Analysis of the human immunodeficiency virus type 1 M group Vpu domains involved in antagonizing tetherin - PubMed (original) (raw)

Analysis of the human immunodeficiency virus type 1 M group Vpu domains involved in antagonizing tetherin

Sarah J Petit et al. J Gen Virol. 2011 Dec.

Abstract

Zoonosis of chimpanzee simian immunodeficiency virus cpz to humans has given rise to both pandemic (M) and non-pandemic (O, N and P) groups of human immunodeficiency virus type-1 (HIV). These lentiviruses encode accessory proteins, including Vpu, which has been shown to reduce CD4 levels on the cell surface, as well as increase virion release from the cell by antagonizing tetherin (CD317, BST2). Here, we confirm that O group Vpus (Ca9 and BCF06) are unable to counteract tetherin or downregulate the protein from the cell surface, although they are still able to reduce cell-surface CD4 levels. We hypothesize that this inability to antagonize tetherin may have contributed to O group viruses failing to achieve pandemic levels of human-to-human transmission. Characterization of chimeric O/M group Vpus and Vpu mutants demonstrate that the Vpu-tetherin interaction is complex, involving several domains. We identify specific residues within the transmembrane proximal region that, along with the transmembrane domain, are crucial for tetherin counteraction and enhanced virion release. We have also shown that the critical domains are responsible for the localization of M group Vpu to the trans-Golgi network, where it relocalizes tetherin to counteract its function. This work sheds light on the acquisition of anti-tetherin activity and the molecular details of pandemic HIV infection in humans.

PubMed Disclaimer

Figures

Fig. 1.

M group Vpu has evolved to antagonize tetherin through its TM and TM proximal domains. (a) Alignment of NL4-3 M group and Ca9 and BCF06 O group Vpus, as well as schematics of the Vpu chimeras. (b) Titres of HIV-1 released from 293T cells cotransfected with a titration of NL4-3, Ca9 or BCF06 Vpu encoding plasmids, HIV-1 vectors and 100 ng human tetherin. (c, d, e, f and g) Titres of HIV-1 released from 293T cells cotransfected with 500 ng [(g) 200, 500, 700 and 1000 ng] of various Vpu encoding plasmids, HIV-1 vectors and 100 ng human tetherin. P24 staining of Western blots of viral supernatants and cell lysates reflect viral titres and indicate equal Gag expression. Vpu expression was measured by detecting the C-terminal HA-tag and β-actin was measured for loading control. Results are representative of three separate experiments.

Fig. 2.

M and O group Vpus downregulate CD4 and tetherin differently from the cell surface. (a) 293T cells were cotransfected with a CD4 expression vector and pIRES2eGFP plasmids expressing GFP alone (no Vpu) or together with the indicated Vpu constructs, and CD4 surface expression was measured by FACS. Shown are relative levels of CD4 cell surface expression (black line) relative to those measured in cells transfected with the GFP only control vector (dotted line). (b) 293T cells were cotransfected with an ectodomain HA-tagged human tetherin vector and pIRES2eGFP plasmids expressing GFP alone (no Vpu) or together with the indicated Vpu constructs and HA tetherin surface expression was measured by FACS. Shown are the relative levels of tetherin cell surface expression (black line) relative to those measured in cells transfected with the GFP only control vector (dotted line). Results are mean±

sem

of three separate experiments. (c) Western blot of HA-tagged Vpu in the pIRES contruct showing expression of the various Vpu constructs, as well as a β-actin control.

Fig. 5.

O group Vpu does not relocalize tetherin to the TGN. Confocal images of 293T cells cotransfected with the various Vpu constructs and human tetherin and stained with immunofluorescently labelled anti-HA (Alexa-488) and anti-tetherin (Alexa-594) antibodies. Cell nucleus is detected with DAPI and merged images are in the fourth column. Each image is representative of at least four different fields positive for each HA-construct, taken at random. The PCC was calculated for each HA-construct. Values represent mean±

sem

Fig. 3.

M and O group Vpus’ localization reflect their anti-tetherin activity. Confocal images of 293T cells transfected with the various Vpu constructs and stained with immunofluorescently labelled anti-HA (Alexa-488) and anti-p230 _trans_-Golgi (anti-Golgin, rhodamine). Cell nucleus is detected with DAPI and merged images are in the fourth column. Each image is representative of at least four different fields positive for each HA-construct, taken at random. The PCC was calculated for each HA-construct. Values represent mean±

sem

Fig. 4.

O group Vpu disrupts the Golgi network. Confocal images of 293T cells cotransfected with the various Vpu constructs and stained with immunofluorescently labelled anti-HA (Alexa-488) and anti-TGN46 (rhodamine). Cell nucleus is detected with DAPI and merged images are in the fourth column. Each image is representative of at least four different fields positive for each HA-construct, taken at random. The PCC was calculated for each HA-construct. Values represent mean±

sem

References

1. Cao H., Thompson H. M., Krueger E. W., McNiven M. A. (2000). Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin. J Cell Sci 113, 1993–2002 - PubMed
1. Carreno S., Engqvist-Goldstein A. E., Zhang C. X., McDonald K. L., Drubin D. G. (2004). Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network. J Cell Biol 165, 781–788 10.1083/jcb.200403120 - DOI - PMC - PubMed
1. Dubé M., Roy B. B., Guiot-Guillain P., Mercier J., Binette J., Leung G., Cohen E. A. (2009). Suppression of Tetherin-restricting activity upon human immunodeficiency virus type 1 particle release correlates with localization of Vpu in the trans-Golgi network. J Virol 83, 4574–4590 10.1128/JVI.01800-08 - DOI - PMC - PubMed
1. Dunn K. W., Kamocka M. M., McDonald J. H. (2011). A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300, C723–C742 10.1152/ajpcell.00462.2010 - DOI - PMC - PubMed
1. Gao F., Bailes E., Robertson D. L., Chen Y., Rodenburg C. M., Michael S. F., Cummins L. B., Arthur L. O., Peeters M., et al. & other authors (1999). Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 397, 436–441 10.1038/17130 - DOI - PubMed

Analysis of the human immunodeficiency virus type 1 M group Vpu domains involved in antagonizing tetherin - PubMed (original) (raw)