Glycosaminoglycans-Specific Cell Targeting and Imaging Using Fluorescent Nanodiamonds Coated with Viral Envelope Proteins

Understanding virus–host interactions is crucial for vaccine development. This study investigates such interactions using fluorescent nanodiamonds (FNDs) coated with vaccinia envelope proteins as the model system. To achieve this goal, we noncovalently conjugated 100 nm FNDs with rA27­(aa 21–84), a recombinant envelope protein of vaccinia virus, for glycosaminoglycans (GAGs)-specific targeting and imaging of living cells. Another recombinant protein rDA27­(aa 33–84) that removes the GAGs-binding sequences was also used for comparison. Three types of A27-coated FNDs were generated, including rA27­(aa 21–84)-FND, rDA27­(aa 33–84)-FND, and hybrid rA27­(aa 21–84)/rDA27­(aa 33–84)-FND. The specificity of these viral protein–FND conjugates toward GAGs binding was examined by flow cytometry, fluorescence microscopy, and gel electrophoresis. Results obtained for normal and GAGs-deficient cells showed that the recombinant proteins maintain their GAG-targeting activities even after immobilization on the FND surface. Our studies provide a new nanoparticle-based platform not only to target specific cell types but also to track the fates of these immobilized viral proteins in targeted cells as well as to isolate and enrich GAGs-associated proteins on cell membrane

Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells

Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light <i>via</i> imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (<3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors <i>via</i> digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 μm²/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus–receptor interactions with nanometer clarity

Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells

Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light <i>via</i> imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (<3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors <i>via</i> digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 μm²/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus–receptor interactions with nanometer clarity

High Protein Diet and Huntington's Disease

<div><p>Huntington’s disease (HD) is a neurodegenerative disorder caused by the <i>huntingtin</i> (<i>HTT</i>) gene with expanded CAG repeats. In addition to the apparent brain abnormalities, impairments also occur in peripheral tissues. We previously reported that mutant Huntingtin (mHTT) exists in the liver and causes urea cycle deficiency. A low protein diet (17%) restores urea cycle activity and ameliorates symptoms in HD model mice. It remains unknown whether the dietary protein content should be monitored closely in HD patients because the normal protein consumption is lower in humans (~15% of total calories) than in mice (~22%). We assessed whether dietary protein content affects the urea cycle in HD patients. Thirty HD patients were hospitalized and received a standard protein diet (13.7% protein) for 5 days, followed by a high protein diet (HPD, 26.3% protein) for another 5 days. Urea cycle deficiency was monitored by the blood levels of citrulline and ammonia. HD progression was determined by the Unified Huntington’s Disease Rating Scale (UHDRS). The HPD increased blood citrulline concentration from 15.19 μmol/l to 16.30 μmol/l (<i>p</i> = 0.0378) in HD patients but did not change blood ammonia concentration. A 2-year pilot study of 14 HD patients found no significant correlation between blood citrulline concentration and HD progression. Our results indicated a short period of the HPD did not markedly compromise urea cycle function. Blood citrulline concentration is not a reliable biomarker of HD progression.</p></div

Crystal Structure of Vaccinia Viral A27 Protein Reveals a Novel Structure Critical for Its Function and Complex Formation with A26 Protein

<div><p>Vaccinia virus envelope protein A27 has multiple functions and is conserved in the <i>Orthopoxvirus</i> genus of the poxvirus family. A27 protein binds to cell surface heparan sulfate, provides an anchor for A26 protein packaging into mature virions, and is essential for egress of mature virus (MV) from infected cells. Here, we crystallized and determined the structure of a truncated form of A27 containing amino acids 21–84, C71/72A (tA27) at 2.2 Å resolution. tA27 protein uses the N-terminal region interface (NTR) to form an unexpected trimeric assembly as the basic unit, which contains two parallel α-helices and one unusual antiparallel α-helix; in a serpentine way, two trimers stack with each other to form a hexamer using the C-terminal region interface (CTR). Recombinant tA27 protein forms oligomers in a concentration-dependent manner <i>in vitro</i> in gel filtration. Analytical ultracentrifugation and multi-angle light scattering revealed that tA27 dimerized in solution and that Leu47, Leu51, and Leu54 at the NTR and Ile68, Asn75, and Leu82 at the CTR are responsible for tA27 self-assembly <i>in vitro</i>. Finally, we constructed recombinant vaccinia viruses expressing full length mutant A27 protein defective in either NTR, CTR, or both interactions; the results demonstrated that wild type A27 dimer/trimer formation was impaired in NTR and CTR mutant viruses, resulting in small plaques that are defective in MV egress. Furthermore, the ability of A27 protein to form disulfide-linked protein complexes with A26 protein was partially or completely interrupted by NTR and CTR mutations, resulting in mature virion progeny with increased plasma membrane fusion activity upon cell entry. Together, these results demonstrate that A27 protein trimer structure is critical for MV egress and membrane fusion modulation. Because A27 is a neutralizing target, structural information will aid the development of inhibitors to block A27 self-assembly or complex formation against vaccinia virus infection.</p></div

Influence of dietary protein content on the urea cycle in Huntington’s disease (HD) patients.

<p>Blood citrulline (A) and ammonia (B) levels in HD patients given the standard protein diet (13.7%) and high protein diet (26.3%). The data for the 23 non-HD subjects (Con) were taken from a previous report [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127654#pone.0127654.ref013" target="_blank">13</a>]. All HD patient data were plotted. Data are presented as mean ± standard deviation (SD) and were analyzed by paired <i>t</i> test. *<i>p</i> < 0.05.</p

AUC-SV and SEC/MALS analysis of tA27 protein showing that tA27 protein formed concentration-dependent dimers.

<p>(A) AUC analyses of tA27-WT protein. Normalized <i>c</i>(<i>s</i>) distribution plots for tA27-WT and mutants. The figure shows the distribution plots for tA27-WT at pH 7.5 (<i>black</i>), tA27-TM-N (<i>green</i>), tA27-TM-C (<i>orange</i>), tA27-6A (<i>purple</i>) and tA27-WT at pH 3.0 (<i>red</i>) obtained from the fitting of SV data using a continuous <i>c</i>(<i>s</i>) distribution model. (B) SEC/MALS profiles of tA27-WT protein at concentrations of 1 mg/ml (<i>red</i>) and 9.5 mg/ml (<i>green</i>). BSA was used as control (<i>black</i>). Thin line segments represent the calculated molar masses; the numbers denote the corresponding molecular weights of each peak (left ordinate axis) in kDa. Solid lines represent normalized UV absorbance (280 nm, right ordinate axis), and dashed lines represent light scattering.</p

Overall structure of tA27.

<p>(A) A schematic representation of the vaccinia virus A27 domain structure. A27 comprises three domains, including the heparin binding domain (HBD), the coiled-coil domain (CCD) for hexameric assembly and interacting with A26, and the leucine zipper domain (LZD) for binding with A17. Expressed tA27 for crystallization studies contains residues from 21 to 84, with mutations in two adjacent cysteine residues (C71A and C72A). The magenta cylinder denotes the region of tA27 structure that corresponds to the α-helix. Black dashed lines indicate the disordered regions. The basic residues in HBD (<i>cyan</i>) and the critical segment for specific heparin binding (<i>underlined</i>) are shown. The residues in the N-terminal region (NTR) interface involved in trimer assembly are presented in blue, and residues in the C-terminal region (CTR) interface involved in hexamer assembly are in green. Purple and green dots denote the mutation sites for <i>in vitro</i> and <i>in vivo</i> studies. (B) This representation of hexameric tA27 is composed of trimer 1 (bottom), shown as ribbon diagram and surface model in blue (chain A), cyan (chain B), and magenta (chain C); and trimer 2 (top), presented as surface model and ribbon diagram in light blue (chain A′), light cyan (chain B′), and light magenta (chain C′). The disordered region, including the HBD, is indicated by black dotted lines. Residues 71 and 72 are colored in red. The filled circles indicate the C-terminus and the white circles denote the N-terminus of tA27. (C) The trimeric structure of Influenza virus HA2. A representative trimer-of-hairpins structure of the viral fusion protein of the influenza virus (Flu) HA2 (PDB code 1HTM) is shown <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003563#ppat.1003563-Bullough1" target="_blank">[60]</a>. For Flu HA2, the interior N-terminal coiled-coil structures are colored in similar blue and the exterior C-terminal antiparallel helices are in similar green. The right part of the figure shows the model rotated 90°.</p

Analysis of vaccinia A27 orthologs.

<p>(A) Multiple sequence alignment of A27 orthologs. The alignment contains group A from Parapoxvirus, including Bovine papular stomatitis virus (BPSV) and Orf virus (ORFV) group B from Orthopoxvirus including Vaccinia virus (VV), Variola virus (VARV), Monkeypox virus (MPXV), Cowpox virus (CPXV), and Ectromelia virus (ECTV) group C from Capripoxvirus, including Lumpy skin disease virus (LSDV), Goatpox virus (GTPV), Sheeppox virus (SPPV), and Suipoxvirus with Swinepox virus (SWPV); and group D from Leporipoxvirus, including Rabbit fibroma virus (RFV) and Myxoma virus (MYXV). Note the heparin binding domain (HBD, <i>orange</i>), coiled-coil domain (CCD, <i>purple</i>), and leucine zipper domain (LZD, <i>grey</i>) regions. The conserved residues surrounding the CCD are marked with orange boxes for NTR and green boxes for CTR. The two cysteine residues in the CCD are denoted by red boxes and dots. Identical and similar amino acid residues are shaded in black and gray, respectively. Dashes denote the sequence gaps introduced to optimize the amino acid sequence alignment. The accession numbers are: BPSV (NP958013); ORFV (AAR98199); VV (YP233032); VARV (ABF23908); MPXV (NP536566); ECTV (NP671648); CPXV (NP619946); LSDV (NP150551); GTPV (ABS72324); SPPV (NP659689); SWPV (NP570274); RFV (NP052004); and MYXV (NP051829). (B) The protein evolution of A27 orthologs. The respective A27 protein lengths of Groups A, B, C, and D are represented by black, cyan, orange and green lines, respectively. The inserted residues are marked with triangles, and the corresponding conserved regions of CCD are boxed in red dashes. (C) Computer modeling of vaccinia A27 orthologues with the X-ray structures of vaccinia virus tA27 show that the CCD regions are structurally conserved, as predicted by molecular modeling, in group A (BPSV, <i>black</i>), group C (LSDV, <i>orange</i>), and group D (RFV, <i>green</i>). The predicted modeling structures were produced by the Phyre server <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003563#ppat.1003563-Kelley1" target="_blank">[61]</a>.</p

WRΔA27L, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A induce cell-to-cell membrane fusion on L cells at neutral pH.

<p>(A) L cells expressing GFP or RFP (1∶1 mixture) were either mock-infected or infected with WR, WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A viruses at an MOI of 50 PFU/cell at 37°C for 30 min and monitored for cell-to-cell fusion at a neutral pH, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003563#s4" target="_blank">Materials and Methods</a>. Cell images were photographed at 2 h post-infection. (B) Quantification of cell-to-cell fusion at neutral pH. The percentage of cells containing both GFP and RFP fluorescence was quantified as cell-cell fusion using Axio Vision Rel. 4.8 with a Zeiss Axiovert fluorescence microscope.</p