Characterization of the effects of a 7 amino-acid deletion in p6^gag to the HIV-1 proteins expression, release and maturation.

<p>MT2 cells were infected with wild type (wt) or deleted-type (7d) recombinant viruses. After 12, 24, 36 and 48 hours, supernatant was collected and pelleted by ultracentrifugation. (A) Western blot analysis of the cell lysates (left panel) and viral lysates (right panel) from cells infected with wt or 7d viruses. (B) The relative expression levels of PR and RT in the viral lysates of cells infected with wt or 7d virus. The total arbitrary densitometer units of PR and RT were standardized by p24 and normalized to those of wt in parallel experiments. The images were analyzed with Image J software. (C) The ratios of p24 vs. Pr55 (maturation index) in the viral lysates at different time points after infection were calculated. The total arbitrary densitometer units of each hours post infection were normalized to those of wt in parallel experiments. All results were representative of two independent experiments. (D) Electron microscopic (EM) examination of the viral particles of cells infected with wt or 7d recombinant viruses. MAGIC-5 cells were fixed and processed for transmission EM at different time points after they were infected with wt or 7d viruses. Scale bar indicates 200 nm. (E) Quantification of relative proportions of mature vs. immature virions released at different time points in the cells infected with wt or 7d viruses using EM. The method of virion quantification has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114441#pone.0114441-Fujii1" target="_blank">[43]</a>.</p

Comparisons of the changes of CD4 cell count and HIV-1 viral loads after the first clinical visit among the following four groups of treatment naïve patients.

<p>Men who have sex with men (MSM, 357 patients for CD4 cell count and 371 patients for viral loads analysis) vs. injection drug users (IDUs, 129 patients for CD4 cell count and 128 patients for viral loads analysis) (Fig. 1A and 1B); patients infected with CRF07_BC vs. infected with subtype B (Figs. 1C and 1D). A generalized estimating equation model was used for the analyses.</p

V3 amino acid sequences and predicted phenotypes of different HIV-1 isolates in Taiwan.

<p>A dash indicated a deletion or lack of an insertion.</p>a<p>The phenotype prediction based on 2 amino acid insertion/deletion between position 14 and 15, as well as variable amino acid positions (11, 18, 19, 23, 24 and 25) of V3 regions.</p>b<p>Geno2pheno (<a href="http://coreceptor.bioinf.mpi-inf.mpg.de/index.php" target="_blank">http://coreceptor.bioinf.mpi-inf.mpg.de/index.php</a>), false-positive rate of 0.01.</p>c<p>Position-Specific Scoring Matrix (PSSM) (<a href="http://indra.mullins.microbiol.washington.edu/webpssm/" target="_blank">http://indra.mullins.microbiol.washington.edu/webpssm/</a>).</p><p>V3 amino acid sequences and predicted phenotypes of different HIV-1 isolates in Taiwan.</p

Demographic data, risk factors and clinical characteristics of HIV-1-infected patients recruited in the study.

<p>Demographic data, risk factors and clinical characteristics of HIV-1-infected patients recruited in the study.</p

Univariate and multivariate generalized estimating equations models of factors associated with CD4 cell counts.

a<p>The mode of transmission was removed from multivariate model II.</p><p>Univariate and multivariate generalized estimating equations models of factors associated with CD4 cell counts.</p

Univariate and multivariate generalized estimating equations model of factors associated with HIV-1 viral loads.

a<p>The mode of transmission was removed from multivariate model II.</p><p>Univariate and multivariate generalized estimating equations model of factors associated with HIV-1 viral loads.</p

Distribution of HIV-1 subtypes and circulating recombinant forms (CRFs) in different groups of patients recruited in this cohort study.

a<p>Includes 4 heterosexual female infected with subtype B, 3 heterosexual female infected with CRF01_AE, and 3 IDUs infected with CRF07_BC.</p><p>Distribution of HIV-1 subtypes and circulating recombinant forms (CRFs) in different groups of patients recruited in this cohort study.</p

The interaction between Alix protein and wild type/mutant Gag. MAGIC-5 cells were infected with wt or 7d recombinant virus for 48 hours.

<p>The Alix and Gag proteins were analyze by TIRF-SR with rabbit anti-Alix polyclonal antibody and mouse anti-p24 monoclonal antibody. Red spots indicate Alix protein. Green spots indicate either wild type or 7d Gag protein. The proportion of co-localization of Alix and Gag protein was quantified using Volocity 3D Image Analysis Software.</p

Characterization of the effects of a 7 amino-acid deletion in p6^gag to the HIV-1 proteins expression, release and maturation.

<p>MT2 cells were infected with wild type (wt) or deleted-type (7d) recombinant viruses. After 12, 24, 36 and 48 hours, supernatant was collected and pelleted by ultracentrifugation. (A) Western blot analysis of the cell lysates (left panel) and viral lysates (right panel) from cells infected with wt or 7d viruses. (B) The relative expression levels of PR and RT in the viral lysates of cells infected with wt or 7d virus. The total arbitrary densitometer units of PR and RT were standardized by p24 and normalized to those of wt in parallel experiments. The images were analyzed with Image J software. (C) The ratios of p24 vs. Pr55 (maturation index) in the viral lysates at different time points after infection were calculated. The total arbitrary densitometer units of each hours post infection were normalized to those of wt in parallel experiments. All results were representative of two independent experiments. (D) Electron microscopic (EM) examination of the viral particles of cells infected with wt or 7d recombinant viruses. MAGIC-5 cells were fixed and processed for transmission EM at different time points after they were infected with wt or 7d viruses. Scale bar indicates 200 nm. (E) Quantification of relative proportions of mature vs. immature virions released at different time points in the cells infected with wt or 7d viruses using EM. The method of virion quantification has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114441#pone.0114441-Fujii1" target="_blank">[43]</a>.</p

Comparison of the replication kinetics of different HIV-1 isolates from patients infected with CRF07_BC or subtype B (A and B) as well as recombinant HIV-1 virus with or without a 7 amino-acid deletion in the p6^gag protein.

<p>(A) PBMCs were infected with fixed amounts of different HIV-1 subtype B (square) or CRF07_BC (circle) isolates and cultural supernatants were collected at days 4, 7, 11, 14, 18 and 21 post-infection. (B) The representative replicative curves of CRF07_BC and subtype B isolates deduced from Fig. 2A. (C) MT2 cells were infected with wild type (wt) or recombinant mutant virus with a 7 amino-acid deletion at the p6^gag (7d). Supernatants were collected at days 2, 4, 6, 8, 10, 12, and 14 post-infection. Viral replication was monitored through p24 antigen production. One-way analysis of variance (ANOVA) and Tukey's post hoc test were used to estimate the differences between subtypes, or between the recombinant viruses.</p