Assembly of a Genome Packaging Motor on Viral DNA Influences Packaging Mechanism

Abstract

During virus assembly, a single genome is packaged into a preformed capsid by a terminase enzyme. Presently, the mechanistic details of the packaging motor are unknown. Single molecule experiments have demonstrated that viral DNA packaging motors are extremely powerful, capable of packaging dsDNA to over 20 atmospheres. Therefore, the mechanistic details of DNA packaging are of interest from both a biological (drug target), and engineering (nanomachine) standpoint. Bacteriophage Lambda (λ) is a model system for the study of dsDNA viruses, including herpesvirus and many bacteriophage. λ terminase is a multifunction enzyme complex with catalytic activities required to (1) site-specifically bind the viral genome, (2) carry out nuclease and helicase activities on the viral genome, (3) bind the empty procapsid, and (4) hydrolyze ATP and translocate viral DNA into the procapsid. The holoenzyme consists of proteins gpA and gpNu1 in a 1:2 ratio called the heterotrimeric protomer. The functional motor is composed of four protomers that assemble into a ring at the packaging initiation site of viral DNA.Here we describe physio-kinetic studies on terminase protomer assembly into a catalytically-competent genome maturation and packaging complex. Assembly of the protomer on viral DNA is much faster than previously thought and affords a motor that packages DNA with high specificity. Furthermore, the packaging ATPase activity of the protomer is down-regulated until both the viral genome and empty procapsid are mated by terminase in preparation for packaging. The ring-tetramer can be artificially assembled in vitro in the absence of DNA and is functional to package DNA. We find that there are important differences between ring and protomer with assembly of a motor on DNA influencing the packaging mechanism. Our results suggest that the protomeric unit is the biologically relevant species in vivo.