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Chimeric antigen receptor (CAR) T cell therapy is a relatively new and powerful way of transforming T cells with receptors needed to recognize and kill diseased cells. Traditionally, it involves extraction of T cells from a patient, ex vivo transformation of them with CARs, expansion, and subsequent re-infusion into the patient. Recent developments aim to avoid this lengthy, costly patient-specific procedure by using var- ious viral and non-viral vector particles for direct in vivo delivery of CAR-encoding genes. In this paper we highlight several fundamental connections between in vitro and in vivo aspects of this process. We dis- cuss the proposed use of in vitro-reconstituted virus-like particles (VLPs), prepared from purified CAR- encoding mRNA and viral capsid protein, and functionalized with a T cell-targeting antibody. We compare and contrast these particles – and their use as gene vectors – with the several modalities currently employed that involve in cellulo generation of lentiviral or AAV vectors or in vitro complexation of nucleic acids with cationic polymers or lipid vesicles. We report the unique stoichiometric preciseness and ther- modynamic stability of VLPs formed from anti-HIV-glycoprotein CAR-encoding mRNA and the capsid pro- tein from a plant virus, and quantify the extent to which these monodisperse spherical VLPs are RNase resistant and lead to strong CAR expression in T cells. Further, in vitro cell-killing experiments are pro- posed, in which these CAR VLP-transformed T cells are mixed with HIV-infected cells, to be followed by in vivo experiments involving injection of the particles into HIV-infected humanized mice. 

In the presence of polyvalent cations, long double-stranded DNA (dsDNA) in dilute solution undergoes a single- molecule, first-order, phase transition (‘‘condensation’’), a phenomenon that has been documented and analyzed by many years of experimental and theoretical studies. There has been no systematic effort, however, to determine whether long single- stranded RNA (ssRNA) shows an analogous behavior. In this study, using dynamic light scattering, analytical ultracentrifugation, and gel electrophoresis, we examine the effects of increasing polyvalent cation concentrations on the effective size of long ssRNAs ranging from 3000 to 12,000 nucleotides. Our results indicate that ssRNA does not undergo a discontinuous conden- sation as does dsDNA but rather a ‘‘continuous’’ decrease in size with increasing polyvalent cation concentration. And, instead of the 10-fold decrease in size shown by long dsDNA, we document a 50% decrease, as demonstrated for a range of lengths and sequences of ssRNA. 

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles(VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chloroticmottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying- mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic. 

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA–protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)—for which RNA–protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA–protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA–protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA–protein interactions, while the growth process is driven less by RNA–protein interactions and more by protein–protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses. 

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV. 

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content—specifically, RNAs 3 and 4—assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific “packaging signals” throughout the viral RNA to package their monopartite genomes.