Journal: AIDS Research and Human Retroviruses

Article Title: Transmitted Antiretroviral Drug Resistance and Thumb Subdomain Polymorphisms Among Newly HIV Type 1 Diagnosed Patients Infected with CRF01_AE and CRF07_BC Virus in Guangdong Province, China

doi: 10.1089/aid.2011.0320

Figure Lengend Snippet: Clinical Information and Resistance-Related Amino Acid Substitutions of Patients Who Harbored Primary Resistance to Antiretroviral Drugs

Article Snippet: HIV-1 RNA was extracted from 200 μl of HIV-1-positive plasma using the DaAn Gene nucleotide extraction kit (DaAn Gene Diagnostic Center of Sun Yat-sen University, Guangzhou, China) according to the manufacturer's instructions.

Techniques: Cell Counting

Journal: AIDS Research and Human Retroviruses

Article Title: Transmitted Antiretroviral Drug Resistance and Thumb Subdomain Polymorphisms Among Newly HIV Type 1 Diagnosed Patients Infected with CRF01_AE and CRF07_BC Virus in Guangdong Province, China

doi: 10.1089/aid.2011.0320

Figure Lengend Snippet: The phylogenetic tree was created based on the neighbor-joining method and the Kimura two-parameter distance estimation method with the protease (PR) and reverse transcriptase (RT) sequences of HIV-1 obtained from newly diagnosed drug-naive patients in this study. The reference sequences of recombinant types used in the comparative phylogenetic analysis were CRF01_AE: U51188, AF197340, and AF197341; CRF_07BC: AF286226, AF03396, and AF286230. The simian immunodeficiency virus sequence AF103818 was used as the outgroup sequence. ▲ represents the sequences of CRF01_AE; △ represents the sequences of CRF_07BC. The bootstrap values (1025 replicates) over 70% are marked at the major branches of the tree. The scale bar represents 0.02 nucleotide substitution per site between the sequences.

Article Snippet: HIV-1 RNA was extracted from 200 μl of HIV-1-positive plasma using the DaAn Gene nucleotide extraction kit (DaAn Gene Diagnostic Center of Sun Yat-sen University, Guangzhou, China) according to the manufacturer's instructions.

Techniques: Reverse Transcription, Recombinant, Virus, Sequencing

Journal: PLoS ONE

Article Title: HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

doi: 10.1371/journal.pone.0095352

Figure Lengend Snippet: (A) SDS-PAGE of purified GST-Nef fusion (lane 1) and GST (lane 2) proteins stained with Coomassie Blue. Molecular weights (MW) are shown on the left. (B) WB of the lysate of E. coli expressing HIV-1 Protease (PR) (lane 1) and a lysate control (LC) (lane 2). *Denotes the detection of two nonspecific bands only in the LC. (C) Protease activity measured by the cleavage of a specific FRET substrate over a 2-hour interval. Substrate cleavage allows emission of light and is represented by the y-axis. All conditions were tested in triplicate. RLU – Relative light units.

Article Snippet: The clarified supernatant was used in the enzymatic reaction, which was performed using the HIV-1 PR assay kit from ProteinOne.

Techniques: SDS Page, Purification, Staining, Expressing, Activity Assay

Journal: PLoS ONE

Article Title: HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

doi: 10.1371/journal.pone.0095352

Figure Lengend Snippet: HIV-1 and HIV-1 Δ Nef viruses were produced in Hek-293T cells treated with increasing concentrations of LPV or in MOLT4 cells treated with increasing concentrations of DRV. The infectivity of the viral progeny was measured in TZM-bl cells, and dose-response curves and IC50 values were fitted using Hill 4-parameter non-linear regression. (A) The concentration-response curves for HIV-1 and HIV-1 Δ Nef in the presence of LPV. (B) The concentration-response curves for HIV-1 and HIV-1 Δ Nef in the presence of DRV. Representative of three experiments.

Article Snippet: The clarified supernatant was used in the enzymatic reaction, which was performed using the HIV-1 PR assay kit from ProteinOne.

Techniques: Produced, Infection, Concentration Assay

Journal: PLoS ONE

Article Title: HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

doi: 10.1371/journal.pone.0095352

Figure Lengend Snippet: HIV-1 and HIV-1 Δ Nef virions were produced in Hek-293T cells and separated using a 30–70% continuous sucrose density gradient. Ten fractions were collected from top to bottom and numbered #1 through #10 accordingly. The fractions were analyzed to determine the content and distribution of IN (top panel) and CA (lower panel). (A) Protein content of soluble proteins (fractions #1–4), immature particles (#5–7) and mature particles (#8–10) for the HIV-1 (left panels) and HIV-1 Δ Nef viruses (right panels). (B) The CA content of cell-free supernatants before fractionation. (C) Quantification of the amount of CA in fractions #8–10. (D) Quantification of the amount of IN in fraction #8–10. * p = 0.061, ** p = 0.036. The WB and ELISA results presented are representative of three experiments, CA and IN values are triplicates.

Article Snippet: The clarified supernatant was used in the enzymatic reaction, which was performed using the HIV-1 PR assay kit from ProteinOne.

Techniques: Produced, Fractionation, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

doi: 10.1371/journal.pone.0095352

Figure Lengend Snippet: HIV-1 and HIV-1 Δ Nef virions were produced in MOLT cells and separated using a 30–70% continuous sucrose density gradient. Twelve fractions were collected from top to bottom and numbered #1 through #12 accordingly. The fractions were precipitated with 20% TCA and analyzed by WB. (A) Infectivity levels of the viral progeny produced in MOLT cells. (B) Lysates of HIV-1- and HIV-1 Δ Nef-transfected cells, showing equivalent levels of viral protein expression. (C) Cell-free supernatants of HIV-1- and HIV-1 Δ Nef-transfected cells, showing equivalent levels viral release. (D) CA content of cell-free supernatants before separation by the density gradient as measured using a p24-ELISA. (E) CA (top panel), IN (middle pannel) and MA (bottom pannel) protein content of mature particles. The top three panels represent the fractions for HIV-1, and the bottom three panels represent the fractions for HIV-1 Δ Nef. (F) Quantification of the amount of CA in mature fractions after densitometry, * p = 0.002. (G) Quantification of the amount of MA in mature fractions after densitometry, ** p = 0.059 (H) Quantification of the amount of IN in mature particles after densitometry. The results presented are representative of three experiments.

Article Snippet: The clarified supernatant was used in the enzymatic reaction, which was performed using the HIV-1 PR assay kit from ProteinOne.

Techniques: Produced, Infection, Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

doi: 10.1371/journal.pone.0095352

Figure Lengend Snippet: HIV-1 and HIV-1 Δ Nef virions were produced in MOLT cells and separated using a 9.6–18% continuous optiprep density gradient. Twelve fractions were collected from top to bottom and numbered #1 through #12 accordingly. The fractions were precipitated with 20% TCA and analyzed by WB to determine the contents and distributions of CA (top panel), MA (middle panel) and IN (bottom panels). Left panels represent the fractions for HIV-1, and the right panels represent the fractions for HIV-1 Δ Nef. (A) The protein contents of the mature particles for the HIV-1 (left) and HIV-1 Δ Nef viruses (right). (B) Numbers of blue-foci of non-fractionated supernatant (sn) and non-preciptated fractions for each virus. *Denotes the detection of a nonspecific band. The result presented is the mean of three experiments.

Article Snippet: The clarified supernatant was used in the enzymatic reaction, which was performed using the HIV-1 PR assay kit from ProteinOne.

Techniques: Produced