Complete Title of Thesis:
"Determinants of Host Range in the Spike Glycoprotein of the Mouse Hepatitis Virus"
Prepared under the direction of: Kathryn V. Holmes, Ph.D.
The host range of the murine coronavirus, mouse hepatitis virus (MHV), is naturally limited to mice and murine cell lines by interactions between the spike glycoprotein (S) and the murine glycoprotein receptor, CEACAM1a. Twenty-one amino acid (aa) substitutions and a 7 aa insert appear to be responsible for the extended host range of the variant, MHV/BHK. To investigate whether these changes altered the host range of MHV-A59, I used targeted RNA recombination (TRR) to generate isogenic viruses that differed only in S. Only viruses with both the 21 aa substitutions and 7 aa insert (S21BHK+i) infected murine, hamster, feline and monkey cell lines. S21BHK+i viruses also infected murine cells in the presence of blocking anti-mCEACAM1a antibodies. S21BHK+i viruses formed altered plaques on murine cell monolayers, yet these viruses bound and were neutralized by mCEACAMIa like MHV-A59. Thus, relatively few changes in S permit MHV-A59 to interact with alternative receptors on murine and nonmurine cells, but do not significantly alter the interaction of the virus with its original murine receptor.
Substitution of Thr62 or Leu65 reduces the binding of soluble S330 and an MHVJHM variant to mCEACAM1a, respectively. I identified five additional residues in S that may determine the host specificity of MHV. I used isogenic viruses that differ only in S to examine the importance of these residues in the host specificity of MHV-A59 in the context of a virion. Viruses with substitutions at Thr62 and Leu65 were readily recovered from and grew to high titers in murine cells. Viruses with Ser33Gly, Leu79Met/Thr82Met, or Lys183Gly substitutions were recovered from hamster cells over-expressing mCEACAMIa [BHK+CEACAM1a(l41R)]. Viruses with Tyr162His, Tyr162Glu, or Tyr162Ala substitutions were not recovered from murine or BHK+CEACAM1a(l41R) cells, although viruses with Tyr162Phe substitutions were readily recovered from murine cells. None of the mutant viruses infected rat, hamster or human cell lines. Thus, a single aa substitution in S can inhibit the interaction of MHVA59 with its murine receptor, but is not sufficient to permit interaction of the virus with alternative receptors on non-murine cells.