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. 2011 Oct;85(20):10627-38.
doi: 10.1128/JVI.00757-11. Epub 2011 Aug 17.

Inhibition of retromer activity by herpesvirus saimiri tip leads to CD4 downregulation and efficient T cell transformation

Dior Kingston  1 , Heesoon Chang, Armin Ensser, Hye-Ra Lee, Jongsoo Lee, Sun-Hwa Lee, Jae Ung Jung, Nam-Hyuk Cho
Affiliations

Inhibition of retromer activity by herpesvirus saimiri tip leads to CD4 downregulation and efficient T cell transformation

Dior Kingston et al. J Virol. 2011 Oct.

Abstract

The mammalian retromer is an evolutionally conserved protein complex composed of a vacuolar protein sorting trimer (Vps 26/29/35) that participates in cargo recognition and a sorting nexin (SNX) dimer that binds to endosomal membranes. The retromer plays an important role in efficient retrograde transport for endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR), a receptor for lysosomal hydrolases, and other endosomal proteins. This ultimately contributes to the control of cell growth, cell adhesion, and cell migration. The herpesvirus saimiri (HVS) tyrosine kinase-interacting protein (Tip), required for the immortalization of primary T lymphocytes, targets cellular signaling molecules, including Lck tyrosine kinases and the p80 endosomal trafficking protein. Despite the pronounced effects of HVS Tip on T cell signal transduction, the details of its activity on T cell immortalization remain elusive. Here, we report that the amino-terminal conserved, glutamate-rich sequence of Tip specifically interacts with the retromer subunit Vps35 and that this interaction not only causes the redistribution of Vps35 from the early endosome to the lysosome but also drastically inhibits retromer activity, as measured by decreased levels of CI-MPR and lower activities of cellular lysosomal hydrolases. Physiologically, the inhibition of intracellular retromer activity by Tip is ultimately linked to the downregulation of CD4 surface expression and to the efficient in vitro immortalization of primary human T cells to interleukin-2 (IL-2)-independent permanent growth. Therefore, HVS Tip uniquely targets the retromer complex to impair the intracellular trafficking functions of infected cells, ultimately contributing to efficient T cell transformation.

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Figures

Fig. 1.
Fig. 1.
Vps35 interacts with the amino-terminal glutamic acid-rich domain of Tip. (A) Amino acid sequence alignment of the first 24 amino acids of Tip isolates of various HVS subgroup C strains. Glutamic acid residues that were changed to glutamine in the Tip E/Q mutant are boxed. (B) Silver-stained SDS-PAGE from protein purification of GST and TipN42-GST. The 90-kDa band was identified as Vps35. (C) AU1-tagged Tip was immunoprecipitated (IP) from transfected 293T cells expressing myc-tagged Vps35, Xpress-tagged p80, and Lck, followed by Western blot analysis for interacting proteins. Numbers for whole-cell lysates (WCL) and IP correspond to the same conditions. Lane 1, vector; lane 2, wt Tip; lane 3, Tip Δ24 mutant; lane 4, Tip Δ2 mutant; and lane 5, Tip mLBD. (D) Immunoprecipitation of myc-tagged Vps35 in the presence of AU1-Tip (lanes 1 and 2) or AU1-Tip E/Q (lanes 3 and 4) from transfected 293T cells. Numbers for WCL and IP correspond to the same conditions. Control lanes 1 and 3 were not transfected with Vps35 (denoted by minus signs).
Fig. 2.
Fig. 2.
Tip binds to amino acids 1 to 120 and 500 to 650 of Vps35. (A) Schematic diagram of Flag-tagged Vps35 deletion mutants used in binding assays. The plus and minus signs indicate binding and no binding, respectively, and the lines below the figures indicate the Tip-binding domains of Vps35. (B) Retromer binding activity of Tip mutants. At 48 h posttransfection with AU1-Tip wt and Flag-Vps35 wt or its deletion mutants, 293T cells were used for immunoprecipitation with anti-AU1 antibody, followed by Western blot analysis with anti-AU1 (Tip) or anti-Flag (Vps35) antibody. Lanes 1 to 9 express wt Tip, while lane 10 expresses only the vector. Vps35 construct expression was as follows: lanes 1 and 10, aa 1 to 796; lane 2, aa 1 to 500; lane 3, aa 1 to 307; lane 4, aa 1 to 230; lane 5, aa 1 to 120; lane 6, aa 120 to 307; lane 7, aa 120 to 796; lane 8, aa 500 to 796; and lane 9, aa 650 to 796.
Fig. 3.
Fig. 3.
Binding domains of Vps26 and SNX1 on Vps35. Vps35-binding activity of Vps26 (A) and SNX1 (B). At 48 h posttransfection with vectors expressing Vps26 or SNX1 and Flag-Vps35 wt or its deletion mutants, transfected 293T cell lysates were used for immunoprecipitation with anti-Vps26 or anti-SNX1 antibody, followed by Western blot analysis with anti-Vps26 or anti-SNX1 and anti-Flag (Vps35) antibodies. (A) Lanes 1 to 9 express Vps26, while lane 10 expresses only the vector. Vps35 construct expression was as follows: lanes 1 and 10, aa 1 to 796; lane 2, aa 1 to 500; lane 3, aa 1 to 307; lane 4, aa 1 to 230; lane 5, aa 1 to 120; lane 6, aa 120 to 307; lane 7, aa 120 to 796; lane 8, aa 500 to 796; and lane 9, aa 650 to 796. (B) Lane 1 expresses only the vector, while lanes 2 to 10 express SNX1. Vps35 construct expression was as follows: lanes 1 and 10, aa 1 to 796; lane 2, aa 1 to 500; lane 3, aa 1 to 307; lane 4, aa 1 to 230; lane 5, aa 1 to 120; lane 6, aa 120 to 307; lane 7, aa 120 to 796; lane 8, aa 500 to 796; and lane 9, aa 650 to 796.
Fig. 4.
Fig. 4.
Vps35 redistributes to lysosomal compartments in the presence of Tip. (A) Immunofluorescence confocal microscopy of Jurkat T cells expressing Tip or its mutants (blue) and Vps35 (green). (B) Jurkat T cells transiently expressing Vps35 (red) costained with the early endosomal marker, EEA1 (green; top), or with the lysosomal marker, Lamp1 (green; bottom). (C and D) Triple staining of Jurkat T cells for Vps35 (green), EEA1 or Lamp2 (red), and wt Tip or the Tip E/Q mutant (blue). Zoomed images of merged images from the stained cell are shown in the far right column. White arrows indicate the areas of colocalization.
Fig. 5.
Fig. 5.
Tip induces the reduction of CI-MPR levels and β-glucuronidase activity but not retromer subunit levels. Jurkat T cells stably expressing Tip and its mutants were used to determine retromer activity as assayed by CI-MPR protein levels and CI-MPR-dependent hydrolase activity. (A) Immunofluorescence confocal microscopy of endogenous CI-MPR in Jurkat stable T cell lines expressing Babe vector, Tip, or the Tip Δ24 or Tip E/Q mutant. (B) Western blot analysis of CI-MPR and β-tubulin in Jurkat stable T cells lines expressing Babe vector, Tip, Tip Δ24 mutant, Tip mSH3B, or Tip E/Q. (C) Equal amounts of protein from stably transfected Jurkat T whole-cell lysates were used to determine the activity of β-glucuronidase and acid phosphatase. Results are expressed as percent activity compared to Jurkat/Babe vector cells with means plus SD from 2 to 8 independent experiments. (D) Western blot analysis of whole-cell lysates of Jurkat stable T cell lines expressing Babe vector, Tip, or Tip Δ24 or Tip E/Q mutant, probing for steady-state levels of the endogenous retromer subunits Vps35, SNX1, Vps26, and Vps29.
Fig. 6.
Fig. 6.
Tip does not interfere with the assembly of the retromer complex. (A) Vps35-Vps26-SNX1 complex was pulled down with the N terminus of Tip. 293T cells were transfected with vectors expressing GST or TipN42-GST. GST pulldowns were performed 48 h posttransfection, followed by Western blot analysis with antibodies against Vps35, SNX1, and Vps26. (B) Vps26 and Vps29 were coimmunoprecipitated with Vps35 in the presence of Tip. Endogenous Vps26 or vimentin (control) was immunoprecipitated from cell lysates of stably transfected Jurkat T cells expressing the Babe vector, wt Tip, or the Tip Δ24 mutant, followed by Western blot analysis using antibodies against Vps35, Vps26, and Vps29. (C) SNX1 colocalized with Vps35 in the presence of wt Tip. Jurkat T cells were electroporated with vectors expressing SNX1 and Vps35 alone or with wt Tip-AU1 or Tip E/Q-AU1. Cells were prepared for immunofluorescence and confocal microscopy 48 h postelectroporation. SNX1 (green), Vps35 (red), wt Tip or Tip E/Q (blue).
Fig. 7.
Fig. 7.
Downregulation of CD4 is dependent on Tip interaction with Vps35. (A) Multicolor flow cytometric analysis of Jurkat stable T cell lines expressing vector, wt Tip, or Tip Δ24 or Tip E/Q mutant with antibodies against TCR (top), CD4 (middle), and CD45 (bottom). (B) Western blot analysis of whole-cell lysates from the indicated Jurkat stable cell lines, probing for CD4 and β-tubulin (as a loading control). (C) Quantitative real-time PCR analysis of CD4 mRNAs. For the relative quantification of CD4 mRNAs, 18S rRNA was used for normalization. (D) Confocal microscopy of Jurkat T cells transiently expressing CD4 (red) and vector (top), wt Tip (green; middle), or the Tip Δ24 mutant (green; bottom). (E) Recovery of CD4 surface expression after trypsin treatment. Jurkat stable T cell lines expressing vector, wt Tip, or Tip Δ2, Tip Δ24, or Tip E/Q mutant were treated with trypsin to remove cell surface-associated CD4, washed, and incubated at 37°C with RPMI 1640. Cells were harvested at the indicated time points and stained with anti-CD4 antibody, followed by flow cytometric analysis.
Fig. 8.
Fig. 8.
Tip Vps35-binding mutant viruses can transform T cells in the absence of IL-2. (A) Schematic diagram of cosmid-based recombinant virus generation and PCR analysis of recombinant viruses used in transformation assays. HVS wt C488 (parental virus) was used as a positive control. Recombinant viruses were generated to encode wt Tip or Tip 3Δ24 or Tip E/Q mutant. PCR amplification of cosmid overlapping regions (ORF3, ORF75, and StpC/Tip) were recombined to produce full-length virus. (B) Micrographs of aggregated, transformed T cells in cultures lacking IL-2. Recombinant wt virus is denoted (R).
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