Fig 2: HIV-1 Tat alters the processing of APP and increases the levels of β-CTF and Aβ42.(A) Level of β-CFT was increased in Lenti-Tat, or Lenti-mTat infected cells while the amount of total APP was not changed. U-87 MG cells were transduced with mock, Lenti-Tat, or Lenti-mTat virus and incubated for 14 days. The cell number was counted, and equal numbers of cells from each sample were analyzed in 16.5% Tris-Tricine gels to detect CTF or 8% SDS-PAGE for the detection of APP and β-actin. Both α- and β-CTF were detected with an anti-APP C-terminal antibody (A8717, top panel), and β-CTF was detected with 6E10 (upper middle panel). The total amounts of APP (22C11, lower middle panel) and β-actin (bottom panel) were not changed by the expression of Tat or mutant Tat protein. (B) Aβ42 level was increased in Lenti-Tat and Lenti-mTat infected U-87 MG cells. Average of Aβ42 concentration for 9 days was calculated from individual Aβ42 concentration. The Aβ42 concentrations in Lenti-Tat- or Lenti-mTat-infected cells were increased by 5.58±0.83 (mean ± SE, P<0.005)-fold or 3.64±0.38 (mean ± SE, P<0.005)-fold, respectively, compared with mock-infected cells. (C) Treatment with the neprilysin inhibitor thiorphan further increased the level of Aβ42 in the presence of Tat. Mock or Lenti-Tat-infected U-87 MG cells were cultured for 3 days in the absence or presence of 10 µM thiorphan. Culture supernatant was harvested from each cell culture and analyzed by Aβ42 ELISA. Error bars represent the mean ± SD. *P<0.05, **P<0.001.

Fig 3: HIV-1 Tat promotes localization of APP to lipid rafts.(A) The amounts of Triton X-100-resistant APP increased in Lenti-Tat infected U-87 MG cells. U-87 MG cells were transduced with mock virus or with Lenti-Tat virus, and incubated for 20 days. Cells were harvested and lysed in the presence of 1% Triton X-100 and the soluble and insoluble fractions were separated by centrifugation. WCE; whole cell extracts, Sol; soluble fraction, Insol; insoluble fraction. (B) U-87 MG cells were transduced with mock virus or Lenti-Tat virus and incubated for 14 days. Cells were harvested and lysed in the presence of 1% Triton X-100 and subjected to 5% and 35% discontinuous sucrose density gradient ultracentrifugation. Fractions of 0.5 ml were harvested from the top to the bottom and analyzed by Western blotting for APP, flotillin-1, transferrin receptor and Tat. The majority of APP was found in the non-raft fractions in mock –infected cells (Left panel) while large amount of APP was moved to lipid raft fractions in Lenti-Tat infected cells (Right panel).

Fig 4: HIV-1 Tat increases Aβ levels and the number and size of amyloid plaques in the APP/PS1 mouse hippocampus.Mock (A) or syn-Tat (B) was injected stereotaxically into APP/PS1 mouse hippocampi. At 2 months after injection, half of the brain was frozen-sectioned and stained with anti-Tat antibody, and then visualized under an inverted phase fluorescence microscope. Scale bar = 50 µm. (C) Mouse hippocampi (n = 9) injected with mock (syn) or Tat (syn-Tat) were dissected, homogenized 4 months after injection, and Aβ42 levels in the hippocampus extracts were examined by ELISA. The Aβ42 concentration for each mouse is shown on the y-axis, and the injected virus is indicated on the x-axis. (D) Equal volumes of syn-Tat and Lenti-Tat were mixed and injected unilaterally into the mouse hippocampus (n = 5). After 4 months, the hippocampus was dissected from mock (con)- and mixed Tat (Tat)-virus-injected hemispheres, and Aβ42 levels were investigated by ELISA. Aβ42 levels were 1.6-fold higher in the Tat-expressing hemisphere of mouse hippocampi (P<0.001). (E) Representative Western blots for the detection of Aβ. Twenty micrograms of mouse hippocampus extract were separated on 16.5% Tris-Tricine PAGE gels followed by Western blotting with anti-APP (6E10) antibody. Five micrograms of hippocampus extract were used for Western blotting for APP and actin. (F, G) Representative photographs of the Aβ plaque burden in APP/PS1 mice are shown. Brain sections of APP/PS1 mice injected with mock (F) or Lenti-Tat (G) virus were stained with anti-Aβ antibody (6E10). Scale bar = 50 µm.

Fig 5: HIV-1 Tat interacts with APP both in vitro and in vivo.(A) GST and GST-Tat were purified on glutathione-Sepharose beads. The beads were boiled to elute the bound proteins, which were then run on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue (left panel). GST pulldown assay with SK-N-MC neuroblastoma cell lysates shows a strong interaction between APP and GST-Tat (right panel). SK-N-MC neuroblastoma cell extracts incubated with GST- or GST-Tat-coated beads for 3 hours. The beads were washed three times with PBS and the eluted proteins were analyzed by western blotting with an anti-APP antibody (22C11). (B) Coimmunoprecipitation of Tat and APP in HEK 293FT cells transfected with Tat and/or Myc-tagged APP695 vectors. Proteins were precipitated with anti-Tat or anti-APP (6E10) antibodies and immunoblotted with anti-Tat or anti-Myc antibodies. (C) Coimmunoprecipitation of U-87 MG cell lysates transduced with mock, Lenti-Tat, or Lenti-mTat virus. APP was precipitated with an APP antibody (6E10), and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (22C11) or anti-Tat antibodies. Reciprocally, Tat was precipitated with anti-Tat antibody, and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (A8717) or anti-Tat antibody. (D) Purified recombinant APP interacts with GST-Tat. Purified recombinant APP (500 ng) was incubated with GST- or GST-Tat-coated beads and the eluted proteins were analyzed by western blotting with an APP antibody. A large amount of recombinant APP bound to the GST-Tat beads. (E) Tat interacts strongly with APP. SK-N-MC neurobalstoma cell lysates were incubated with GST, GST-Tat, or GST-Tat beads, and washed three times in buffer containing 137, 200, 300, 400 or 500 mM NaCl. APP remained associated with GST-Tat under high-salt conditions. (F) The cysteine-rich domain of Tat is important for association with APP. Deletion mutants were produced as GST-fusion proteins and subjected to GST-pulldown assays with SK-N-MC cell lysates. L, load; B; bound.