signal The SV40 large T-antigen has been used as a model protein to study nuclear localization signals (NLSs). It is imported into the nucleus by its interaction with importin α. The NLS sequence is PKKKRKV. Interaction with pRb via the LXCXE motif SV40 large TAg, other polyomavirus large T antigens, adenovirus E1a proteins, and oncogenic human papillomavirus E7 proteins share a structural motif that encodes a high-affinity pRb-binding domain. A diagnostic pattern for a high-affinity pRb-binding domain was refined using an artificial intelligence pattern-induction program running on a massively parallel supercomputer (Connection Machine-2). The motif is characterized by an Asp, Asn or Thr residue followed by three invariant amino acids, interspersed with non-conserved amino acids (designated by x, where x cannot be a Lys or Arg residue). A negatively charged region frequently follows carboxy-terminal to the pRb-binding domain. {Asp/Asn/Thr} – Leu – x – Cys – x – Glu – x – ... {negatively charged region} Hydrophobic and electrostatic properties are highly conserved in this motif. For example, a local hydrophobicity maximum occurs in the vicinity of the invariant Leu residue. A net negative charge occurs within 3 residues amino-terminal to the invariant Leu residue; furthermore, positively charged amino acids (Lys or Arg) are not found within the Leu – x – Cys – x – Glu sequence, nor in the positions immediately flanking this sequence. The pRb-binding motif and negatively charged region match to a segment of SV40 TAg beginning at residue 102 and ending at residue 115 as shown below: – Asn – Leu – Phe – Cys – Ser – Glu – Glu – Met – Pro – Ser – Ser – Asp – Asp – Glu – Functional studies of TAg proteins bearing mutations within this segment (amino acid positions 106 to 114, inclusive) demonstrate that certain deleterious mutations abolish malignant transforming activity. For example, mutation of the invariant Glu at position 107 to Lys-107 completely abolishes transforming activity. Deleterious mutations within this segment (amino acid positions 105 to 114, inclusive) also impair binding of the mutant TAg protein species to pRb, implying a correlation between transforming activity and the ability of TAg to bind pRb. A detailed computerized bioinformatics analysis, as well as an x-ray crystallography study, have demonstrated the biophysical basis for the interaction between this region of TAg and pRb. TAg residues 103 to 109 form an extended loop structure that binds tightly