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Department of Microbiology, A Leader in Microbiology and Microbial Pathogenesis Research and Training.

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Brian C. Turner, Ph.D.

KV Holmes Lab


 

THESIS


Complete Title of Thesis:
"Interactions of Murine Coronavirus with its Murine CEACAM1 Receptors"


Prepared under the direction of: Kathryn V. Holmes, Ph.D.


SUMMARY

Murine coronavirus MHV causes epizootics in laboratory mice and inhibits immune responses to tumors, skin transplants and infectious agents by an unknown mechanism long after virus has been cleared. I postulated that dendritic cells (DC) might play a role in MHV-induced immune modulation. Bone marrow-derived DC (BMDC) and a murine dendritic cell line were found to express the CEACAM1a receptor for MHV, be susceptible to MHVA59 infection, and produce infectious virus. Incubation of BMDC with anti-CEACAM1a monoclonal antibody, MAb-CC1, prevented MHV-A59 infection. Thus, MHV productively infects murine DC through a receptormediated pathway. Infection of DC in vivo might play a role in MHV-induced immune modulation.

All MHV-susceptible inbred strains of mice are homozygous for the CEACAM1a allele. To identify amino acids involved in coronavirus receptor activity, I performed structure-based mutagenesis on the predicted virus-binding face of the receptor and challenged hamster cells expressing mutant receptors with neurotropic (MHV-JHM) and hepatotropic (MHV-A59) viruses. Alanine substitution at projecting I41 of mCEACAM1a blocked receptor activity for MHV-JHM, but only partially inhibited MHV-A59 infection. Alanine substitutions at both I41 and R96 blocked MHV-A59 receptor activity. I41 and R47 were critical for MAb-CC1 binding, showing that virus and MAb-CC1 have overlapping binding sites on CEACAM1a.

Residue 47 is biologically very important for MHV receptor activity. An R47F substitution in mCEACAMIa increased receptor activity 3 fold and rescued mCEACAM1a[1,4] I41A and I41A/R96A receptor activity. The SJL/J strain of inbred mice is highly resistant to MHV and is homozygous for murine CEACAM1b, which differs from mCEACAM1a in the virus binding domain. To determine the molecular basis for virus resistance I mutagenized mCEACAM1b and challenged the mutant receptors with MHV strains. CEACAM1b is a weak receptor for MHV-A59, but does not function for MHV-JHM. H47A or H47R substitution in mCEACAM1b increased receptor activity MHV-A59 and conferred MHV-JHM receptor activity.

My research suggests that projecting hydrophobic residues from mCEACAM1a proteins may fit into a putative hydrophobic pocket in the MHV spike glycoprotein that is variable among MHV strains. These findings will aid in engineering MHV-resistant mice and in designing novel anti-viral therapies that interfere with receptor-spike interactions.