Structural Correlates of Rotavirus Cell Entry

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration

Auto-tethering as a selection mechanism for recognition of multimeric substrates by the AAA+ unfoldase ClpX

Thesis: Ph. D. in Biochemistry, Massachusetts Institute of Technology, Department of Biology, 2008.Cataloged from PDF version of thesis.Includes bibliographical references.The CIp/Hsp1OO enzymes, which belong to the AAA+ family of ATPases, use their unfoldase activity to degrade and remodel multimeric substrates in the bacterial cell. The mechanical energy exerted by CIp/Hsp1OO enzymes drives forward essential transitions in important biological processes. However, with a potentially destructive and energetically expensive enzymatic activity, mechanisms must be employed to ensure that Clp/Hsp1OO enzymes act on the desired substrate at the right time and in the right location. The remodeling of stable complexes by Clp/HsplOO enzymes must be directed toward the correctly assembled form of substrates in the cell, and therefore strategies must exist that guide Clp/Hsp1OO enzymes to correctly distinguish between multimeric and monomeric forms of a substrate. In this work I explore how substrate multimerization modulates recognition by the enzyme, using the AAA+ unfoldase CIpX and its multimeric substrate the Mu transpososome. Phage Mu transposase tetramerizes in the cell to form the Mu transpososome, which mediates replicative transposition of the phage. After transposition is completed, the Mu transpososome forms an extremely stable tetramer that needs to be destabilized by CIpX to allow it to facilitate phage Mu genome amplification. How CIpX is guided to the correctly assembled stable transpososome is the subject of my work. I find that multimerization of the phage Mu transposase to form the tetrameric Mu transpososome exposes residues that make contact with the CIpX only in the context of the tetrameric complex. These unique contacts recruit CIpX to the stable transpososome with high affinity. The dual role of subunits in the transpososome in providing high affinity CIpX binding sites as well as CIpX substrate degradation signals is referred to in this work as auto-tethering. Additionally, I show that the N terminal domain of CIpX, which plays a role in substrate selection, is important in facilitating discrimination between different multimeric forms of MuA by CIpX. CIpX destabilizes the tetrameric transpososome by unfolding only one of the subunits within the complex. However, it is not known which subunit within the transpososome is unfolded, nor is it clear whether it is the same or different subunits that facilitate high affinity binding of CIpX to the complex. I am currently performing experiments to determine the geometry of unfolding and auto-tethering using an altered specificity mutant of MuA, which binds to Mu DNA binding sites in the transpososome containing compensatory mutations. This work can shed light on the division of labor required to mediate auto-tethering in the transpososome as well as in other multimeric substrates of CIpX.by Aliaa H. Abdelhakim.Ph. D. in Biochemistr

OCT Angiography: A Non-Invasive Method For Assessing Optic Nerve Head Vasculitis In Bartonella Neuroretinitis

Bartonella henselae, an endotheliotropic pathogen, causes optic disc vasculitis with resulting neuroretinitis and peripapillary retinal exudation. We used OCT angiography (OCTA), fluorescein angiography (FA) and red-free photography to assess the disc vasculature in a rare case of bilateral Bartonella neuroretinitis

Relief of Cystoid Macular Edema-Induced Focal Axonal Compression with Anti-Vascular Endothelial Growth Factor Treatment

Purpose: To evaluate the mechanical compression of retinal nerve fiber layer (RNFL) by intraretinal cysts in macular edema and its relief with anti-vascular endothelial growth factor (anti-VEGF) treatment

Chronic, Painful Abducens Palsy May Require Angiography

"Posteriorly-draining cavernous-carotid fistulas (CCFs) ("white-eyed shunts") can present with painful cranial nerve palsies. We report a case of posteriorly-draining CCF presenting as a persistently painful, chronic left abducens palsy.

Association of productive (A) and non-productive (B) particles with AP-2 clathrin adaptor, dynamin, and Rab5.

<p>“Productive particles” are those that bind and uncoat as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004355#ppat-1004355-g003" target="_blank">Figure 3</a>; “non-productive particles”, those that fail to uncoat within 30 mins of binding. Of 62 productive particles followed in cells transfected with σ2-eGFP (<b>A</b>, left), 50 did not colocalize with σ2 at any time, while 12 colocalized with σ2 at an early time point. Likewise, of 62 productive particles followed in Rab5-eGFP transfected cells (<b>A</b>, right), only 11 appeared to colocalize with Rab5, while of 166 non-productive particles (<b>B</b>, right), a substantial majority ended up in Rab5 endosomes. Cells stably transfected with σ2-eGFP or Rab5-eGFP or transiently transfected with dynamin-eGFP.</p

Fluorescent labeling of recoated TLP.

<p><b>A</b>. Images of particles, adsorbed to coverslip, at each of three wavelengths and overlay. <b>B</b>. Analysis of recoated, purified TLPs by 12% SDS-PAGE. <b>C</b>. Fluorescent focus assay measuring effect of labeling on reacoated particle infectivity, with labeling as shown. <b>D</b>. Images of doubly labeled (VP7, green; DLP, red) recoated particles, 10 min and 45 min following addition to BSC-1 cells. Uncoated particles (red) are evident at the later time point. <b>E</b>. Time lapse sequence of triply labeled particles uncoating in BSC-1 cells. Time frames at 5 sec intervals from an arbitrary time point following addition of particles to cell. Pseudo-colors: VP7, blue; VP4, green; DLP, red. <b>F</b>. Graphic representation of time lapse sequence in panel E; images recorded at 1 sec intervals. Intensities (colors as in E) evaluated in a box moving with the DLP; orange curve: velocity of DLP.</p