Journal: BMC Infectious Diseases

Article Title: The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

doi: 10.1186/s12879-017-2184-4

Figure Lengend Snippet: a Schematic representation of HIV-1 Gag protein domains. The four major domains of Gag (MA, CA, p7, and p6) are depicted including the two linker sequences p1 and p2. HIV-1 Gag interacts with the ESCRT complex proteins Tsg101 and Alix to regulate viral budding. The sequence of subtype B NL4-3 gag p6 is presented and the sequence motifs PTAP and YPXnL, which serve as the binding motif for Tsg101 and Alix, respectively, are highlighted using the square boxes. Con_C represents the subtype C gag p6 consensus amino acid sequence. The dashes represent sequence identity and the dots sequence deletion. b A comparative analysis of the PTAP sequence duplication in subtypes B and C. In subtype B, a partial PTAP duplication consisting of three amino-acid residues (APP) is common. In contrast in subtype C, a sequence duplication of 14 amino acids is common. A 14 amino acid sequence duplication of subtype C derived from the primary clinical isolate T004 is presented. The amino acid sequences, the original and the duplicated sequences, in Gag and Gag-Pol are depicted. The arrows represent the length of sequence duplication and the direction of polymerization by the reverse transcriptase while synthesizing the cDNA from the viral RNA. The core PTAP motifs are highlighted using the square boxes. The sequences flanking the 3 or 14 aa residues are shown in gray

Article Snippet: RNA was extracted from 150 μl of plasma samples using a commercial Viral RNA isolation kit (NucleoSpin® RNA Virus, Ref. No. 740956.50, MACHEREY-NAGEL GmbH & Co. KG, Germany).

Techniques: Sequencing, Binding Assay, Derivative Assay, Reverse Transcription

Journal: BMC Infectious Diseases

Article Title: The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

doi: 10.1186/s12879-017-2184-4

Figure Lengend Snippet: Multiple sequence alignment of the PTAP sequences of eight primary viral isolates containing PTAP duplication. The sequences were derived from the plasma viral RNA collected at multiple longitudinal time-points and by sequencing the PCR fragments. In three subjects (T004, 2012, 2032), the PTAP motif duplication involved the duplication of 14 amino acids. In the other five subjects, the duplicated sequence length was shorter containing 12 (2018), 11(2020), 9 (2037) or 8 (T014, and 2006) residues. Of note, in all the sequences, the ‘PTAP’ core motif was intact in the original and the duplicated sequences. Note that in two subjects (2014 and 2018) a mixed infection of single- and double-PTAP viral strains could be seen by conventional sequencing. In subject T014, PTAP duplication was observed only at a single time-point M24. For subject 2006, the plasma sample was available only at the baseline. The vertical dashed line demarcates the original and the duplicated PTAP motifs. The arrows indicate the direction of the RT polymerization. Cons: The consensus subtype C gag sequence of 14 amino acids. The same 14 amino acid consensus sequence was used twice in the analysis for convenience. *For these five samples, only gag p6 , not the full-length gag , was amplified. These sequences have not been deposited in the Genbank and are not part of the phylogenetic tree presented in Fig. . D: the duplicated PTAP motif, O, the original PTAP motif

Article Snippet: RNA was extracted from 150 μl of plasma samples using a commercial Viral RNA isolation kit (NucleoSpin® RNA Virus, Ref. No. 740956.50, MACHEREY-NAGEL GmbH & Co. KG, Germany).

Techniques: Sequencing, Derivative Assay, Clinical Proteomics, Infection, Amplification

Journal: PLoS Pathogens

Article Title: The alpha-1 subunit of the Na + ,K + -ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells

doi: 10.1371/journal.ppat.1007963

Figure Lengend Snippet: Macropinocytosis was assayed by monitoring the uptake of dextran (10,000 MW) conjugated to AF568 (dextran-AF568). All incubations with dextran-AF568 were preceded by serum-starvation for 16 h. (A) RSV induces macropinocytosis. A549 cells were mock-infected or infected with wt RSV (MOI = 5 PFU/cell) in medium containing dextran-AF568 (cyan). At 5 h p.i., cells were fixed with 4% PFA and nuclei counterstained with DAPI (blue), and imaged on a Leica SP5 confocal microscope with a 40x Objective NA 1.3 and 2.0x zoom. (B) Co-localization of ATP1A1, RSV N, and dextran-AF568 in RSV-infected A549 cells . Cells were infected with RSV in the presence of dextran-AF568 as described above, incubated for 5 h, fixed with 4% PFA, permeabilized with 0.1% Triton X-100, subjected to immunofluorescence staining with an anti-ATP1A1 rabbit MAb (ab76020) and an anti-RSV-N mouse MAb (ab94806), followed by AF488-conjugated goat anti-rabbit and AF647-conjugated goat anti-mouse secondary antibodies. Z-stacks were acquired on Leica SP8 confocal microscope with 63x objective, NA 1.4 and 3.0x zoom. Arrows indicate co-localization of ATP1A1 (green) and RSV N (red) in dextran-AF568-positive (cyan) vesicles. (C) Co-localization of RSV F and RSV N with dextran-AF568 in RSV-infected A549 cells. Cells were infected with RSV in the presence of dextran-AF568, incubated for 5 h, fixed, and permeabilized as described in B. The cells were then subjected to immunostaining: RSV F was detected with AF488-conjugated anti-RSV F MAb #1129 , and RSV N was detected with an allophycocyanin (APC)-conjugated anti-RSV N MAb (NB100-64752APC, Novus Biologicals, LLC). Image acquisition and analysis were performed as described above for B. Arrows indicate RSV F (green) and RSV N (red) in dextran-AF568-positive (cyan) vesicles. All scale bars are 10 μm. (D–F) Quantification of dextran-AF568 uptake during RSV infection. (D) A549 cells were transfected with ATP1A1 siRNA2 or Neg. siRNA 1, incubated for 48 h p.t., and inoculated with wt RSV in dextran-AF568-containing medium, or (E ) A549 cells were pre-treated with ouabain or PST2238 for 16 h and inoculated with wt RSV in dextran-AF568-containing medium, or (F ) A549 cells were infected with wt RSV or rgRSV dSH/dG in dextran-AF568-containing medium. For all treatments (D-F) cells were fixed 5 h p.i., counterstained with DAPI and z-stacks were acquired on a Leica SP8 confocal microscope with 63x objective NA 1.4, 1.0x zoom. For each treatment, the uptake of dextran-AF568 in vesicles greater than 1.0 μm 3 was quantified as described in detail in the Materials and Methods section. Mean values are reported relative to RSV-infected cells transfected with Neg. siRNA 1 (D), or mock-treated infected cells (E), or wt RSV-infected cells (F). Error bars indicate the standard deviation of at least three independent experiments. The statistical significance of difference was determined for (D) and (E) by one-way analysis of variance with Tukey’s multiple comparison post-test and for (F) by a two-tailed unpaired t-test. P-values are shown for each comparison.

Article Snippet: Simultaneous staining of RSV-F and RSV-N was performed with conjugated primary antibodies: specifically, anti-RSV-F mouse MAb #1129 [ ] that we conjugated with AF488 [Antibody Labeling Kit (Thermo Fisher Scientific, Waltham, MA)]; and a commercially-available anti-RSV N mouse MAb conjugated with allophycocyanin (APC) (NB100-64752APC, Novus Biologicals, Littleton, CO).

Techniques: Infection, Microscopy, Incubation, Immunofluorescence, Staining, Immunostaining, Transfection, Standard Deviation, Comparison, Two Tailed Test