Tetherin inhibits HIV-1 release by directly tethering virions to cells David Perez– Caballero, Trinity Zang, Alaleh Ebrahimi, Matthew W.McNatt, Devon A.Gregory, Marc C.Johnson, and Paul D. Bieniasz The Rockefeller University, New York, NY 10016, USA 3Howard Hughes Medical Institute, Aaron Diamond AIDS Research Center, New York, NY 10016, USA 4Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211, USA

HIV

Basic information TETHERIN Human cells possess different restriction factors that inhibit the release of many retroviruses and other enveloped virus particles. To counter such inhibition, HIV and other viruses produce certain proteins that neutralize these restriction factors. HIV – Vpu (Viral protein U) required for efficient release of virions . In the absence of Vpu, fully matured virions are unable to bud out and are tethered to the plasma membrane which are later endocytosed by the cell (Neil et al,2006). Tethered virions can be released by proteases like subtilisin- surface protein. IFNα also induces tethering which can be counteracted by Vpu (Neil et al,2007). Microarray analyses – CD317 membrane protein- TETHERIN

Structural features of tetherin TypeII Single pass dimeric transmembrane protein, encoding a transmembrane domain at its N-terminus, and GPI anchor at C-terminus. Membrane anchors are linked by an extracellular coiled coil domain. Two N-Glycosylation sites : N65 and N92 Three Cystein disulphide bonds : C53, C63 and C91

Transient and stable expression of N-glycosylation mutants Stable transfection of 293T using LHCX retroviral vectors Treatment with peptide N glycosidase F

Modification of tetherin by addition of GPI anchor Co-transfection of GFP expression plasmid

Dimer formation by cysteine mutants excepting the triple mutant Tetherin has same configuration as it is schematically represented.

Determination of anti- viral activity of tetherin mutants Dystrophia myotonica protein kinase Urokinase plasminogen activator receptor

Construction of an artificial tetherin like protein mimicking tetherin activity The loss in activity of tetherin could be rescued by replacing similar domains from unrelated heterologous proteins having no sequence homology. TfR : transferrin receptor DMPK: Dystrophia myotonica protein kinase uPAR: urokinase plasminogen activator receptor

Determination of anti-viral activity of artificial tetherin Pr55 gag precursor p24 mature capsid protein Vpu does not have an effect on artifial tetherin

Anti- viral activity of artificial tetherin mutants Infectivity assay

Anti- viral activity of artificial tetherin mutants Subtilisin treatment could release the trapped virions (Neil et al 2006).

Anti- viral activity of artificial tetherin mutants Since artificial tetherin having no sequence homology with native tetherin but could mimic its antiviral activity, its likely that tetherin functions by directly tethering virions.

Incorporation of tetherin into virion particles TEM analysis of HT1080 stably expressing tetherin-HA and infected with HIV-1(del vpu).

Incorporation of tetherin mutants into HIV1 particles. Suggestes that tetherin does get incorporated with either of its membrane anchors.

Configuration of tetherin in virions Suggestes tetherin gets incorporated into virions using its N-terminus as a parallel homodimer.

Effect of Vpu on virions derived from HIV-1(wt) and HIV-1(del Vpu) Vpu excludes tetherin from virons by targeting its transembrane domain

Tetherin associated with budding virions Virions that are in the act,but have have not completed budding was generated by expressing HIV Gag with a mutation in the PTAP L domain Analysed by conducting scanning electron microscope and backscatter electron detection Disadvantage of not able to observe tetherin-dependent tethering events Advantage that interaction between tetherin deletion mutants and budding particles could be observed

Models for Tetherin Incorporation Model 1- the TM domains of a tetherin dimer are incorporated into the virion envelope and the GPI anchors remain embedded in the host-cell membrane. Model 2- the reverse situation occurs. Model 3-only one of a pair of disulfide-linked tetherin molecules has both membrane anchors incorporated into the virion envelope. Model 4- one disulfide linked tetherin dimer incorporated into the virion envelope interacts with another dimer in the host-cell membrane via coiled-coil-based interactions

To summarize …. N and C-terminal anchors are responsible for tetherin activity. The overall configuration of tetherin rather than primary sequence is critical for anti-viral activity. Tetherin does get incorporated into HIV-1 particles as a parallel homodimer using either of its two membrane anchors. Tetherin functions directly by attaching to virion envelopes by either of its membrane anchors and is likely sufficient to tether enveloped virus particles that bud through the plasma membrane.

Localisation of tetherin in ER

Expression of artificial tetherin

Opti prep to show incorporation of tetherin in virions.

Surface expression of tetherin proteins

Secretion of tPA tetherin derived protein lacking both membrane anchors is dependent on glycosylation sites

Surface expression of wild type and artificial tetherin proteins

Inhibition of Ebola VLP release by artificial tetherin

Incorporation of inactive and partly active tetherin mutants into HIV virions.

Expression and release of gag