Intra- and inter-run variation of the SG-PERT assay.

<p>(A) Standard curve composed of a pre-made six 10-fold serial dilution series of replication-competent HIV-1 containing supernatant measured in 12 independent SG-PERT experiments. For each experiment obtained Cq values are plotted versus the RT activity in each sample. RT activity values were determined by running a dilution series of recombinant HIV-1 RT in parallel. Standard deviation on the obtained crossing point values is indicated for each dilution. (B) RT activity values obtained for 8 repeated measurements of different HIV-1 samples (sample number 1 to 6) within the same run. The average RT activity value for each sample is indicated by a red line, error bars represent standard deviation on the obtained RT activity values. Numbers indicate intra-run variation for each sample, expressed as percentage of the average RT activity values (coefficient of variation). (C) RT activity values obtained for different HIV-1 samples (sample number 1 to 11) in at least 3 independent SG-PERT experiments. The average RT activity value for each sample is indicated by a red line, error bars represent standard deviation on the obtained RT activity values. Numbers indicate inter-run variation for each sample, expressed as percentage of the average RT activity values (coefficient of variation). Experiments were performed on the LightCycler® 480.</p

Sensitivity and specificity of the SG-PERT assay.

<p>(A) Melting curves of PCR products obtained by SG-PERT assay on the LightCycler® 480 when using 10¹¹ or 10⁴ pU recombinant HIV-1 RT or nuclease-free water (non-template control = <i>NTC</i>) as input for the assay, as indicated. (B, D) Amplification curves of indicated amount of (B) recombinant HIV-1 RT (pU), (D) replication competent HIV-1 (NL4-3 strain) (ng p24/mL) or nuclease-free water (<i>NTC</i>) obtained by SG-PERT on the LightCycler® 480. (C,E) Relation between input of (C) recombinant HIV-1 RT, (E) replication competent HIV-1 (<i>HIV-1</i>), HIV-1 based lentiviral vectors (<i>HIV-1 based vector</i>) or Moloney Murine Leukemia-based retroviral vectors (<i>MoMLV</i>) and obtained cycle of quantification (Cq) values by SG-PERT on the LightCycler® 480. Viral titers in the undiluted samples in (E) (value of “0” on x-axis) were 3,100 ng p24/mL for the replication competent HIV-1 virus, 1.12×10⁷ transducing units/mL (TU/mL) for the HIV-1 based viral vector and 4.9×10⁵ TU/mL for the MoMLV-based vector. Only input levels within linear range of the assay were included for correlation analysis.</p

SIV_MAC transduction of human peripheral blood mononuclear cells or of monocyte derived dendritic cells is less efficient than by HIV-1.

<p>(A) VSV G-pseudotyped HIV-1_NL4-3GFP (black squares) and SIV_MAC239GFP (white circles) two-part vectors were generated by plasmid transfection of 293T cells. Vector stocks were normalized by titer on CRFK cells, and then used to challenge human peripheral blood mononuclear cells. (B) VSV G-pseudotyped, HIV-1_NL4-3 (black squares) and SIV_MAC239 (white circles) three-part vectors were generated by plasmid transfection of 293T cells. In each case, the viral genomic RNA was designed to transduce an identical SFFV-GFP reporter gene. Vector stocks were normalized by titer on CRFK cells, and then used to challenge monocyte derived dendritic cells (DCs). 2 days post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as percent GFP⁺ (infected) cells (Y axis) versus CRFK infectious units (IU) x 1,000 (X axis). Shown are representative data with cells from 4 independent blood donors.</p

The decrease in T cell transduction efficiency by SIV_MAC is not explained by differences in reporter gene expression.

<p>(A) CRFK cells (left panel) and Jurkat T cells (right panel) were transduced with VSV G-pseudotyped, single-cycle, two-part HIV-1_NL4-3GFP or SIV_MAC239-GFP vectors, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005050#ppat.1005050.g001" target="_blank">Fig 1</a>_. Virus stocks were normalized by reverse transcriptase activity prior to transduction. 48 hrs after transduction, cells were visualized by phase contrast and fluorescence microscopy. Shown are representative fields for each condition at 100x magnification. Fluorescence intensity of individual T cells transduced with SIV_MAC239-GFP is at least as strong as that in cells transduced with HIV-1_NL4-3GFP. (B) VSV G-pseudotyped, HIV-1_NL4-3 (black squares) and SIV_MAC239 (white circles) three-part vectors were generated by plasmid transfection of 293T cells. In each case, the viral genomic RNA was designed to transduce an identical SFFV-GFP reporter gene. Vector stocks were normalized by titer on CRFK cells, and then used to challenge Jurkat T cells. 48 hrs post vector challenge, the percentage GFP-expressing cells was determined by FACS. Data is plotted as percent GFP⁺ (infected) cells (Y axis) versus CRFK infectious units (IU) x 1,000 (X axis).</p

Alignments of amino acid sequences of CA proteins encoded by selected HIV-2 isolates and SIV from the Los Alamos databases.

<p>Dots denote amino acid identity with the ROD CA; dashes denote gaps introduced to optimize alignment. HIV-2 CRF01_AB-specific amino acid residues are in red. Arrows indicate key residues at 119, 159, and 178, and the position (in the corresponding DNA sequence) of the <i>Hind</i>III restriction site used in the constructs. HIV-2A, HIV-2B, and HIV-2AB denote HIV-2 group A, HIV-2 group B, and HIV-2 CRF01_AB, respectively.</p

Structural models of the HIV-2 capsid C-terminal domain in dimeric form (A) and the HIV-2 GH123 capsid hexamer (B and C).

<p>(A) The C-terminal domain dimer model (from the amino acid position 150 to 219) of HIV-2 capsid (CA) is based on the viral sequence of NMC842. HIV-2 CRF01_AB-specific amino acid substitutions are shown in red. (B and C) The space-filling model of CA hexamer from the top (B) and side (C) is shown. Positions of HIV-2 CRF01_AB-specific amino acid substitutions are shown in red. L4/5 and 120P are shown in green and blue, respectively.</p

HIV-2 CRF01_AB CA C-terminal domain-specific sequence also affects viral sensitivity to human TRIM5α.

<p>(A) Schematic representation of chimeric viral CAs. Black and white bars show 842 and GH123/G CA peptide sequences, respectively. An arrow denotes the position (in the corresponding DNA sequence) of the <i>Hind</i>III restriction site used in the construct. (B and C) CEM-SS cells were infected with recombinant SeV encoding human (Hu: black squares) or CM SPRY(-) (white circles) TRIM5α. Nine hours after infection, cells were superinfected with 842 (B) and 842Hind (C). Culture supernatants were assayed for levels of p25 (CA). Error bars show actual fluctuations between levels of p25 (CA) in duplicate samples from one of three independent experiments.</p