Phylogenetic analysis of HIV-1 <i>env</i> C2-V3-C3 sequences of monocyte- and CD4+ T cell-derived HIV-1.

<p>The neighbor-joining trees of 1175 M and 1175 T (A) as well as 1690 M and 1690 T (B) were shown, respectively. The <i>env</i> C2-V3-C3 sequences of provirues as well as primary isolate before and after MDM selection were acquired from limiting dilution PCR. MDM selection experiments were using seven-day old MDM prepared from 3 healthy donors, respectively, at MOI = 0.01. On Day 22 post-infection, supernatants of infected MDM were subjected to sequence analysis. The viral nucleotide sequences were used to construct neighbor-joining trees based on nucleotide pairwise distances. Reference sequences (not labeled) are HIV-1 subtype B sequences from GenBank database (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>).</p

Viral isolation from Subject 1175 and 1690.

<p>Clinical information of study Subject 1175 (A) and 1690 (B). For each subject, the first visit with confirmative diagnosis of seroconversion, along with the corresponding HIV-1 RNA viral load, is plotted at Month 0. Subsequently, longitudinal visits, viral loads, treatment and sampling time points are shown. (C) The monocyte purification strategy. Non-monocytes were magnetically labeled and removed from PBMCs of 2 healthy donors. The eluent monocytes were positively sorted and subjected to a four-hour attachment purification followed by two intensive washes with PBS. Percentage of T cell contamination before and after attachment purification was shown in the shaded quadrants of each flow cytometry graph. Monocyte- and CD4+ T cell-derived HIV-1 were obtained from subject 1175 (D) and 1690 (E) by being co-cultivated with CD8+ T cell-depleted donor PBMC. Longitudinal HIV-1 p24 concentrations in supernatants were shown for each co-cultivation. The high limit cut-off of HIV-1 p24 concentration is 20 ng/ml and lower limit for detection (LLD) is 0.01 ng/ml. The time points for viral expansion and stock preparation were indicated with dash lines with arrowheads. TCID₅₀ titers of each viral expansion of 1175 T and 1175 M (D), as well as 1690 T and 1690 M (E), are shown in each corresponding box.</p

Intracellular viral DNA persistence in MDM acute infection and longitudinal reverse transcriptase activity assay.

<p>(A) HIV-1 DNA synthesis kinetics in MDM. Seven-day old MDM from 3 healthy donors were infected by 1690 M and 1690 T, respectively, at MOI = 0.01. Cells were harvested at 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 168 h post-infection. Intracellular HIV-1 DNA copy numbers were determined. *<i>P</i><0.05, donor n = 3. (B) Viral resistance to azidothymidine. Seven-day old MDM were infected by 1690 M and 1690 T, respectively, at MOI = 0.01. On Day 6 post-infection, supernatants were removed and MDM were exposed to AZT at serially diluted concentrations as indicated. The inhibition rates calculated by relative p24 production were shown for the time points of Day 8, 12 and 16 post-infection, respectively. Data were shown as mean ± SEM, donor n = 3.</p

Observation of replication kinetics in MDM.

<p>HIV-1 p24 production kinetics in MDM was shown for1690 M (A) and1690 T and 1175 M and 1175 T (B). Seven-day old MDM infection at MOI = 0.01 for 4 h before continuously cultured in fresh media. Supernatant HIV-1 p24 concentrations were determined and shown longitudinally (ng/mL, mean ± SEM, donor n = 4). (C) Supernatant viral RNA copy numbers of MDM infections. Supernatant samples were acquired on Day 22 post-infection and data were shown as copies/mL (donor n = 3). (D) Viral replication in PHA-PBMC. Donor PBMCs were stimulated by PHA for 48 h before BaL, 1690 M, 1690 T, 1175 M and 1175 T infections at MOI = 0.01. HIV-1 p24 concentrations in culture supernatants on Day 7 post-infection were shown (ng/mL, mean ± SEM). *<i>P</i><0.05, donor n = 4 compared with the BaL-infected group. <i>E,</i> Intracellular staining of HIV-1 p24. MDM were infected for 14 d before cyto-immunochemistry staining with CD14 (red), DAPI (blue) and HIV-1 p24 (green). Scale bar = 20 µm.</p

HIV-1 Env V3 amino acid sequences with computational and biological prediction of co-receptor usage.

a<p>10 to15 end-point diluted PCR products were directly sequenced from each virus, and the Env V3 regions of 1690 M, 1690 T, 1175 M and 1175 T, aligned to the HXB2 strain, were shown. The positions 11 and 25 of the Env V3 region are indicated in bold. “−” Represents a sequence gap.</p>b<p>Non-syncytia formation and syncytia formation HIV-1 are abbreviated as NSI and SI, respectively.</p>c<p>CCR5- and CXCR4- tropic viruses were indicated by R5 and X4, respectively.</p