Vpu-mediated tetherin antagonism of ongoing HIV-1 infection in CD4+ T-cells is not directly related to the extent of tetherin cell surface downmodulation

AbstractTetherin is a host cell restriction factor that acts against HIV-1 and other enveloped viruses. The antiviral activity of tetherin is antagonized by the HIV-1 protein Vpu, that downregulates tetherin from the cell surface.Here, we report the specific detection of cell surface tetherin levels in primary activated CD4+ T-cells and in CD4+ T-cell lines. Differences were observed regarding tetherin cell surface expression, Vpu-mediated tetherin downmodulation and promotion of virus release. However, Vpu expression in all T-cell lines resulted in a 2-fold increase in numbers of infected cells after three days. This implies a Vpu-mediated effect in ongoing infection and possibly in cell-to-cell viral spread that is independent of the extent of Vpu-mediated tetherin cell surface downmodulation. Endogenous cell surface tetherin levels in T-cell lines were also downmodulated following infection with Vpu-deleted virus, suggesting an additional Vpu-independent mechanism of tetherin cell surface downmodulation following HIV-1 infection in T-cell lines

Tetherin restricts direct cell-to-cell infection of HIV-1

<p>Abstract</p> <p>Background</p> <p>Tetherin (BST-2/CD317/HM1.24) is an interferon (IFN)-inducible factor of the innate immune system, recently shown to exert antiviral activity against HIV-1 and other enveloped viruses by tethering nascent viral particles to the cell surface, thereby inhibiting viral release. In HIV-1 infection, the viral protein U (Vpu) counteracts this antiviral action by down-modulating tetherin from the cell surface. Viral dissemination between T-cells can occur <it>via </it>cell-free transmission or the more efficient direct cell-to-cell route through lipid raft-rich virological synapses, to which tetherin localizes.</p> <p>Results</p> <p>We established a flow cytometry-based co-culture assay to distinguish viral transfer from viral transmission and investigated the influence of tetherin on cell-to-cell spread of HIV-1. Sup-T1 cells inducible for tetherin expression were used to examine the impact of effector and target cell tetherin expression on virus transfer and transmission. Using this assay, we showed that tetherin inhibits direct cell-to-cell virus transfer and transmission. Viral Vpu promoted viral transmission from tetherin-expressing cells by down-modulating tetherin from the effector cell surface. Further, we showed that tetherin on the target cell promotes viral transfer and transmission. Viral infectivity in itself was not affected by tetherin.</p> <p>Conclusion</p> <p>In addition to inhibiting viral release, tetherin also inhibits direct cell-to-cell spread. Viral protein Vpu counteracts this restriction, outweighing its possible cost of fitness in cell-to-cell transmission. The differential role of tetherin in effector and target cells suggest a role for tetherin in cell-cell contacts and virological synapses.</p

Identification of an env-defective HIV-1 mutant capable of spontaneous reversion to a wild-type phenotype in certain T-cell lines

BACKGROUND: Attempts to eradicate HIV from cellular reservoirs are vital but depend on a clear understanding of how viral variants are transmitted and survive in the different cell types that constitute such reservoirs. Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms. METHODS: The ability of HIV containing an env G367R mutation in cell-free and cell-associated viruses to cause infection and to revert to wild-type was measured using several T cell lines. To determine factors that might potentially influence the reversion of G367R, we studied each of entry inhibitors, inhibitors of cellular endocytosis, and modulators of cell growth and activation. RESULTS: We demonstrate that an HIV-1 variant containing a G367R substitution within the CD4 binding site of gp120 was non-infectious as free virus in culture but was infectious when infected cells were co-cultured with certain T cell lines or when cells were transfected by a relevant proviral plasmid. Differences in viral infectivity by cell-associated G367R viruses were determined by the type of target cell employed, regardless which type of donor cell was used. Reversion was slowed or inhibited by entry inhibitors and by inhibitors of cellular endocytosis. Interleukin 2 was able to block G367R reversion in only one of the T cell lines studied but not in the other, while phorbol 12-myristate 13-acetate (PMA) inhibited G367R reversion in all the T cell lines. CONCLUSIONS: Env-defective HIV may have a different phenotype as cell-free versus cell-associated virus. The persistence of defective forms can potentially lead to the emergence of virulent forms. The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells. This is the first demonstration of a mutation in the HIV envelope, i.e. G367R, that can compromise infection by cell-free virus but less severely by cell-associated virus and that does so in a cell type-dependent manner

Peatland initiation and carbon accumulation in the Falkland Islands

The Falkland Islands in the South Atlantic Ocean contain extensive peatlands at the edge of their global climatic envelope, but the long-term carbon dynamics of these sites is poorly quantified. We present new data for ten sites, compile previously-published data and produce a new synthesis. Many peatlands in the Falkland Islands developed notably early, with a fifth of basal 14 C dates pre-Holocene. Falkland Islands peats have high ash content, high carbon content and high bulk density compared to global norms. In many sites carbon accumulation rates are extremely low, which may partly relate to low average rainfall, or to carbon loss through burning and aeolian processes. However, in coastal Tussac peatlands carbon accumulation can be extremely rapid. Our re-analysis of published data from Beauchene Island, the southernmost of the Falkland Islands, yields an exceptional long-term apparent carbon accumulation rate of 139 g C m −2 yr −1 , to our knowledge the highest recorded for any global peatland. This high accumulation might relate to the combination of a long growing-season and marine nutrient inputs. Given extensive coverage and carbon-dense peats the carbon stock of Falkland Islands peatlands is clearly considerable but robust quantification will require the development of a reliable peat map. Falkland Island peatlands challenge many standard assumptions and deserve more detailed study

The HIV-1 Vpu Viroporin Inhibitor BIT225 Does Not Affect Vpu-Mediated Tetherin Antagonism

Among its many roles, the HIV-1 accessory protein Vpu performs a viroporin function and also antagonizes the host cell restriction factor tetherin through its transmembrane domain. BIT225 is a small molecule inhibitor that specifically targets the Vpu viroporin function, which, in macrophages, resulted in late stage inhibition of virus release and decreased infectivity of released virus, a phenotype similar to tetherin-mediated restriction. Here, we investigated whether BIT225 might mediate its antiviral function, at least in part, via inhibition of Vpu-mediated tetherin antagonism. Using T-cell lines inducible for tetherin expression, we found that BIT225 does not exert its antiviral function by inhibiting Vpu-mediated tetherin downmodulation from the cell surface, the main site of action of tetherin activity. In addition, results from a bioluminescence resonance energy transfer (BRET) assay showed that the Vpu-tetherin interaction was not affected by BIT225. Our data provide support for the concept that tetherin antagonism and viroporin function are separable on the Vpu transmembrane and that viroporin function might be cell-type dependent. Further, this work contributes to the characterization of BIT225 as an inhibitor that specifically targets the viroporin function of Vpu