VISA

Most viruses have as their genome – not double-standed (ds)DNA, as is the case with all other evolving organisms, but rather – single-stranded (ss)RNA. Further, because ssRNA is much more compact and compressible than dsDNA, it is possible to make ssRNA-genome virus particles from their purified components, ssRNA and capsid protein, by simply mixing these components in physiological buffer solution. Each infectious particle consists of a single RNA molecule inside a shell comprised of hundreds of copies of the capsid protein. These remarkable facts have facilitated a large number of experimental and theoretical investigations of the underlying co-assembly process – the self-assembly of perfectly-ordered/icosahedrally-symmetric nucleocapsids. And it is widely believed that this “test-tube”/in vitro phenomenon mimics the formation of a new generation of virus particles in their host cell, following the replication of their ssRNA genome and the synthesis of a hundreds-of-times-larger number of capsid proteins. But, in fact, there is to date essentially no experimental or theoretical program that sets out to bridge the gap between the knowledge bases acquired from in vitro studies of virus self-assembly on the one hand and intracellular viral replication on the other. In the present project, we propose a first set of systematic steps towards connecting these two sets of phenomena. In particular, we argue that the difference between in vitro and intracellular (cytoplasmic) self-assembly of virus particles from their RNA and capsid protein is twofold: (1) in cytoplasm these basic components are embedded in a concentrated solution of proteins and nucleic acids that is highly viscous and whose osmotic effects alter the conformations of and interactions between the viral RNA and capsid proteins; and (2) in the cellular context, the capsid proteins are being actively synthesized, i.e., through ATP consumption, by the ribosomal machinery so that a nearly-constant influx of capsid proteins drives the virus self-assembly process towards a nonequilibrium state. To connect the in vitro and cellular contexts, we propose to extend our earlier measurements of virus self-assembly from purified RNA and capsid protein components by: (1) carrying these reactions in the controlled presence of calibrated osmolytes and by synthesizing capsid protein in cytoplasmic extracts where messenger (m)RNA is translated into capsid protein which in turn packages it into nucleocapsids; and (2) monitoring capsid protein and nucleocapsid production directly in live cells. These processes will be investigated and quantified by a mix of state-of-the-art: experimental techniques including solid-state NMR, time-resolved synchrotron X-ray scattering, cryotransmission electron and super-resolution fluorescence microscopies; and analytical theory and coarse-grained computer simulations. All of this work will be carried out in parallel for a mammalian and a plant virus – Hepatitis B virus (HBV), which has a DNA genome but whose genetic information is packaged in the cytoplasm in mRNA form, and cowpea chlorotic mottle virus (CCMV), respectively, both well characterized by in vitro studies – in order to provide for the first time a general foundation for and understanding of virus self-assembly in cellular environment.