Cellular transcription factor Sp1 recruits simian virus 40 capsid proteins to the viral packaging signal, ses.
Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP1(5)) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP1(5)-VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP1(5)-VP2/3-DNA-Sp1 complex.
Gordon-Shaag
Ariela
A
Department of Hematology, The Hebrew University-Hadassah Medical School and Hadassah University Hospital, Jerusalem 91120, Israel.
Ben-Nun-Shaul
Orly
O
Roitman
Vered
V
Yosef
Yael
Y
Oppenheim
Ariella
A
D001665:Binding Sites; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D004251:DNA Transposable Elements / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D012045:Regulatory Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000235:genetics*
D036022:Capsid Proteins; D004251:DNA Transposable Elements; D004279:DNA, Viral; D016329:Sp1 Transcription Factor; C062481:VP1 protein, polyomavirus; C085902:VP3 protein, Simian virus 40