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. 2009 Feb 24;106(8):2868-73.
doi: 10.1073/pnas.0813223106. Epub 2009 Feb 5.

Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion

Eri Miyagi  1 , Amy J Andrew, Sandra Kao, Klaus Strebel
Affiliations

Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion

Eri Miyagi et al. Proc Natl Acad Sci U S A. .

Abstract

HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression of Bst-2 in primary cells and cell lines is IFN-inducible. (A) HeLa and 293T cells were cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of IFNα (1 ng/mL) for 24 h. Whole-cell lysates were subjected to immunoblotting by using a Bst-2-specific polyclonal rabbit antibody. The same blot was subsequently probed with a tubulin-specific antibody (tub) as internal reference for sample loading. (B) HeLa cells, MDM, and PBMC were cultured in the absence of IFNα (lanes 1, 3, and 7) or were treated for 24 h with 0.1 ng/mL (lane 4), 1 ng/mL (lanes 2, 5, and 8), or 10 ng/mL (lane 6) IFNα. For comparison, PBMC were stimulated with CD3/CD28 antibodies as described in Materials and Methods (lane 9). Whole-cell lysates were processed for immunoblotting as described in A except that actin was used as internal reference for sample loading. (C) CEMx174, A3.01, H9, and Jurkat cells were analyzed with or without prior IFNα treatment (1 ng/mL) as in A.
Fig. 2.
Fig. 2.
Vpu reduces Bst-2 levels in transfected HeLa cells and in HIV-infected macrophages. (A) HeLa cells were transfected with 1 μg of pEGFP-N1 (Clontech) with (+) or without (−) 1 μg of pcDNA-Vphu. Total DNA was adjusted to 5 μg with empty-vector DNA. Cells were harvested 24 h after transfection and split into two fractions. One fraction was used to prepare whole-cell lysates (total); the other fraction was sorted for GFP-positive cells [GFP(+)] before cell lysis with a FACS Aria (BD Biosciences) as reported in ref. . Cell lysates were analyzed by immunoblotting with antibodies to Bst-2 (Upper) or Vpu (Lower). Proteins are identified on the right. (B) HeLa cells were transfected as in A, stained with Bst-2 antibodies as described in Materials and Methods, and analyzed on a FACS Calibur. Transfected cells were gated for GFP-positive cells. As a control, untransfected HeLa cells were stained with preimmune serum (control). (C) Human MDM were infected with HIV-1 Ada as described in ref. (lane 2). Uninfected macrophages were cultured in parallel as a control (lane 1). Cells were harvested 24 days after infection, and whole-cell lysates were analyzed by immunoblotting for Bst-2, viral capsid protein (CA), or Vpu. The blot was subsequently reprobed with tubulin-specific antibody to control for sample loading.
Fig. 3.
Fig. 3.
Vpu does not reduce Bst-2 levels and does not induce cell surface down-regulation of Bst-2 during replication in CEMx174 cells. (A) CEMx174 cells were infected with equal reverse transcriptase units of WT NL4-3, NL4-3/Udel, NL4-3/Urd, and NL4-3/U26 virus stocks produced in 293T cells. Virus replication was monitored by measuring the virus-associated reverse transcriptase activity in the culture supernatants. Error bars reflect mean error from duplicate independent infections. (B and C) On day 4 after infection, ≈40% of the cells were removed from the infected cultures and divided into two aliquots. One aliquot of each sample was used for preparation of whole-cell extracts, followed by immunoblotting (B); the other part of the cells was used for Bst-2 cell surface staining followed by staining for intracellular p24 as described in Materials and Methods (C).
Fig. 4.
Fig. 4.
Packaging of endogenous Bst-2 into HIV-1 virions. HeLa cells were transfected with 5 μg each of WT pNL4-3 (lanes 2, 5, and 8) or NL4-3/Udel (lanes 3, 6, and 9). A mock-transfected culture was analyzed in parallel (lanes 1, 4, and 7). Viral fractions (virus 1 and virus 2) were collected as described in the text. Cell lysates and concentrated viral supernatants were subjected to immunoblotting with antibodies to Bst-2 (Top). Viral capsid protein (CA) was identified with an HIV-positive patient serum (Middle). The blot was reprobed with an antibody to tubulin (tub) as internal reference (Bottom). Proteins are identified on the right.
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