified, and averaged to obtain detailed pictures, showing both the conversion of virus into its expanded form and the passage of RNA into intact liposomes. Unexpectedly, the virus and membrane surfaces were located ∼50 Å apart, with the 5-fold axis tilted away from the perpendicular, and the solvent spaces between them were spanned by either one or two long “umbilical” density features that lie at an angle to the virus and membrane. The thinner connector, which sometimes appears alone, is 28 to 30 Å in diameter and has a footprint on the virus surface located close to either a 5-fold or a 3-fold axis. The broader connector has a footprint near the quasi-3-fold hole that opens upon virus expansion and is hypothesized to include RNA, shielded from enzymatic degradation by polypeptides that include the N-terminal extension of VP1 and capsid protein VP4. The implications of these observations for the mechanism of RNase-protected RNA transfer in picornaviruses are discussed.

A nano-fibrillated mesoporous carbon as an effective support for palladium nanoparticles in the aerobic oxidation of alcohols "on pure water".

A novel nano-fibrillated mesoporous carbon (IFMC) was successfully prepared via carbonization of the ionic liquid 1-methyl-3-phenethyl-1H-imidazolium hydrogen sulfate (1) in the presence of SBA-15. The material was shown to be an efficient and unique support for the palladium nanoparticle (PdNP) catalyst Pd@IFMC (2) in aerobic oxidation of heterocyclic, benzylic, and heteroatom containing alcohols on pure water at temperatures as low as 40 °C for the first time and giving almost consistent activities and selectivities within more than six reaction runs. The catalyst has also been employed as an effective catalyst for the selective oxidation of aliphatic and allylic alcohols at 70-80 °C. The materials were characterized by X-ray photoelectron spectroscopy (XPS), N(2) adsorption-desorption analysis, transmission electron microscopy (TEM), and electron tomography (ET). Our compelling XPS and ET studies showed that higher activity of 2 compared to Pd@CMK-3 and Pd/C in the aerobic oxidation of alcohols on water might be due to the presence of nitrogen functionalities inside the carbon structure and also the fibrous nature of our materials. The presence of a nitrogen heteroatom in the carboneous framework might also be responsible for the relatively uniform and nearly atomic-scale distribution of PdNPs throughout the mesoporous structure and the inhibition of Pd agglomeration during the reaction, resulting in high durability, high stability, and recycling characteristics of 2. This effect was clearly confirmed by comparing the TEM images of the recovered 2 and Pd@CMK-3.

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Background: Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis. Results: Two distinct interactions are involved in formation of the DNA polymerase/DNA helicase complex. Conclusion: The multiple interactions between DNA polymerase and DNA helicase account for the high processivity of leading strand synthesis. Significance: Understanding of the replication process in bacteriophage T7 facilitates studies in more complex systems. Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We present the first structural model of a bacteriophage T7 DNA helicase-DNA polymerase complex using a combination of small angle x-ray scattering, single-molecule, and biochemical methods. We propose that the protein-protein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. DNA primase facilitates binding of DNA helicase to ssDNA and contributes to formation of the DNA helicase-DNA polymerase complex by stabilizing DNA helicase.

Spacer capture and integration by a type I-F Cas1?Cas2-3 CRISPR adaptation complex

Significance CRISPR-Cas systems provide prokaryotic adaptive immunity against invading genetic elements. For immunity, fragments of invader DNA are integrated into CRISPR arrays by Cas1 and Cas2 proteins. Type I-F systems contain a unique fusion of Cas2 to Cas3, the enzyme responsible for destruction of invading DNA. Structural, biophysical, and biochemical analyses of Cas1 and Cas2-3 from Pectobacterium atrosepticum demonstrated that they form a 400-kDa complex with a Cas14:Cas2-32 stoichiometry. Cas1–Cas2-3 binds, processes, and catalyzes the integration of DNA into CRISPR arrays independent of Cas3 activity. The arrangement of Cas3 in the complex, together with its redundant role in processing and integration, supports a scenario where Cas3 couples invader destruction with immunization—a process recently demonstrated in vivo. CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1–Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas14–Cas2-32 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1–Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1–Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3′-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1–Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1–Cas2-3 adaptation complexes and their integration mechanism.

Mechanism of Action and Capsid-Stabilizing Properties of VHHs with an In Vitro Antipolioviral Activity

ABSTRACT Previously, we reported on the in vitro antiviral activity of single-domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in vitro setting and showed 50% effective concentrations (EC50s) in the nanomolar range. In the present study, we further investigated the mechanism of action of these VHHs. All five VHHs interfere at multiple levels of the viral replication cycle, as they interfere both with attachment of the virus to cells and with viral uncoating. The latter effect is consistent with their ability to stabilize the poliovirus capsid, as observed in a ThermoFluor thermal shift assay, in which the virus is gradually heated and the temperature causing 50% of the RNA to be released from the capsid is determined, either in the presence or in the absence of the VHHs. The VHH-capsid interactions were also seen to induce aggregation of the virus-VHH complexes. However, this observation cannot yet be linked to their mechanism of action. Cryo-electron microscopy (cryo-EM) reconstructions of two VHHs in complex with poliovirus type 1 show no conformational changes of the capsid to explain this aggregation. On the other hand, these reconstructions do show that the binding sites of VHHs PVSP6A and PVSP29F overlap the binding site for the poliovirus receptor (CD155/PVR) and span interfaces that are altered during receptor-induced conformational changes associated with cell entry. This may explain the interference at the level of cell attachment of the virus as well as their effect on uncoating. IMPORTANCE The study describes the mechanism of neutralization and the capsid-stabilizing activity of five single-domain antibody fragments (VHHs) that have an in vitro neutralizing activity against poliovirus type 1. The results show that the VHHs interfere at multiple levels of the viral replication cycle (cell attachment and viral uncoating). These mechanisms are possibly shared by some conventional antibodies and may therefore provide some insight into the natural immune responses. Since the binding sites of two VHHs studied by cryo-EM are very similar to that of the receptor, the VHHs can be used as probes to study the authentic virus-cell interaction. The structures and conclusions in this study are original and raise interesting findings regarding virus-receptor interactions and the order of key events early in infection.

Biochemical and electron microscopic characterization of the F1F0 ATP synthase from the hyperthermophilic eubacterium Aquifex aeolicus

The F1F0 ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI‐mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits α, β, γ, δ and ε of F1 and subunits a and c of F0. Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F1 and F0. In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the δ subunit.

Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord

Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting.