Protein fragment complementation assay for studying viral protein-protein interactions

Herpesviruses cause highly prevalent infections associated with
usually mild symptoms resulting in life long latency. However, they
can provoke fatal disease in susceptible individuals such as
immunocompromised patients either in the context of primary
infection or after reactivation from latency. Despite of their
emerging medical importance, herpesvirus infections can so far only
be controlled by antiviral therapy targeting viral DNA replication,
accompanied with side effects and occurrence of resistant strains.
In this work, a novel platform for drug discovery was established
that is based on a protein complementation assay (PCA), which can
be used to study viral protein-protein interactions in a simple
cell based assay. In a PCA two inactive fragments of a reporter
enzyme are fused to two interacting proteins. Interaction of the
proteins leads to proximity of the enzyme fragments, followed by
reconstitution of the reporter enzyme activity. Members of the UL34
and UL31 families are conserved herpesvirus proteins. They interact
with one another forming the nuclear egress complex (NEC), which is
essential for the export of viral capsids from the cell nucleus.
This crucial protein-protein interaction might serve as a potential
drug target for anti-herpesvirus chemotherapy. In this work the
mutual binding sites of the two proteins were localized and studied
for their conservation. A PCA was established for M50 and M53 – the
NEC proteins of the murine cytomegalovirus (MCMV) - by fusion of
the N- and C-terminal part of the TEM-1 ß-lactamase of E. coli. The
assay was validated and applied to representative members of the
three herpesvirus subfamilies. Cross-complementation assays showed
that partners derived from the same subfamily can replace each
other in the PCA, however, homologues from different subfamilies
can not. This cross-complementation reflects the in vivo situation:
the human cytomegalovirus (HCMV), but not the HSV-1 or PrV
homologues are able to rescue the M50 or M53 null phenotype in the
viral context of MCMV. The lack of complementation between the
subfamilies is due to their diverged binding sites, which are
located in all cases within the first conserved region of the UL31
family proteins. The study of the binding site in UL34 family
members revealed a bipartite binding motif. With the aim of a
future high-throughput inhibitor screen an in vitro NEC-PCA was
established using purified fusion proteins of HCMV.