title: Amino acid residues that are important for Hyal2 function as a receptor for jaagsiekte sheep retrovirus.
abstract: BACKGROUND
  Infection by jaagsiekte sheep retrovirus (JSRV) and by enzootic nasal tumor virus (ENTV) depends on cell-surface expression of the virus entry receptor, hyaluronidase 2 (Hyal2). Human Hyal2 binds the envelope (Env) proteins of these viruses and is functional as a receptor, but Hyal2 from mice does not bind Env nor does it mediate entry of either virus. Here we have explored the amino acid determinants that account for the difference in receptor function.
  
  
  RESULTS
  Analysis of human-mouse Hyal2 chimeric proteins showed that amino acid differences responsible for the difference in Hyal2 receptor activity were localized to the central third of Hyal2. Human Hyal2 mutants containing single or double amino acid replacements with the respective mouse amino acids were generated across this region and were assayed for activity. None of the single or double mutation reduced the receptor activity of human Hyal2 by more than 10-fold, whereas mouse Hyal2 activity is reduced 1,000-fold from that of human Hyal2. While the 3-dimensional structures of mammalian Hyal2 proteins are unknown, bee venom hyaluronidase shows significant amino acid similarity to human and mouse Hyal2 and its structure has been determined. Many mutations having the largest negative effects on human Hyal2 function mapped to a small region of the bee venom hyaluronidase close to but not overlapping the active site of the enzyme, suggesting that this site represents the binding site for Env. Analysis of synonymous and non-synonymous nucleotide substitutions in the coding sequences of multiple mammalian Hyal2 proteins shows that the proteins are undergoing strong selection for amino acid conservation. We found no evidence for positive selection of amino acid changes that might reflect evolution of mammalian hosts to resist JSRV or ENTV infection.
  
  
  CONCLUSIONS
  These results show that the greatly reduced receptor activity of mouse Hyal2 in comparison to that of human Hyal2 is determined by multiple amino acid changes acting in concert. In particular, no one amino acid change blocks infection. However, the most important amino acids map to a small patch on a predicted 3-dimensional Hyal2 structure, which may represent the binding site for Env.
authors.affiliation: SAIC-Frederick, National Cancer Institute, Frederick, Maryland 21702, USA. duh@mail.ncifcrf.gov
authors.forename: Fuh-Mei
authors.initials: FM
authors.lastname: Duh
authors.forename: Clarissa
authors.initials: C
authors.lastname: Dirks
authors.forename: Michael I
authors.initials: MI
authors.lastname: Lerman
authors.forename: A Dusty
authors.initials: AD
authors.lastname: Miller
mesh_terms: D000818:Animals; D001514:Bee Venoms / Q000201:enzymology; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000502:physiology; D017124:Conserved Sequence; D003460:Crystallization; D058851:GPI-Linked Proteins; D015686:Gene Products, env / Q000737:chemistry; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000737:chemistry* / Q000502:physiology; D022141:Jaagsiekte sheep retrovirus / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D051381:Rats; D011916:Rats, Inbred F344; D011991:Receptors, Virus / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D001514:Bee Venoms; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D015686:Gene Products, env; D011991:Receptors, Virus; C473096:Hyal2 protein, human; D006821:Hyaluronoglucosaminidase