Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus associated with significant morbidity and mortality in immunocompromised and immunosuppressed individuals. The two major immediate early proteins of HCMV, immediate early protein 1 & 2 (IE1/IE2) are nuclear localised proteins expressed immediately after viral infection. These multifunctional proteins are potent transactivators of viral gene expression and are key regulators of host and viral protein function during lytic infection.
IE1/IE2 share a common N-terminal region of 85 amino acids and diverge at the C-termini due to alternative splicing and polyadenylation. A predicted nuclear localisation signal (NLS) in the N-terminus of IE1 and IE2 has been identified, with two additional NLSs predicted in exon 5 of IE2. However, the mechanistic basis regulating nuclear localisation of these two proteins and the physiological importance of encoding multiple NLS remains to be elucidated.
To identify the structural basis of binding of the NLS of IE1/IE2 to nuclear import receptors (importins) each NLS was crystallised with importin α before the atomic structure of the complex was resolved. In addition, the specificity of the IE1/IE2 NLS for specific importin family members was determined by fluorescence polarisation and electromobility shift assays, with enhanced affinity for importin α3 identified. The crystal structure of the shared IE1/IE2 NLS in complex with multiple importin family members elucidated that this NLS was bipartite in nature with key basic residues binding in both the major and minor binding site of the importin molecules. The NLS sequences unique to IE2 exhibited monopartite binding to the major site of importin alpha. This biochemical and structural analysis provides a platform for understanding viral-host structure function relationships during CMV infection as well as providing insights into the contribution of specific importin α isoforms to regulating the initiation of the viral gene expression cascade