The Teaching of Singing in Meiji Period : Mainly on the contents of teaching

Additional file 3: Fig. S4. Spontaneous gp120 shedding from cell surface. The susceptibility of gp41 mutants to spontaneously shed gp120 was determined by flow cytometry and ELISA as described previously [79]. Briefly, culture medium of transiently transfected envelope expressing cells was exchanged for fresh medium containing Brefeldin A (BioLegend) and 0.2 % Sodium azide. Cells were then incubated for 15 h at 37˚C, 5 % CO2. (a) Level of envelope expression before and after incubation was compared by staining with 2G12. (b) Amount of gp120 released during incubation was determined by gp120 capture ELISA. As a positive control, cells expressing WT envelope was incubated with 20 µg/ml sCD4, which trigger gp120 shedding. Cells expressing SIV Env (SIV) and no Env (No Env) were used as negative control. The results are shown as the means ± standard errors of four replicas

Diversity of the gp120 subpopulations carrying a V3 loop with net charge of +7 or +3.

<p>Full-length gp120 sequences of the HIV-1 CRF01_AE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037530#pone.0037530-Buonaguro1" target="_blank">[42]</a> were extracted from a public database, and divided into subgroups on the basis of the net charge of the V3 loop (+2∼+8). The divided sequences were used to calculate the Shannon entropy, <i>H(i)</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037530#pone.0037530-Shannon1" target="_blank">[63]</a>, within each subpopulation, and the <i>H(i)</i> values were plotted on the 3-D structure of gp120 (PDB code: 2B4C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037530#pone.0037530-Huang1" target="_blank">[1]</a>). The results for the gp120 subgroups that have V3 loops with +7 (left panel) and +3 (right panel) charges are shown as representative. The numbers of sequences used to calculate the <i>H(i)</i> were 9 and 81 for +7 and +3, respectively. (A) Distribution of <i>H(i)</i> in the gp120 monomer. (B) Distribution of <i>H(i)</i> around the CD4 binding site.</p

Neutralization sensitivity of the isogenic V3 recombinant HIV-1 to anti-gp120 monoclonal antibodies.

#<p>Neutralization epitope in the Gp120 outer domain before CD4 binding.</p>$<p>Neutralization epitope induced in Gp120 after CD4 binding.</p>*<p>Epitopes outside of the CD4 binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037530#pone.0037530-diMarzoVeronese1" target="_blank">[37]</a>.</p>@<p>The effect of each antibody on viral infectivity was tested in duplicate.</p

MD simulation of the identical gp120 outer domain carrying a V3 loop with net charge of +7 or +3.

<p>(A) Schematic representation of the gp120 open reading frame along with the amino acid sequences. The net charge indicates the number of positively charged amino acids (R, K, and H) minus the number of negatively charged amino acids (D and E) in the V3 loop. A light blue box indicates the outer domain used for the MD simulations. A pink box indicates the V3 loop. The numbers indicate amino acid positions at the outer domain (amino acids 1 to 233 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037530#pone-0037530-g001" target="_blank">Figure 1A</a> correspond to amino acids 256 to 489 in the gp120 of HIV-1_LAI) or the V3 loop. An open black box in the V3 loop sequence indicates a potential site for the N-linked glycosylation. (B–D) Left panels: Gp120_LAI-NH1V3; Right panels: Gp120_LAI-TH09V3. +7 and +3 indicate the net charges of V3 loops of the recombinant proteins. (B) Time course of RMSD during MD simulations. The RMSD values indicate the structural fluctuations of the outer domain in aqueous solution. The numbers in the horizontal axes indicate the time of MD simulation. (C) Distribution of RMSF in the gp120 outer domain. The RMSF values indicate the atomic fluctuations of the main chains of individual amino acids during 10–30 ns of MD simulations. The numbers in the horizontal axes indicate amino acid positions at the outer domain. (D) Superimposition of Gp120 models at 10, 15, 20, 25, and 30 ns of MD simulation. A green asterisk indicates approximate position of a potential N-glycosylation site at the V3 stem. A green arrow indicates the site of the disulfide bond at the V3 base.</p

Comparison of the averaged 3-D models during MD simulation.

<p>(A) Superposition of the averaged structures obtained with the 40,000 snapshots obtained from 10–30 ns of MD simulations using ptraj module in Amber 9. Red and Blue ribbons: loops of Gp120_LAI-NH1V3 and Gp120_LAI-TH09V3 with V3 net charges of +7 and +3, respectively. (B–D) Configuration and structural dynamics of the CD4 binding loop. The distance between the Cα of Gly115 and the Cα of Gly221 in the CD4 binding loop was calculated to monitor configurational changes (B). The distance was monitored during the 10–30 ns of MD simulation (C) and the average distance with variance was plotted (D). +7: Gp120_LAI-NH1V3; +3: Gp120_LAI-TH09V3.</p

Growth of GH123 and its mutant viruses in the presence of CM TRIM5α.

<p>MT4 cells were infected with CM-TRIM5α-SeV (black circles) or CM-SPRY(–)-SeV (white circles) then superinfected with GH123 mutant viruses. Culture supernatants were periodically assayed for levels of virus capsid. Error bars show actual fluctuations between measurements of capsid in duplicate samples. A representative of two independent experiments is shown.</p

Growth of SIVmac239 and its mutant viruses in the presence of CM TRIM5α.

<p>MT4 cells were infected with CM-TRIM5α-SeV (black circles) or CM-SPRY(–)-SeV (white circles) then superinfected with SIVmac239 mutant viruses. Culture supernatants were periodically assayed for levels of virus capsid. Error bars show actual fluctuations between measurements of capsid in duplicate samples. A representative of three independent experiments is shown.</p

Effects of an aspartic acid-to-alanine substitution at the 97th position of the HIV-2 CA on viral growth in the presence or absence of CM TRIM5α.

<p>MT4 cells were infected with CM-TRIM5α-SeV (black circles) or CM-SPRY(–)-SeV (white circles) then superinfected with GH123 mutant viruses. Culture supernatants were periodically assayed for levels of viral capsid. Error bars show actual fluctuations between measurements of capsid in duplicate samples. A representative of three independent experiments is shown.</p

The probability of forming a hydrogen bond between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7 of the CA in 60,000 trajectories during 5–20 nanoseconds of MD simulations and the sensitivity phenotype.

<p>The probability of forming a hydrogen bond between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7 of the CA in 60,000 trajectories during 5–20 nanoseconds of MD simulations and the sensitivity phenotype.</p

Surface structure of the HIV-2 capsid N-terminal domain.

<p>Surface structure of the GH123 and mutant GH123 CAs visualized with PyMOL. Red color indicates the 120th amino acid of the GH123 and mutant GH123 CAs.</p