Reconstructing the Cell Entry Pathway of an Extinct Virus

Abstract

Approximately 8% of the human genome comprises endogenous retroviruses (ERVs), remnants of ancient germline infections. ERVs represent the best “molecular fossils” of viral infection of the host or its ancestor. Knowledge of each step of ERV biology will help provide insight into the long-term evolutionary history of virus-host relationships. Many characteristics of ERVs have, however, been obscured by the accumulation of mutations over evolutionary time, hampering our ability to study their interactions with the host. We rationalized that the properties of the youngest human ERVs, HERV-K(HML-2), would most closely resemble those of the infectious progenitor virus, and focused on its envelope protein (Env) to understand cell entry and tropism. We generated a recombinant vesicular stomatitis virus expressing an ancestral HERV-K Env and show that entry proceeds by a dynamin-dependent but clathrin-independent pathway with subsequent endosomal acidification. HERV-K Env imparts a broad species and tissue tropism, indicating that cellular factors required for HERV-K entry are both evolutionarily conserved and ubiquitously expressed. A genetic screen for host factors required for HERV-K entry identified multiple genes essential for biosynthesis of heparan sulfate, a ubiquitously expressed cell surface molecule. Cells specifically engineered to lack heparan sulfate or cell surface sulfation were resistant to HERV-K mediated infection as were cells chemically treated to inhibit sulfation. HERV-K Env mediated infection was specifically sensitive to inhibition by soluble heparan sulfate. Using quantitative single particle binding assays we show that cell surface sulfation facilitates virus attachment, and using purified recombinant protein we demonstrate a direct interaction between HERV-K Env and heparin in vitro. The ability to bind heparin is conserved among the phylogenetically distinct HERV-K elements, HERV-K 108 and Xq21.33. Collectively this data reconstructs a model for entry in which HERV-K binds heparan sulfate, internalizes via dynamin-dependent endocytosis and fuses from within acidified endosomes. Our findings suggest that endogenization of HERV-K, and likely other ERVs, was a consequence of utilizing ubiquitously expressed entry factors, rather than germ cell specific tropism.