CAML does not modulate tetherin-mediated restriction of HIV-1 particle release

Abstract

Background: Tetherin/BST-2 is a recently-identified potent restriction factor in human cells that restricts HIV particle release following particle formation and budding at the plasma membrane. Vpu counteracts tetherin's restriction of particle release in a manner that has not yet been fully defined. We recently identified calcium-modulating cyclophilin ligand (CAML) as a Vpu-interacting protein that also restricts particle release. We hypothesized that CAML may act to enhance tetherin-mediated restriction of particle release and thereby explain how two distinct factors could be responsible for Vpu-responsive restriction.

Methodology/principal findings: Endogenous levels of tetherin in human cells correlated well with their restriction pattern and responsiveness to Vpu, while levels of cellular CAML protein did not. Tetherin but not CAML was inducible by interferon in a wide variety of human cells. Stable depletion of human CAML in restrictive HeLa cells had no effect on cell surface levels of tetherin, and failed to relieve tetherin-mediated restriction. Stable depletion of tetherin from HeLa cells, in contrast, rendered HeLa cells permissive and Vpu-unresponsive. Tetherin but not CAML expression in permissive human cells rendered them restrictive and Vpu responsive. Depletion of CAML had no influence on cell surface levels of tetherin.

Conclusions/significance: We conclude that tetherin restricts particle release and does not require CAML for this effect. Furthermore, these results do not support a major role for CAML in restricting HIV particle release in human cells.

Figure 1. Tetherin is interferon-inducible; CAML is not interferon-inducible.

A) Western blot for endogenous tetherin was performed using rabbit polyclonal antisera before or 48 hours after interferon-induction. Note that induction was demonstrated in each case, and baseline levels of tetherin were highest in HeLa cells. Lanes were normalized by total protein quantitation. D) Western blotting for endogenous CAML was performed in the same panel of cells. Note that no induction was demonstrated, and all cell types expressed detectable CAML. Cell lines are indicated above the blots, and IFN treatment is indicated with a plus sign. M  =  molecular mass markers.

Figure 2. CAML depletion does not inhibit tetherin-mediated restriction of particle release.

293T cells were transduced with an shRNA-encoding lentivirus expressing control shRNA or CAML shRNA and selected with puromycin. Selected cell populations were then transfected with tetherin expression vector and NLUdel proviral DNA. Cells (C) and supernatants (S) harvested at 48 hours for Western blot analysis. Particles present in supernatants were pelleted through 20% sucrose prior to loading. Analysis was performed using infrared detection, allowing simultaneous assessment of CAML in cell lysates by rabbit polyclonal antisera (red) and HIV Gag proteins by anti-p24 monoclonal antibody (green). Co-transfection of cells with a tetherin expression construct is indicated by plus signs. M  =  molecular mass markers.

Figure 3. Direct comparison of CAML and tetherin effect on particle release in 293T cells and HT1080 cells.

A) 293T cells were transfected with increasing amounts of CAML or tetherin expression vectors and NL4-3 (left) or NLUdel provirus (right). Cells and particles in supernatants were harvested at 48 hours post-transfection and analyzed by Western blotting using an anti-p24 monoclonal antibody. Total amount of transfected CAML or tetherin plasmid DNA per 35 mm² well is indicated above the blots. B) HT1080 cells were employed in experiments identical to those described for 293T cells. No consistent effect of CAML on virus release was noted in either cell type, while tetherin potently inhibited virus release. Molecular mass markers are indicated on the left of each panel.

Figure 4. Depletion of CAML in HeLa cells fails to relieve restriction to particle release.

A stably-transduced cell population demonstrating marked CAML knockdown (lanes 3, 4, 7, 8) was transfected with NL4-3 or NLUdel proviral DNA and compared with control shRNA transduced cells (lanes 1, 2, 5, 6). CAML protein is shown in red. In the presence of Vpu, particle release was equivalent in CAML knockdown or control cells (lanes 1–4). In the absence of Vpu, the restriction to particle release was present and was not affected by CAML depletion (lane 8 vs. lane 6).

Figure 5. Tetherin depletion alone rescues particle release in HeLa cells.

A) Two independently-derived knockdown cell populations (labeled 1,2) were employed in which knockdown of endogenous tetherin was nearly complete. Asterisk indicates a background band present in all lanes. B) Particle release defect in control shRNA-transduced HeLa cells (C) vs. knockdown cells (1, 2) is shown. Particle release was inhibited in control cells expressing NLUdel virus, while both knockdown cell populations restored particle release (NLUdel, 1 and 2 vs. C).

Figure 6. CAML does not regulate cell surface levels of tetherin.

A rabbit polyclonal anti-tetherin antibody was used to quantify tetherin on the cell surface of HeLa cells by flow cytometry. A) Unstained cell control (white) and tetherin surface staining (filled histogram) using primary rabbit anti-tetherin followed by anti-rabbit APC staining. B) HeLa cells transduced with a control shRNA lentiviral vector (filled histogram) compared with unstained control cells (white). Dashed line indicates histogram of tetherin shRNA-transduced cells, demonstrating that cell surface levels were diminished by tetherin shRNA. C) Cell surface levels of tetherin in control shRNA transduced cells (filled histogram) did not differ from cells in which CAML was significantly depleted using CAML-specific shRNA (dashed lines). D) Vpu-EGFP transfected HeLa cells demonstrate cell surface downregulation of tetherin, as indicated by left shift of EGFP-positive population. E) CAML knockdown HeLa cells demonstrate cell surface downregulation of tetherin by Vpu-EGFP, similar to that shown in D.