Evidence for a dominant-acting, capsid-specific, restriction activity in Jurkat T cells.

<p>(A) Jurkat and HeLa cells stably expressing the ALV-A receptor (TvA) or TagRFP-657, as indicated, were fused by treatment with PEG and transduced with ALV-A Env-pseudotyped HIV-1_NL4-3-GFP, or with isogenic vector bearing the SIV_MAC239 CA^1-202. Shown are flow cytometry dot plots obtained 48 hrs post-transduction. HeLa-TagRFP-657 cells are only permissive to infection with ALV-A Env-pseudotyped vectors after fusion with Jurkat-TvA. Infected heterokaryons were visualized as GFP and TagRFP-657 double-positive cells. As a positive transduction control, TagRFP-657 and TvA were also co-expressed in HeLa cells, as indicated. The percentage of transduced cells are indicated. (B) Bar graph showing the infectivity of the SIV_MAC239 CA^1-202-bearing vector relative to the isogenic vector bearing HIV-1 CA, for the HeLa, Jurkat and heterokaryons. Data from the flow cytometry data shown in A, and two repeat experiments, is shown with the standard deviation.</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

The capsid of SIV_MAC, HIV-2, or SIV_SM is sufficient to decrease HIV-1 transduction efficiency in a T cell-specific manner.

<p>(A) Chimeric vectors were generated in which the coding sequence for HIV-1 CA amino acid residues 1 to 202 of the two-part HIV-1_NL4-3GFP vector (white squares) was replaced with sequence encoding the corresponding amino acid residues from HIV-2_ROD (grey triangles) or SIV_MAC239 (black circles). VSV G-pseudotyped vector was generated for each by transfection of 293T cells. Stocks were normalized by RT and used to challenge CRFK cells (B) or HeLa cells (C). Stocks were then normalized for CRFK transduction activity and used to challenge Jurkat T cells (D). 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as percent GFP⁺ (infected) cells (Y axis) versus RT activity (B and C), or versus CRFK infectious units (IU) x 1000 (X axis). (E) Chimeric vectors were generated in which the coding sequence for HIV-1 CA amino acid residues 1 to 202 of the HIV-1 <i>gag-pol</i> expression vector (white) was replaced with sequence encoding the corresponding amino acid residues from various HIV-2 isolates (grey) or SIV_SME041 (black). Three-part, VSV G-pseudotyped, SFFV-GFP bearing vectors were generated for each CA chimera by transfection of 293T cells. Stocks were then normalized for CRFK transduction activity and used to challenge Jurkat T cells. 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as CRFK normalized transduction of Jurkat cells, relative to the parental HIV-1 vector (F). Accession numbers for the different CA coding sequences are as follows: HIV-2(AB), 731744; HIV-2(A), GH123; HIV-2(D), L33083; HIV-2(E), L33087; HIV-2(F), U75441; HIV-2(H), AY5308; SIV_SME041, HM059825.</p

As₂O₃ specifically increases SIV_MAC infectivity in human blood cells.

<p>TE671 cells (A), Jurkat T cells (B), human PBMC (C), or human CD4⁺ T cells (D) were transduced with two-part, VSV G-pseudotyped HIV-1_<b>NL4-3</b>-GFP or SIV_<b>MAC</b>GFP vectors using a predetermined quantity of virus such that 1% of cells were infected. As_<b>2</b>O_<b>3</b> was added 1 hr prior to vector challenge and maintained for 12 hrs post-infection, at the concentrations indicated on the X axis. 48 hrs post-challenge the percentage of GFP-expressing cells was determined. The Y axis shows the fold increase relative to infection without As_<b>2</b>O_<b>3</b>.</p

The block to SIV_MAC infection of Jurkat T cells occurs after formation of 2-LTR circles.

<p>CRFK and Jurkat (A), or Hela and Jurkat (B), or PBMCs (C) were infected with VSV G-pseudotyped HIV-1_NL4-3-GFP, or with isogenic vector bearing the SIV_MAC239 CA residues 1 to 202. 24 hrs post-infection, DNA was collected from the cells and subjected to qPCR using primers specific for full-length linear viral cDNA, 2-LTR circles, or proviral DNA, as indicated. Shown is the abundance of signal from vector bearing the SIV_MAC239 CA^1-202, relative to the amount of signal from HIV-1_NL4-3-GFP. In each case, infection was performed in the presence of an RT inhibitor to control for background levels of signal.</p

The transduction defect associated with SIV_MAC CA is independent of the virus entry pathway.

<p>A two-part, <i>env</i>-minus HIV-1 vector with GFP in place of nef (black squares), or an isogenic vector in which CA^1-202 coding sequences were replaced with those from SIV_MAC239 (white circles), were produced by 293T transfection. Each vector was pseudotyped with Env glycoprotein from either HIV-1_HXB2(A), HIV-2_MCN (B), ecotropic MLV (C), or ALV-A (D) and transduction efficiency was measured on HeLa cells bearing human CD4 (A and B), the mCAT1 ecotropic receptor (C), or the avian TVA receptor (D), and then used to challenge Jurkat cells bearing the same receptors. 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS.</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

SIV_MAC, HIV-2, and SIV_SM transduction of human T cells is less efficient than transduction by HIV-1.

<p>(A) Transduction efficiency of VSV G-pseudotyped two-part vectors for HIV-1_NL4-3GFP (white squares), HIV-2_RODGFP (grey triangles), or SIV_MAC239GFP (black circles) on Jurkat T cells. (B) Chimeric vectors were generated in which <i>gag-pol</i> of SIV_MAC239GFP (white squares) was replaced with <i>gag-pol</i> from SIV_SME543 (grey triangles) or SIV_SM041 (black circles). In each case (A and B), VSV G-pseudotyped vectors were generated by plasmid transfection of 293T cells. Vector stocks were normalized by titer on CRFK cells, and then used to challenge Jurkat T cells. 48 hrs 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 1000 (X axis).</p