Abstract
Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resolution analysis of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, respectively. Multicolor super-resolution microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters containing on average 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochemical and super-resolution analysis revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resolution microscopy analysis of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions.
Highlights
Viruses heavily depend on the host cell machinery for their replication, they face numerous blockades imposed by cellular proteins at several distinct steps in their life cycle
Viral proteins within virions were visualized with nanometer resolution yielding new insight into the structure of the human immunodeficiency virus 1 (HIV-1)
Our super-resolution analysis was extended to tetherin, a cellular restriction factor that inhibits the release of several enveloped viruses
Summary
Viruses heavily depend on the host cell machinery for their replication, they face numerous blockades imposed by cellular proteins at several distinct steps in their life cycle. HIV-1 Vpu interacts with the tetherin transmembrane domain [6,16] and counteracts tetherin by degradation and removal from the cell surface [2,17,18,19]. Through these combined activities, Vpu impairs incorporation of tetherin into virions and restriction [13]. Detailed analysis of tetherin distribution in the plasma membrane, of the role of lipid rafts in HIV-1 tetherin interactions and of the orientation and number of tetherin molecules involved in restriction is still lacking
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