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A Very Late Viral Protein Triggers the Lytic Release of SV40 1,2 1 1,2* Robert Daniels , Dorota Sadowicz , Daniel N. Hebert 1 Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts, United States of America, 2 Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts, United States of America How nonenveloped viruses such as simian virus 40 (SV40) trigger the lytic release of their progeny is poorly understood. Here, we demonstrate that SV40 expresses a novel later protein termed VP4 that triggers the timely lytic release of its progeny. Like VP3, VP4 synthesis initiates from a downstream AUG start codon within the VP2 transcript and localizes to the nucleus. However, VP4 expression occurs ;24 h later at a time that coincides with cell lysis, and it is not incorporated into mature virions. Mutation of the VP4 initiation codon from the SV40 genome delayed lysis by 2 d and reduced infectious particle release. Furthermore, the co-expression of VP4 and VP3, but not their individual expression, recapitulated cell lysis in bacteria. Thus, SV40 regulates its life cycle by the later temporal expression of VP4, which results in cell lysis and enables the 50-nm virus to exit the cell. This study also demonstrates how viruses can generate multiple proteins with diverse functions and localizations from a single reading frame. Citation: Daniels R, Sadowicz D, Hebert DN (2007) A very late viral protein triggers the lytic release of SV40. PLoS Pathog 3(7): e98. doi:10.1371/journal.ppat.0030098 Introduction new SV40 gene product called VP4 that is expressed ;24 h after the late structural proteins V