Functional dissection of the prototype foamy virus glycoprotein heparan sulfate binding site

The foamy virus (FV) glycoprotein complex (GPC) facilitates exceptionally broad species and tissue tropism. While cell surface heparan sulfate (HS) serves as a known attachment factor, it is not essential for viral entry. Recent high-resolution structures of GPCs from various FV species identified an evolutionarily conserved, positively charged surface patch (PCSP) on the receptor-binding domain (RBD) as a putative HS-binding site (HSBS). To date, only the gorilla FV (SFVggo) HSBS has been functionally characterized, demonstrating the role of basic PCSP residues in HS-dependent attachment. Experimental evidence supporting a universal role for the GPC PCSP across other FV species is currently lacking.

The prototype FV (PFV) GPC PCSP consists of four central residues surrounded by five peripheral, positively charged residues. Using charge-switch mutagenesis, we investigated the functional role of eight PCSP residues. The central residues-K₃₄₃, K₃₅₅, R₃₅₇, and K₃₆₈-proved essential for HS-dependent attachment and infection across various target cells. Individual mutations of these residues reduced attachment and infectivity in HT1080 cells by 50- to 100-fold. Among peripheral residues, only K₃₅₆ contributed significantly to these processes on different HS-expressing target cells. Notably, all mutant PFV GPCs maintained levels of attachment and infectivity in HS-deficient cells similar to those of the wild-type, though these levels were 10- to 30-fold lower than in HS-expressing parental cells but well above background.

The minimal HSBS of the PFV GPC is defined by four central, evolutionarily conserved positively charged residues. Substituting these with negatively charged amino acids abolishes HS-dependent attachment and severely reduces specific infectivity. The minor impact of the peripheral residue mutation K₃₅₆E, combined with the lack of evolutionary conservation among most peripheral positively charged residues in primate FV species, suggests these residues play only a secondary role in HS interaction. Furthermore, the residual infectivity of PCSP mutants in HS-deficient cells confirms that HS is an important attachment factor but not an essential entry receptor. The functional homology between PFV and SFVggo GPCs strongly suggests that this conserved PCSP constitutes a universal HS-binding site across all FV species.