1. Characterization and detection of artificial replication-competent lentivirus of altered host range 
Harry I Segall, Eunsun Yoo, Richard E Sutton 
Molecular Therapy 
Volume 8, Issue 1, Pages (July 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Spread of recombinant RCL. (A) Structure of RCLs, with parent HIV isolate NL4-3 at top. Delimited bars indicate deletions in provirus. (B) Spread of HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr in 293T cells using increasing amounts of virus, ‡ denotes>90% cell death. (C) Cell supernatant titers (eYFP+ cells) as a function of passage number, beginning with 10 IU of RCLs HIV-VSV G-IRES-eYFPΔEnv (circles) and HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr (diamonds). As expected, titers for replication-defective HIV-IRES-eYFPΔEnvΔVifΔVpr (VSV G) virus (squares) remained undetectable. (D) Single eYFP+ cluster of cells infected with HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr after a 1:20 split, with corresponding bright field. (E) Three eYFP+ clusters of cells transduced with replication-defective HIV-IRES-eYFP after a 1:20 split, with corresponding bright field. Note both the reduced number of positive cells and the diminished fluorescence compared to (D).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 1
Spread of recombinant RCL. (A) Structure of RCLs, with parent HIV isolate NL4-3 at top. Delimited bars indicate deletions in provirus. (B) Spread of HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr in 293T cells using increasing amounts of virus, ‡ denotes>90% cell death. (C) Cell supernatant titers (eYFP+ cells) as a function of passage number, beginning with 10 IU of RCLs HIV-VSV G-IRES-eYFPΔEnv (circles) and HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr (diamonds). As expected, titers for replication-defective HIV-IRES-eYFPΔEnvΔVifΔVpr (VSV G) virus (squares) remained undetectable. (D) Single eYFP+ cluster of cells infected with HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr after a 1:20 split, with corresponding bright field. (E) Three eYFP+ clusters of cells transduced with replication-defective HIV-IRES-eYFP after a 1:20 split, with corresponding bright field. Note both the reduced number of positive cells and the diminished fluorescence compared to (D).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 2
Quantitation of viral spread. (A) Mean cell fluorescence (MCF) ratios for the different viruses as a function of passage number: HIV-VSV G-IRES-eYFPΔEnv (circles), HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr (diamonds), HIV-IRES-eYFPΔEnvΔVifΔVpr (VSV G) (squares). MCFs were based upon positive and negative cell populations, measured by flow cytometry; ‡ denotes>90% cell death. (B) Higher cell density accelerates viral spread. Cells infected with 100 IU (dotted line) or 1000 IU (solid line) of RCL HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr were passaged at three different densities: 1/3, 1/10, and 1/30, and viral spread, as monitored by flow cytometry (%YFP-positive cells), is shown over time (days of passage). (C) Saquinavir blocks RCL spread. Cells (293T) were infected with 104 IU of HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr, and viral spread was measured by flow cytometry (%YFP-positive cells) in the absence (circles) and in the presence of 1.0 μM saquinavir (diamonds) or following removal of saquinavir from cultures after the first 3 days (squares). (D) Quantitation of culture supernatant p24 capsid as a function of passage number: HIV-VSV G-IRES-eYFPΔEnv (circles), HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr (diamonds), HIV-IRES-eYFP (VSV G) (squares); ‡ denotes>90% cell death. (E) Quantitation of proviral copy number (normalized to β-actin) by semiquantitative PCR: HIV-VSV G-IRES-eYFPΔEnv (circles), HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr (diamonds), HIV-IRES-eYFP (VSV G) (squares); ‡ denotes>90% cell death. (F) Spread of an RCL encoding Moloney 4070A amphotropic envelope (HIV-Ampho-IRES-eYFP-ΔEnvΔVifΔVpr), shown as the percentage YFP-positive cells over time in culture and the fluorescence index.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 3
Marker Alka-rescue assay. (A) Schematic of the assay. (B) Viral spread using indicated amounts of HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr, with sensitivity cut-off at 0.1% (as measured by flow cytometry). (C) Culture supernatant AP titer, using indicated amounts of HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr, with sensitivity cut-off of 100 IU/ml.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 4
Sensitivity of the marker Alka-rescue assay. (A) Comparison of detection sensitivity of marker Alka-rescue assay (AP-stained HOS cells; closed circles) with Tat-transactivation assay (X-gal-stained MAGI cells; squares), flow cytometry (YFP-positive cells; triangles), and p24 ELISA (open circles). The graph shows the measured viral titers in supernatant of Alka-rescue (293-G-P-AIB) cells plotted versus the calculated viral titers used for infection of these cells. (B) Detection of viral spread by flow cytometry. Alka-rescue cells (293-G-P-AIB) were infected in triplicate with increasing amounts of RCL (HIV-VSV G-IRES-eYFPΔEnvΔVifΔVpr) and passaged over time. At the indicated time points, the percentage of eYFP-positive cells was determined by flow cytometry. (C) Early detection of viral spread by marker Alka-rescue assay. Culture supernatants from the lowest viral dilution (0.05 IU) that produced a detectable spread by flow cytometry (circles) were used for p24 ELISA (squares) and to transduce naïve HOS cells, which were subsequently stained for AP activity (triangles).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions