The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn 2+ ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.