Anti-tetherin activities in Vpu-expressing primate lentiviruses

Abstract

Background: The anti-viral activity of the cellular restriction factor, BST-2/tetherin, was first observed as an ability to block the release of Vpu-minus HIV-1 from the surface of infected cells. However, tetherin restriction is also counteracted by primate lentiviruses that do not express a Vpu protein, where anti-tetherin functions are provided by either the Env protein (HIV-2, SIVtan) or the Nef protein (SIVsm/mac and SIVagm). Within the primate lentiviruses, Vpu is also present in the genomes of SIVcpz and certain SIVsyk viruses. We asked whether, in these viruses, anti-tetherin activity was always a property of Vpu, or if it had selectively evolved in HIV-1 to perform this function.

Results: We found that despite the close relatedness of HIV-1 and SIVcpz, the chimpanzee viruses use Nef instead of Vpu to counteract tetherin. Furthermore, SIVcpz Nef proteins had activity against chimpanzee but not human tetherin. This specificity mapped to a short sequence that is present in the cytoplasmic tail of primate but not human tetherins, and this also accounts for the specificity of SIVsm/mac Nef for primate but not human tetherins. In contrast, Vpu proteins from four diverse members of the SIVsyk lineage all displayed an anti-tetherin activity that was active against macaque tetherin. Interestingly, Vpu from a SIVgsn isolate was also found to have activity against human tetherin.

Conclusions: Primate lentiviruses show a high degree of flexibility in their use of anti-tetherin factors, indicating a strong selective pressure to counteract tetherin restriction. The identification of an activity against human tetherin in SIVgsn Vpu suggests that the presence of Vpu in the ancestral SIVmus/mon/gsn virus believed to have contributed the 3' half of the HIV-1 genome may have played a role in the evolution of viruses that could counteract human tetherin and infect humans.

Figure 1

HIV-1 but not SIVcpz Vpu overcomes human tetherin restriction. (A) SIVcpz/HIV-1 lineage of the primate lentiviruses, showing three major HIV-1 groups (M, N and O) and the SIVcpz isolates used in this study. SIVcpz TAN3 and ANT were isolated from Pan troglodytes schweinfurthii (P.t.s.) and are less closely related to HIV-1 than SIVcpz strains isolated from Pan troglodytes troglodytes (P.t.t.). Figure adapted from Wain et al. (2007) [35]. (B) Confocal analysis of distribution of GAB1 and ANT Vpu-EGFP fusion proteins and EGFP control, in transiently transfected HeLa cells. (C) HeLa cells (express tetherin) were transfected with pHIV-1-pack (expresses HIV-1 Gag-Pol, Rev), together with either a control CMV expression vector (-), or expression plasmids for human codon-optimized Vpu from HIV-1 (HIV-1 Vpu), or non-codon-optimized EGFP tagged Vpu proteins from HIV-1, SIVcpz GAB1 or SIVcpz ANT. Cell lysates (lys) were probed with indicated antibodies. The Vpu-EGFP proteins from HIV-1, SIVcpz GAB1 and SIVcpz ANT have predicted molecular weights of 47, 33 and 42 kDa, respectively. Intracellular Gag proteins in cell lysates and virus-like particles released into supernatant (VLP) were detected using anti-p24 antibody. Mean-fold enhancement of HIV-1 VLP release in presence of Vpu is shown relative to baseline (control) levels in absence of Vpu for three independent experiments, except for the HIV-1 Vpu-EGFP sample (n = 1).

Figure 2

Activity of HIV-1 and SIVcpz Vpu against chimpanzee tetherin. Anti-tetherin activities of indicated viral proteins were examined by measurement of HIV-1 VLP release, detected by Western blotting of cell lysate and VLP fractions with anti-p24 antibody (left panels) or as mean-fold enhancement of VLP release relative to baseline (control) levels in absence of Vpu or Env proteins (right panels): (A) HIV-1 Vpu and HIV-2 Env activity against human (Hum) and chimpanzee (Cpz) tetherin expressed in 293A cells, (B) Activity of HIV-1 Vpu and SIVcpz GAB1 and SIVcpz ANT Vpu-EGFP proteins against Cpz-tetherin expressed in 293A cells, and (C) Activity of HIV-1 Vpu and SIVcpz GAB1 and SIVcpz ANT Vpu-EGFP proteins against Cpz-tetherin expressed in chimpanzee (Cpz_B) cells. * indicates p24 signal was too low to quantify.

Figure 3

Activity of SIVcpz genomic fragments against human and chimpanzee tetherin. (A) Expression of Env proteins from SIVcpz subgenomic fragments (express Env, Vpu and Rev). (B) Activity of SIVcpz genomic fragments in HIV-1 VLP release assay against human tetherin present in HeLa cells. (C) Activity of SIVcpz genomic fragments in HIV-1 VLP release assay against chimpanzee (Cpz) tetherin expressed in 293A cells. HIV-1 Vpu, HIV-2 Env, and HIV-1 Env were included as positive and negative controls as indicated.

Figure 4

Anti-tetherin activity of SIVcpz Nef. (A) Expression of indicated SIV Nef-EGFP proteins, detected with anti-GFP antibody. Activities of Nef-EGFP proteins against (B) chimpanzee, or (C) human tetherin expressed in 293A cells. Mean fold-enhancement of HIV-1 VLP release in presence of Nef-EGFP is shown relative to baseline (control) levels for n = 2 or 3 independent experiments.

Figure 5

Specificity of interaction between SIVcpz and SIVmac Nef proteins and tetherin. (A) Partial sequence alignment of human, chimpanzee (Cpz) and H(+5)-tetherin proteins, showing the N-terminal cytoplasmic tail, the transmembrane (TM) domain and the start of the extracellular domain. H(+5)-tetherin contains an insertion (DDIWKK) from Cpz-tetherin in place of human tetherin residue E-14. (B) Western blot of expression of indicated tetherin constructs, from lysates of transfected 293A cells. (C) Anti-tetherin activities of SIVmac239 and SIVcpz Nef-EGFP proteins against H(+5)-tetherin expressed in 293A cells. Mean fold-enhancement of HIV-1 VLP release in presence of Nef-EGFP proteins is shown relative to baseline (control) levels for n = 2 independent experiments.

Figure 6

Anti-tetherin activity of SIVsyk lineage Vpu proteins. (A) SIVsyk lineage of the primate lentiviruses, showing viruses that express Vpu (boxed). SIVmon/mus/gsn viruses form the SIVgsn sublineage, while SIVden is less closely related. Figure adapted from Dazza et al. (2005) [28]. (B) Expression of SIVmus/mon/gsn and SIVden Vpu-EGFP proteins detected with anti-GFP antibody. Anti-tetherin activities of indicated Vpu-EGFP proteins were measured by HIV-1 VLP release assays, against (C) human tetherin present in HeLa cells, (D) Mac-tetherin expressed in macaque (LLCMK2) cells, and (E) Hum-tetherin expressed in LLCMK2 cells. HIV-1 Vpu and SIVmac239 Nef-EGFP proteins were included as controls. Mean fold-enhancement of HIV-1 VLP release in presence of anti-tetherin proteins is shown relative to baseline (control) levels, for n = 2 independent experiments.

Figure 7

Specificity of interaction of SIVsyk Vpu proteins and tetherin. (A) Anti-tetherin activities of HIV-1 Vpu and indicated SIVsyk Vpu-EGFP proteins against H(+5)-tetherin expressed in LLCMK2 cells, measured by HIV-1 VLP release assay. Mean fold-enhancement of HIV-1 VLP release in the presence of anti-tetherin proteins is shown relative to baseline (control) levels in their absence, for n = 2 independent experiments. (B) Partial sequence alignment of human, macaque (Mac) and MH-tetherin showing the N-terminal cytoplasmic tail, the transmembrane (TM) domain and the start of the extracellular domain. Mac-tetherin starts at M-11 of the full-length Macaca mulatta tetherin [9]. MH-tetherin has N-terminal cytoplasmic tail and TM domains of Mac-tetherin with a human tetherin extracellular domain. (C) Western blot of expression of indicated tetherin constructs, from lysates of transfected 293A cells. (D) Anti-tetherin activities of HIV-1 Vpu and indicated SIVsyk Vpu-EGFP proteins against MH-tetherin expressed in LLCMK2 cells, measured by HIV-1 VLP release assay. Mean fold enhancement of VLP release in presence of Vpu proteins is shown relative to baseline (control) levels in absence of Vpu, for n = 2 independent experiments.

Figure 8

Ability of Vpu proteins to remove human tetherin from surface of cells. (A) HeLa cells were transfected with a control EGFP expression plasmid, HIV-1 Vpu or Vpu-EGFP fusion proteins from SIVmus/mon/gsn isolates. Non-permeabilized cells were incubated with anti-BST-2 antibody to reveal cell surface tetherin, followed by fixation and permeablization to visualize intracellular proteins. HIV-1 Vpu (untagged) was used as a positive control for tetherin removal from surface and visualized with an anti-Vpu antiserum, while the SIV Vpu-EGFP proteins and EGFP control were visualized using anti-GFP antibody. Both HIV-1 and SIVgsn Vpu proteins removed human tetherin from the cell surface, but SIVmus and SIVmon did not. Vpu-expressing cells are arrowed. (B) HeLa cells were transfected with a control EGFP expression plasmid alone or together with HIV-1 Vpu, or with each of the SIVmus/mon/gsn Vpu-EGFP expression plasmids. Cells were stained with anti-BST-2 antibody and analyzed by FACS. The histograms show cells gated for EGFP expression, with the control EGFP expression vector cells in black, and Vpu samples in blue.