Design and Analysis of a Consensus HERV-K Provirus

<div><p>(A) Diagram of HERV-K(HML-2) provirus. ORFs are depicted as boxes. Proviruses used in the design of HERV-K_CON that contain intact versions of Gag, protease, Pol, and Env are listed under each ORF (*K108 encodes a full-length Pol ORF, but a presumed essential YIDD motif is mutated).</p> <p>(B) Phylogenetic analysis of HERV-K_CON and the ten proviruses used to generate it, constructed using Kimura 2-parameter algorithm in the TreeMaker program after gap-stripping the sequence alignment (<a href="http://www.hiv.lanl.gov/content/hiv-db/CONTAM/TreeMaker/TreeMaker.html" target="_blank">http://www.hiv.lanl.gov/content/hiv-db/CONTAM/TreeMaker/TreeMaker.html</a>).</p> <p>(C) Comparison of HERV-K_CON with the ten HERV-K proviruses. Each contributing provirus was compared to HERV-K_CON using HYPERMUT, and the number of nucleotide differences for each provirus relative to HERV-K_CON is plotted.</p></div

HERV-K_CON Tropism

<div><p>(A) Human, squirrel monkey, feline, hamster, or murine cells were infected with VSV-G pseudotyped HERV-K_CON particles. Two days postinfection, GFP⁺ foci were quantified microscopically, and titers are expressed as number of infectious units (i.u.) per milliliter of virus-containing supernatant applied.</p> <p>(B) Human, squirrel monkey, feline, or murine cells were infected with HERV-K_CON Env pseudotyped HIV-1 particles as in (A). Two days postinfection, GFP-positive foci were quantified.</p> <p>All data are representative of at least three experiments.</p></div

Assembly, Processing, and Release of HERV-K Virus-Like Particles

<div><p>(A–D) 293T cells were transfected with Gag-, Gag-PR–, or Gag-PR-Pol–expressing vectors.</p> <p>(A) Western blot analysis of cell lysates (left) and virions (center and right) using a commercially available antibody to HERV-K Gag. Center shows VLPs from 293T cells transfected with a plasmid-expressing Gag, and right shows VLPs from Gag-PR– and Gag-PR-Pol–expressing 293T cells. Decreasing amounts of virion lysate (0.1, 0.05, or 0.025 μl for Gag; 0.4, 0.2, or 0.1 μl for Gag-PR and Gag-PR-Pol) were loaded to semiquantitatively estimate relative levels of VLP production.</p> <p>(B) Silver stain analysis of a 4% to 20% gradient SDS-PAGE gel loaded with VLPs harvested from 293T cells transfected with plasmids expressing Gag, Gag-PR, Gag-PR-Pol, or empty plasmid control. An asterisk marks a nonspecific 66-kDa protein band, most probably BSA, that is abundant in the culture medium.</p> <p>(C) Silver stain analysis of VLPs harvested from 293T cells containing Gag, Gag-PR, Gag-PR-Pol, or Gag-PR(mut) encoding an active site mutation (DTG-AAA) in protease. An asterisk marks a nonspecific 66-kDa protein band, most probably BSA, that is abundant in the culture medium.</p> <p>(D) Reverse transcriptase activity in culture supernatants of 293T cells transfected with empty pCRV1 (vector) or vectors expressing HERV-K_CON Gag, Gag-PR, or Gag-PR-Pol proteins, as indicated. Enzymatic activity was determined relative to a recombinant HIV-1 reverse transcriptase standard and is representative of three experiments. Supernatants from 293T cells transfected with an HIV-1–based proviral plasmid are included for comparison.</p> <p>(E) Two representative 293T cells transfected with HERV-K_CON Gag and Gag-GFP expression plasmids. Cells were fixed 18 h post-transfection, and nuclei were stained with DAPI (blue) prior to visualization by deconvolution microscopy. Top, Images acquired at the mid-section of the cell to show localization of Gag-GFP proteins; bottom, focused on the bottom of the cell to show accumulated VLPs at the cell–coverslip interface.</p> <p>(F) Gallery of electron micrographs of 293T cells transfected with a Gag-PR–expressing plasmid. Black scale bars in the upper and middle panels represent 500 nm, while scale bars in the lower two panels represent 100 nm.</p></div

Effects of TRIM5 and APOBEC3 Proteins on HERV-K_CON Infectivity

<div><p>(A) Unmanipulated CHO cells or variants stably expressing human TRIM5α, rhesus monkey TRIM5α, or owl monkey TRIM-Cyp were infected with VSV-G pseudotyped retroviral vectors that are sensitive to one or more of the TRIM5 proteins (N-MLV or HIV-1) or TRIM5-resistant controls (B-MLV or HIV-1 carrying SIVmac CA HIV(SCA)), as indicated. Two days postinfection, the percentage of GFP⁺ cells was determined using FACS.</p> <p>(B) The same panel of CHO-derived TRIM5-expressing CHO cell lines were inoculated with HERV-K_CON(VSV-G). Two days postinfection, GFP⁺ foci were quantified.</p> <p>(C) APOBEC3F and APOBEC3G expression plasmids were cotransfected into 293T cells during generation of CHKCG-containing HERV-K_CON(VSV-G) particles. Fresh 293T cells were infected with the resulting viral supernatant, and GFP⁺ foci were quantified 2 d later.</p> <p>(D) Western blot analysis, using the anti–HERV-K Gag antibody, of cell and HERV-K_CON virion lysates generated upon coexpression of APOBEC3F or APOBEC3G, as indicated.</p> <p>All data are representative of at least three experiments.</p></div

Generation of Single Cycle Infectious Virions Containing HERV-K_CON Genomes and Gag-PR-Pol

<div><p>(A) Schematic representation of the HERV-K_CON–based CHKCG genome. Changes to HERV-K_CON are depicted in white boxes. The 5′ LTR was modified to include the CMV promoter inserted in place of the U3 region. The Env ORF, which now contains CMV-GFP, is disrupted.</p> <p>(B and C) Photomicrographs of 293T cell monolayers after infection with CHKCG-carrying HERV-K_CON(VSV-G) pseudotyped virions, which were generated following transfection of 293T cells with the indicated plasmid mixtures.</p> <p>(D and E) Infectious titers of HERV-K_CON(VSV-G) pseudotyped virions generated following transfection with the indicated plasmid mixtures in the absence (D) or presence (E) of K-Rev/Rec and using 293T target cells. GFP-positive foci were enumerated visually and expressed as infectious units per milliliter of virion-containing supernatant.</p> <p>(F) Infectious titers of CHKCG containing HERV-K_CON(VSV-G) using 293T target cells in the presence or absence of 50 μM AZT.</p> <p>All data are representative of at least three experiments.</p></div

Transduction Mediated by HERV-K_CON Gag-PR-Pol and Genomes Results in Stable Proviral Integration

<div><p>(A) Puromycin-resistant colonies of 293T cells infected with either VSV-G pseudotyped (left) or Env defective (right) virions carrying the CHKCP genome. Infected 293T cells were selected in 0.5 μg/ml puromycin for 2 wk and then fixed and stained to reveal colonies of viable cells. Data are representative of at least three experiments.</p> <p>(B) Experimental strategy for detection of HERV-K_CON proviruses in CHO745 cells using PCR primers targeted to HERV-K_CON Gag and LTR sequences, or flanking hamster DNA sequences.</p> <p>(C) PCR amplification of HERV-K_CON<i>gag</i> DNA using Gag-S and Gag-AS primers in four expanded clones of puromycin-resistant CHO745 cells transduced with CHKCP-containing HERV-K_CON(VSV-G) particles.</p> <p>(D) Nucleotide sequences at the 5′ and 3′ ends of integrated CHKCP proviral DNA, revealing six nucleotide duplicated sequences at the CHKCP integration sites.</p> <p>(E) Verification of the presence and absence of an integrated provirus and the empty preintegration site in CHKCP-transduced and naïve CHO745 cells using combinations of HERV-K and hamster DNA targeted PCR primers (see [B] for primer design strategy). DNA templates and PCR primer pairs used are indicated above each lane, and the expected PCR product size is given below each lane. A representative analysis of a single CHKCG-carrying CHO745 cell clone is shown; similar results were obtained with two additional clones. Uninfected CHO745 cells and human 293T cells serve as controls.</p></div

Intracellular HIV-1 Gag-CFP Partly Co-localizes with both Early and Late Endosomal Markers in HeLa Cells

<div><p>(A) HeLa cells expressing HIV-1 Gag-CFP (green) were fixed 18 h post-transfection and stained with a monoclonal antibody specific for human CD63, and a secondary anti-mouse Alexa-Fluor-588 conjugate (red). Nuclei were counter-stained with DAPI (blue).</p><p>(B) HeLa cells expressing HIV-1 Gag-CFP (green) were co-transfected with CherryFP-Rab5a (red), a marker for early endosomal structures. Cells were fixed and images acquired at 18 h post-transfection. The lower set of images presented in (B) are shown at approximately 5-fold higher magnification to give a clearer indication of the juxtaposition of the HIV-1 Gag-CFP and CherryFP-Rab5a signals. White bars in the micrographs indicate a distance of 10 μm, except in the lower set of images in (B), where the bar indicates 2 μm</p></div

Cell Type–Dependent Effects of Vpu on Retroviral Particle Release

<div><p>Human HeLa and HOS cells, together with AGM Vero cells, were transfected with HIV-1 NL4.3 and NL4.3delVpu proviral plasmids, or an MLV Gag-Pol–expression vector, as indicated, in the presence of plasmids expressing either no Vpu (none), codon-optimized Vpu, or an inactive Vpu mutant containing a CD8 TM domain (V8).</p><p>(A and B) Western blot analyses of cell and virion lysates, probed using antibodies to HIV-1 (A) or MLV CA (B).</p><p>(C and D) Results of chemiluminsecent β-galactosidase assays following inoculation of HeLa-TZM indicator cells with supernatants derived from HeLa, HOS, or Vero cells transfected with NL4.3 (white bars) or NL4.3delVpu (black bars) proviral plasmids (C) or plasmids expressing MLV Gag-Pol, a packageable HIV-1 Tat–expression vector, and VSV-G (D). Vpu and control (GFP)–expression plasmids were included, as indicated, and results plotted as relative light units (RLU) ± standard deviation of the mean.</p></div

Immunofluorescent Localization of HIV-1 Gag Expressed from a Proviral Construct and Effects of Vpu and Endocytosis Inhibitors

<div><p>(A) HeLa cells (left and center panels) or HOS cells (right panel) were transfected with NL4.3 or NL4.3delVpu proviral plasmids, as indicated. Gag localization was determined by immunofluorescence using an α-CA antibody as described in Materials and Methods.</p><p>(B) HeLa cells expressing Gag from the NL4.3delVpu proviral plasmid, and either DN (S34N, upper panels) or constitutively active (Q79L center and lower panels) mutants of CherryFP-Rab5a (red), were fixed and subjected to immunofluorescence to detect HIV-1 Gag (green) at 20 h post-transfection. White bars in the micrographs indicate a distance of 10 μm. The lower three images in (B) show expanded views of a portion of the overlay panels (indicated by the dashed squares) to exemplify how HIV-1 Gag and the CherryRab5a proteins are juxtaposed, Note that Gag sometimes appears to be within the lumen of the CherryFP-Rab5a(Q79L)–marked endosomes.</p><p>(C) The numbers of HIV-1 Gag–expressing cells in ten fields exhibiting PM Gag immunofluorescence only (black bars) or additional intracellular accumulations (white bars) after transfection with NL4.3 or NL4.3delVpu proviral plasmids in the presence of the indicated co-expressed cellular proteins was counted, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020039#ppat-0020039-g002" target="_blank">Figure 2</a>C. Data are plotted as a percentage of the total number of cells with visible Gag accumulations.</p></div

Endosomal Accumulation of HIV-1 and MLV Gag-CFP Requires a Functional L-Domain

<p>HeLa cells were transfected with plasmids expressing HIV-1 (A) or MLV Gag-CFP (B), or mutant variants thereof lacking functional L-domains (HIV-1 Gag dLD-CFP and MLV Gag dPY-CFP, respectively) in the presence or absence of co-expressed Vpu, as indicated. The cells were fixed at 18 h post-transfection and images acquired. Representative examples are shown in panels (A) and (B) and a quantitative assessment of Gag-CFP localization, enumerated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020039#ppat-0020039-g001" target="_blank">Figure 1</a>, is shown (C). The numbers of HIV-1 Gag-CFP–expressing cells in ten fields exhibiting Gag-CFP accumulation only at the PM (black bars) or at intracellular sites (white bars) were counted, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020039#ppat-0020039-g002" target="_blank">Figure 2</a>C, and are plotted as a percentage of the total number of cells with Gag-CFP accumulations.</p