Simian Virus 40 Vp1 DNA-Binding Domain Is Functionally Separable from the Overlapping Nuclear Localization Signal and Is Required for Effective Virion Formation and Full Viability

The virion particle of simian virus 40 (SV40), like those of other papovaviruses, packages the double-stranded circular viral DNA in an icosahedrally symmetric capsid. The SV40 capsid is composed of 72 pentamers of the major capsid protein Vp1, and the pentameric units are tied together by the carboxy-terminal arms which extend into neighboring pentamers (20, 27). The amino-terminal 15 amino acids of Vp1 are not visible in the crystal structure due to disorder but probably extend into the virion core to interact with the viral minichromosome (20), which consists of the viral DNA and the four cellular core histones. A lower-resolution view of murine polyomavirus virions identifies molecules of the minor capsid proteins Vp2 and Vp3 (Vp2/3) as prongs that extend from the minichromosome core into the axial cavities of Vp1 pentamers (10). Though much is known about the structure of the virion, how virions are assembled in the cell nucleus during productive infection is still not well defined. Presumed to be important for the assembly is the interaction of the capsid proteins with DNA and with histones. Since all capsid proteins of SV40 bind DNA (4, 26), but only Vp1 of polyomavirus does (2, 22), the interactions involved in the assembly process can be different even among members of the papovavirus family. Defining a functional DNA-binding domain (DBD) for the interaction of each capsid protein with viral DNA may unravel the unique process for the assembly of individual viruses. SV40 assembly is thought to consist of two phases. In the “subvirion” assembly phase, pentamerized Vp1 associates with Vp2/3 in the cytoplasm soon after the proteins' synthesis, and they are transported to the nucleus as such subvirion complexes (9, 21). All of the SV40 Vp1, Vp2, and Vp3 harbor nuclear localization signals (NLSs) (3, 13). Consistent with the capsid proteins' interaction prior to nuclear transport, wild-type Vp2/3 could rescue the nuclear localization of an NLS-defective Vp1 and vice versa (13). This phenotypic complementation would prove useful in our functional dissection of the Vp1 DBD (see below). The second phase of “virion” assembly begins once the capsid proteins enter the nucleus, the site of viral DNA replication and packaging. In a stepwise model for virion formation, the capsid proteins are sequentially added to and arranged on the viral minichromosome, resulting in the condensation and packaging of the viral DNA and the formation of the capsid (1, 5, 15). All three SV40 capsid proteins can bind DNA nonspecifically (4, 26). The cooperative binding of Vp3 and the transcription factor Sp1 to ses, the encapsidation signal of SV40 DNA, has been proposed to provide packaging specificity by nucleating the capsid proteins' addition to the viral minichromosome (6, 11, 23). The DNA-binding and protein-interactive functions of individual capsid proteins may collectively contribute to the packaging or virion assembly process. In this study, we mapped the DBD of SV40 Vp1 and found that it overlaps with the previously identified, amino-terminal bipartite NLS (14), which comprises two clusters of basic residues, 4-lysine-arginine-lysine-6 and 15-lysine-lysine-proline-lysine-18. We also examined whether this DBD is important for the formation of infectious virions in the viral life cycle. Despite the overlap of the DBD with the NLS, the application of phenotypic complementation has allowed us to identify at least the second basic cluster, represented by mutant NLSm2, as a determinant for nuclear virion assembly. Our results are consistent with an important role of the Vp1 DNA-binding activity in the proper packaging of virions.