Similar replication kinetics of HXB/LW and NL4–3 in SCID-hu Thy/Liv mouse and HF-TOC model systems. (A) Replication of HXB/LW and NL4–3 in SCID-hu Thy/Liv mice. Levels of p24 capsid protein associated with 10⁶ cells are shown on the y axis. Each column represents the average values for each virus (numbers in parentheses are numbers of animals), along with standard error bars. (B) Replication of HXB/LW and NL4–3 in HF-TOC. Thymus fragments were infected with equivalent infectious units, and thymocytes were harvested at various times postinfection. Levels of p24 capsid protein associated with 10⁶ cells are shown on the y axis. Data represent the average values at the indicated times (numbers in parentheses are numbers of donors), along with standard error bars. (C) Quantitation of proviral DNA. Human thymocytes from SCID-hu Thy/Liv mice infected with NL4–3 (two SCID-hu Thy/Liv mice; panels NLa and NLb), HXB/LW (three SCID-hu Thy/Liv mice; panels LWa to LWc), or HXB2 (panel HB) were assayed by 10-fold dilution of infected cells into uninfected human cells. Lanes 1, 10,000 sample cells; lanes 2, 1,000 sample cells plus 9,000 normal human cells; lanes 3, 100 sample cells plus 9,900 normal human cells. ACH2 cells (one HIV-1 genome per cell) were used as standard controls (lane 4, 10 ACH2 cells plus 9,990 normal human cells; lane 5, 1 ACH2 cell plus 9,999 normal human cells; lane 6, 10,000 normal human cells). β-Globin primers were used as internal controls.

Repaired nef gene did not alter HXB/LW replication kinetics. (A) The expression of nef in A293T cells transfected with proviruses encoding HXB2, HXB/LWn−, and HXB/LWn+ was detected by Western blot analysis. The same blot was probed with an antitubulin MAb to monitor total protein levels. (B) The expression of nef in H9 T cells infected with HXB2, HXB/LWn−, and HXB/LWn+ was detected by Western blot analysis. NL4–3-infected H9 cells were included as positive controls. The same blot was probed with an antitubulin MAb to monitor total protein levels. (C) Similar levels of HXB/LWn+, HXB/LWn−, and NL4–3 replication in SCID-hu Thy/Liv mice. SCID-hu Thy/Liv organs were infected with equivalent infectious units, and thymocytes were harvested at various times. Levels of p24 capsid protein associated with 10⁶ cells are shown on the y axis. Each column represents the average of three independent experiments (numbers in parentheses are numbers of animals), along with calculated standard error bars. (D) Similar levels of HXB/LWn+, HXB/LWn−, and NL4–3 replication in HF-TOC. Fetal thymus fragments were infected with equivalent infectious units, and thymocytes were harvested at various times. Levels of p24 capsid protein associated with 10⁶ cells are shown on the y axis. Values at each time point represent averages from four independent experiments (numbers in parentheses are numbers of donors), along with calculated standard error bars.

Relative pathogenicity of HXB/LWn+, HXB/LWn−, and NL4–3 correlated to the presence of an intact nef ORF. (A) Pathogenic plot for SCID-hu Thy/Liv mice. p24 capsid protein levels (x axis) were plotted against the total percentage of CD4⁺ thymocytes relative to that in mock infections (y axis) for the time points from 2 to 6 weeks postinfection from the experiments shown in Fig. 3C. The NL4–3 standard regression curve was generated from 32 SCID-hu Thy/Liv mice infected with the NL4–3 virus from 10 different experiments (R² = 0.17). Numbers of mice were as follows: HXB/LWn−, n = 16 (●); HXB/LWn+, n = 18 (□); NL4–3, n = 14 (▵); and NL4–3 standard, n = 32. (B) Pathogenicity plot for HF-TOC. p24 capsid protein levels (x axis) were plotted against the total percentage of CD4⁺ thymocytes (y axis) for all time points from the HF-TOC experiments shown in Fig. 3D. The NL4–3 standard regression curve was generated from 100 HF-TOC assays of 26 different donor thymus tissues infected with the NL4–3 virus (R² = 0.79). Numbers of tissues were as follows: HXB/LWn−, n = 9 (●); HXB/LWn+, n = 8 (□); and NL4–3 standard, n = 100.

Reduced pathogenicity of HXB/LW in human thymus. (A) Replication and pathogenic activities of NL4–3, HXB2, and HXB2/LW in SCID-hu Thy/Liv mice. SCID-hu Thy/Liv mice were injected with equivalent infectious units of each virus, and thymocytes were analyzed at 4 weeks postinfection. Levels of cell-associated p24 antigen in 10⁶ thymocytes were measured. (B) Comparison of CD4⁺ thymocyte depletion in SCID-hu Thy/Liv mice infected with HXB/LW or NL4–3. Thymocytes from the infected SCID-hu Thy/Liv organs shown in Fig. 1A were analyzed by FACS for CD4 and CD8. The total percentages of CD4⁺ thymocytes (CD4⁺ CD8⁻ and CD4⁺ CD8⁺) are shown on the y axis. The percentage of total CD4⁺ cells in mock-infected animals at each time point in each experiment was set to 100%. The results shown are the average values, along with standard error bars. (C) Immunohistochemical staining of NL4–3- and HXB/LW-infected SCID-hu Thy/Liv organs. Frozen sections of NL4–3-infected (upper panels) and HXB/LW-infected (lower panels) SCID-hu Thy/Liv organs (6 weeks postinfection [wpi]) were stained with human anti-HIV serum and hematoxylin-eosin to detect HIV antigens and thymocytes, respectively. Magnifications: ×10 for left panels and ×60 for right panels. (D) Comparison of CD4⁺ thymocyte depletion in HF-TOC infected with HXB/LW or NL4–3. Thymocytes isolated from the infected HF-TOC fragments shown in Fig. 1B were analyzed by FACS for CD4 and CD8. The total percentages of CD4⁺ thymocytes (CD4⁺ CD8⁻ and CD4⁺ CD8⁺) are shown on the y axis. The percentage of total CD4⁺ cells in mock-infected tissues at each time point in each experiment was set to 100%. The results shown are the average values, along with standard error bars.

Repaired nef gene enhanced pathogenesis in HXB/LW-infected thymus. (A) Similar levels of thymocyte depletion in HXB/LWn+- and NL4–3-infected SCID-hu Thy/Liv mice. SCID-hu Thy/Liv mice were injected with equivalent infectious units of virus, and thymocytes were harvested at 4 weeks postinfection and analyzed by FACS. The thymocyte subpopulations were expressed as a percentage of the events in the live gate. The levels of p24 capsid protein associated with 10⁶ thymocytes in each SCID-hu Thy/Liv mouse were as follows: LWn+, 490 ng; LWn− (HXB/LW), 670 ng; and NL4–3, 67 ng. (B) Pathogenesis kinetics in SCID-hu Thy/Liv mice infected with HXB/LWn+, HXB/LWn−, and NL4–3. Thymocytes from the infected SCID-hu Thy/Liv organs shown in Fig. 3C were analyzed by FACS at the times shown on the x axis. The total percentage of CD4⁺ thymocytes relative to that in mock infections is shown on the y axis. The percentage of total CD4⁺ cells in mock-infected animals at each time in each experiment was set to 100%. The results shown are the average values, along with standard error bars. (C) Pathogenesis kinetics in HF-TOC infected with HXB/LWn+, HXB/LWn−, and NL4–3. Thymocytes from the infected HF-TOC fragments shown in Fig. 3D were analyzed by FACS at the times shown on the x axis. The total percentage of CD4⁺ thymocytes relative to that in mock infections is shown on the y axis. The percentage of total CD4⁺ cells in mock-infected animals at each time in each experiment was set to 100%. The results shown are the average values, along with standard error bars.

Model of in vivo reversion of HIV-1 attenuated in vitro. Tissue culture-attenuated viruses carry mutations in multiple genetic determinants of replication and pathogenicity, rendering them unable to replicate efficiently in vivo. After a short passage in vivo, reversions of a replication determinant(s) generate replication-competent isolates that remain attenuated for a pathogenic determinant(s). After further passage in vivo, reversion of a pathogenic determinant(s) results in a pathogenic virus.