Date of Award
Doctor of Philosophy (PhD)
The cellular restriction factor, tetherin, prevents HIV-1 and other enveloped virus particles from being disseminated into the extracellular milieu by infiltrating their envelopes and by physically crosslinking them to the cell surface. However, the mechanisms underlying virion retention have not yet been fully delineated. In this body of work, we employed biochemical assays and engineered tetherin proteins to demonstrate conclusively that virion tethers are composed of the tetherin protein itself, and to elucidate the configuration and topology that tetherin adopts during virion entrapment. We demonstrated that tetherin dimers adopt an “axial” configuration, in which pairs of transmembrane domains or pairs of glycosylphosphatidyl inositol anchors are inserted into assembling particles, while the remaining pair of membrane anchors remains embedded in the infected cell membrane. We used quantitative western blotting to determine that a few dozen tetherin dimers are used to trap each virion particle, and that there is ~3-5 fold preference for the insertion of glycosylphosphatidyl inositol anchors rather than transmembrane domains into tethered virions. Cumulatively, these results demonstrated that axially configured tetherin homodimers are directly responsible for trapping virions at the cell surface. We propose that insertion of glycosylphosphatidyl inositol anchors may be preferred so that effector functions that require exposure of the tetherin N-terminus to the cytoplasm of infected cells are retained. Allied to these efforts, we also endeavored to determine the evolutionary origins of tetherin. We used computational and biochemical approaches to build a case that tetherin arose via duplication of the neighboring PLVAP/PV1 gene. The PV1 and tetherin genes are located adjacent to each other in eutherian mammals and encode proteins with a shared and relatively unusual overall architecture. Phylogenetic analyses provided evidence that tetherin is ~147 MY old and that it has evolved under positive selection. Conversely, PV1 is ~400 MY old and has evolved under purifying selection in therian mammals. Using biochemical assays, we demonstrated that PV1 can be endowed with antiviral activity against HIV-1 when it is engineered to encode a C-terminal GPI anchor. Although we did not detect any sequence homology between PV1 and tetherin, in silico evolution experiments indicated that it is possible to evolve sequences from an ancestral PV1 sequence that bear no significant homology to contemporary PV1, after simulating the evolutionary processes that contributed to the emergence of tetherin. We found that genes encoding proteins with PV1/tetherin-like architecture are quite rare in the human and mouse genomes, and that the majority of these genes that are adjacent to each other likely arose via gene duplication. Moreover, some, but not all, PV1/tetherin-like proteins have the intrinsic ability to trap virions, when appended with a GPI anchor. Finally, we analyzed the organization of genes in the PV1 locus from amphibians to eutherian mammals and identified an ancestral synteny block that supports a model in which tetherin arose from a PV1 duplication, that occurred between the times that monotremes and marsupials diverged from other mammals (~166-147 MYA). Cumulatively, these findings suggest that tetherin arose by gene duplication of PV1 and exemplify the remarkable capacity of our genomes to innovate antiviral factors from pre-existing genetic raw material.
Venkatesh, Siddarth, "Mechanism and Evolutionary Origins of HIV-1 Virion Entrapment by Tetherin" (2014). Student Theses and Dissertations. 220.