Gelbart/Knobler Research Group

Gelbart/Knobler Lab

We are a group of biophysicists and molecular and cellular biologists, trying to figure out how viruses “work” from a physical-science point of view, and how to use reconstituted non-infectious forms of them – virus-like particles – for biotechnology and translational medicine purposes. We focused initially on "viruses as physical objects," determining the solution

conditions necessary to reconstitute them from purified components.

Presently, a large portion of our work is devoted to developing virus-like particles as vectors for RNA gene delivery and therapeutics.

Bill and Chuck started the lab in 2003, after each had worked for decades (!) in fields that have little (actually, nothing) to do with viruses (or anything else biological, for that matter). Bill had carried out theoretical work on the statistical mechanics and optical properties of simple liquids and on phase transitions in liquid crystals and self-assembling systems; and Chuck had done experimental work on critical phenomena in liquid solutions, kinetics of phase transitions, and the physics of molecular monolayers on liquid and solid surfaces.

Research Overview

Physical Chemistry of the Self-Assembly of Simple Viruses

Current projects involving the physical aspects of viruses:

3D Cryo-Electron Microscopy (CryoEM) Reconstructions of RNA Inside Viral Capsids
Distribution of RNA Ends in Multiplet-Capsid Structures
In vitro Synthesis of Enveloped Virus-Like Particles (EVLPs)
RNA Homopolymer Packaging by CCMV - the Role of RNA Secondary Structure on Self-Assembly
Catching the Ends of Fluctuating RNA Outside Intact Capsids
Characterization of Assembly Mechanism through Cryo-Electron Tomography (CryoET) of Individual RNA-Capsid Protein Assembly Intermediates
CryoET Reconstructions of Bromovirus (B1, B2, and B3/4) Virions

Virus particles can be as simple as a molecule of RNA or DNA inside a spherical shell – the “capsid” – made up of multiple copies of a single protein. Further, because viruses only “become alive” when they are inside their hosts, it is possible to study them -- outside their hosts, in a test tube -- as physical objects, i.e., to do the same controlled experiments (and theory) on them that one routinely does with more familiar polymer and colloidal systems.

This approach is best illustrated with the example of one of the simplest viruses we work on – cowpea chlorotic mottle virus (CCMV) – a plant virus with a single-stranded RNA genome. Each particle of CCMV is a one-molecule-thick spherical shell consisting of (exactly!) 180 copies of a single protein, surrounding and protecting single copies of RNA genes of the virus. The 180 capsid proteins are organized into groups of 12 pentamers and 20 hexamers, involving 3 sets of inequivalent positions.

Most remarkably, this icosahedrally-symmetric (soccer-ball-like/”Bucky-ball”-like) structure can form spontaneously: one simply has to mix the purified RNA molecules and purified capsid proteins under the right conditions of pH and ionic strength and a virus forms that is infectious and indistinguishable from those found in infected plants. By changing these solution conditions, and replacing the viral RNA by non-viral RNA (and even by synthetic anionic polymers) and replacing the wildtype capsid protein by genetically- and/or chemically- modified forms of it, we are able to learn a lot about the fundamental physical chemistry of this biological process – the formation of an infectious virus, and also to prepare well-characterized and effective gene delivery systems.

Targeted Delivery of Self-Amplifying RNA Genes

As a result of millions of years of evolution, what viruses do better than anything else - better than anything else they do, or any thing else does - is to protect and deliver genes to specific (targeted) cells. This is why it is impossible not to consider their use as a gene delivery systems for medical purposes. To avoid the complications and potential dangers of using "attenuated" or "inactivated" viruses, however, we pursue a research program that involves the design and synthesis - from purified components in test tubes - of virus-like particles (VLPs) that contain genes of interest in single-stranded RNA (ssRNA) form. The ssRNA form is essential for enabling spontaneously self-assembly of the gene into a VLP when mixed with the right viral capsid protein (CP); this isn't possible, for example, with double-stranded DNA (dsDNA). For self-assembly purposes, the "right" CP is one that will efficiently package any RNA, independent of its sequence, as long as it isn't too long or too short. The CP from tobacco mosaic virus (TMV) also works for this purpose (forming cylindrical capsids), after inserting a "packaging signal" sequence into the heterologous RNA of interest. We have found that CP from cowpea chlorotic mottle virus (CCMV) or brome mosaic virus (BMV) does this job. The other advantage of RNA genes is that it is possible to work with them in messenger-sense and self-amplifying form, enabling them to be strongly translated and replicated in the cytoplasm of targeted host cells before being translated into proteins of interest.

The scenario outlined above - for in vitro reconstituted VLPs consisting of self-amplifying RNA inside a protective protein shell - mimics closely the life cycle of many of the simplest and most common viruses. The figure here shows the replication and protein synthesis scheme for molecule 1 of the two-molecule RNA genome of the insect virus Nodamura, into which we have added a gene of interest (GOI) at the end of the open reading frame (ORF) coding for the RNA-depended RNA polymerase (RdRp). This RNA replicase protein binds the RNA molecule (that encodes it and the GOI) and generates large numbers of reverse-complement (negative-sense) strands that are in turn transcribed by the replicase to make up to hundreds of thousands of copies of the original molecule. T2A encodes a self-cleaving peptide sequence that ensures the independence/function of the RNA replicase and GOI proteins.

Current projects involving the delivery of genetic information in RNA form:

Design and synthesis of self-replicating forms of mRNA (replicons)
Synthesis of "stealth" virus-like particles
In vitro reconstituted VLPs for delivery of microRNA replicons
In vitro reconstituted CCMV VLPs for delivery of mRNA and self-amplifying mRNA
In vitro reconstituted TMV VLPs for delivery of RNA therapeutics
Delivery of self-amplifying adjuvants to trigger interferon production
Targeted delivery of gene therapies through protein fusions and chemical conjugations of ligands to reconstituted VLPs
In cellulo syntheses of RNA-specific TMV and Sindbis virus-like particles
Quantification of in trans vs in cis RNA replication