Date Published: August 6, 2018
Publisher: Public Library of Science
Author(s): Lindsey R. Robinson-McCarthy, Kevin R. McCarthy, Matthijs Raaben, Silvia Piccinotti, Joppe Nieuwenhuis, Sarah H. Stubbs, Mark J. G. Bakkers, Sean P. J. Whelan, Nels C. Elde.
Endogenous retroviruses (ERVs), remnants of ancient germline infections, comprise 8% of the human genome. The most recently integrated includes human ERV-K (HERV-K) where several envelope (env) sequences remain intact. Viral pseudotypes decorated with one of those Envs are infectious. Using a recombinant vesicular stomatitis virus encoding HERV-K Env as its sole attachment and fusion protein (VSV-HERVK) we conducted a genome-wide haploid genetic screen to interrogate the host requirements for infection. This screen identified 11 genes involved in heparan sulfate biosynthesis. Genetic inhibition or chemical removal of heparan sulfate and addition of excess soluble heparan sulfate inhibit infection. Direct binding of heparin to soluble HERV-K Env and purified VSV-HERVK defines it as critical for viral attachment. Cell surface bound VSV-HERVK particles are triggered to infect on exposure to acidic pH, whereas acid pH pretreatment of virions blocks infection. Testing of additional endogenous HERV-K env sequences reveals they bind heparin and mediate acid pH triggered fusion. This work reconstructs and defines key steps in the infectious entry pathway of an extinct virus.
Endogenous retroviruses (ERVs) are remnants of ancient germline infections and comprise approximately 8% of the human genome . The degraded nature of ERV sequences impedes investigation of the properties of the infectious progenitor viruses and the events that led to their endogenization. During evolution, ERV sequences accumulate mutations, consequently the most recently endogenized sequences are the most likely to reflect the properties of the progenitor virus from which they were derived. The most recently endogenized human endogenous retroviruses (HERVs) belong to the HERV-K (HML-2) group. Multiple endogenization events resulted in approximately 90 proviral copies and 1,000 solo long terminal repeats (LTRs) in the reference human genome . The HERV-K (HML-2) group is approximately 30–35 million years old , with evidence of endogenization as recently as 100,000–600,000 years ago [4, 5].
To identify host factors required for HERV-K Env mediated entry, we performed a haploid genetic screen  (Fig 1A and 1B). Briefly, HAP1 cells were mutagenized using a retroviral gene-trap vector, a murine embryonic stem cell virus (MSCV)-based vector which inserts a GFP gene with a strong splice acceptor site randomly throughout the genome to generate a population with inactivating mutations across the genome, and infected with VSV-HERVK. This approach has previously been shown to disrupt >98% of expressed genes . Deep sequencing genomic DNA from cells that survived VSV-HERVK infection identified sites of integration of the gene-trap retrovirus (Fig 1C). Among the genes identified were 11 involved in the biosynthesis of heparan sulfate—a glycosaminoglycan (GAG) ubiquitously expressed on the cell surface. Six of those genes (GPC3, EXT1, EXT2, EXTL3, HS2ST1, and NDST1) are specific to heparan sulfate and heparin and not other GAGs (S1 Fig). For follow up, we selected EXT1 which encodes an enzyme that catalyzes the addition of a glucaronic acid—N-acetylglucosamine (GlcA-GlcNAc) disaccharide onto the growing heparan sulfate chain and SLC35B2 which encodes the Golgi-resident transporter of the universal sulfate donor 3’-phosphoadenosine-5’-phosphosulfate (PAPS) . Three additional genes, myosin X (MYO10), sortilin (SORT1), and CREB binding protein (CREBBP) scored as significant and were also selected for further follow up.
The major conclusion of this study is that heparan sulfate is a direct HERV-K Env attachment factor. Binding of HERV-K Env to heparan sulfate is most sensitive to the loss of 2-O sulfation, implicating this modification in attachment. Combined with earlier work we posit the following model for the entry of the retrovirus HERV-K (Fig 5). Following binding to cell surface heparan sulfate, virus is taken up into cells in a dynamin-dependent, clathrin independent manner with subsequent acidification of the endosome leading to membrane fusion and productive infection. This model is reminiscent of the sialic-acid binding and acid pH requirement for productive influenza virus entry. We cannot, however, rule out the possibility that HERV-K entry may require additional host factors not identified through the haploid genetic screening approach—such as essential host genes, or those with redundant function for HERV-K entry. The low pH-mediated inactivation of VSV-HERVK, and lack of identification of endosomal factors other than acidic pH, raise the possibility that heparan sulfate may act directly as a receptor. Regardless of whether binding to heparan sulfate is sufficient to fulfill both attachment and receptor functions, this study defines heparan sulfate as an important host factor for HERV-K Env-mediated cell entry. The ability of HERV-K Env to bind heparan sulfate underscores that such binding is an ancient property of viruses.