Dr. Joseph Ahearn hired
me as a technician upon my immigration to the US. His lab was studying
the molecular basis of infection of human cells by Epstein-Barr virus (EBV).
To enter B-lymphocytes EBV uses human complement receptor type II (CR2)
(2,3). An extracellular portion of the CR2
molecule consists of the 16 short consensus repeats (SCRs). EBV was
found to have narrow tropism and is capable of infecting only human B cells
but not mouse B lymphocytes (1). Ahearn's lab used
this EBV property to map CR2 domains that are responsible for EBV binding.
We have constructed multiple human-mouse chimerical CR2 molecules and expressed
them in COS cells to assess their ability to bind EBV. EBV binding
to COS cells was detected by incubating transient trasformants with EBV
and then with mouse anti-gp350 MAb and DTAF-conjugated goat anti-mouse
antibodies.
I was responsible for constructing the human-mouse
chimerical molecules. Don Martin was using two-step
PCR technique to make fusion proteins and I was screening for recombinant
clones and their confirmation by sequence analysis. I have also constructed
several chimerical CR2 molecules by conventional subcloning from previously
made chimeras and did several COS cell transfection experiments.
As a result of this effort we demonstrated that the first two N-terminal
SCRs of the human CR2 were necessary and sufficient for EBV binding.
Remarkably, the same SCRs are necessary and sufficient for binding to C3dg,
which is the natural CR2 ligand 1.
Since human CR2 can bind mouse C3dg as well, it was possible to predict
from homology analysis between human and mouse SCRs the individual aminoacids
in human CR2 sequence that are responsible for EBV binding.
REFERENCES
1. Sugden, B. An intricate route to immortality. Cell,
57:5, 1989
2. Fingeroth, J.D. et al. Epstein-Barr virus
receptor of human B-lymphocytes is the C3d receptor CR2. PNAS, 81:4510,
1984
3. Nemerow G.R., Identification and characterization of the EBV receptor
on human B-lymphocytes and its relationship to the C3d receptor (CR2).
J.
Virol., 55:347
Two-step PCR
One part of the chimerical
molecule is made in the first step by PCR using human CR2 as a template.
The upstream PCR primer has a unique XmaI site. The second part of
the chimera is made by using the amplified human fragment from the first
step as an upstream primer for PCR using mouse CR2 as a template and the
downstream primer with XhoI restriction site. Then XmaI-XhoI PCR
fragments were recloned intothe mouse or human complete cDNA and
used for the EBV binding studies.
1. From reviewing the literature it becomes clear that most receptors bind ligands by their N-terminal domains. The simple reason is that probably only N-terminal of the receptors are readily exposed and other parts are buried by glycosilation and neighboring protein molecules. (back to main reading)