Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Microbiology & Plant Pathology
Non Technical Summary
The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that in solution virions are highly dynamic assemblies. We propose to unravel the functional relationship between capsid dynamics and pathogenesis. To this end, we will use Brome mosaic virus (BMV)- an RNA virus pathogenic to plants, as a model system. The three genomic RNAs (RNAs 1, 2, and 3) and a single subgenomic RNA (RNA4) of BMV and CCMV (BMV), are distributed among three physically homogeneous virions. The proposed study employs theAgrobacterium-mediated transient expression approach for independently assembling three virion types. Then, we will employ state-of-the-art techniques to identify the dynamics of the three capsid types and their mutants with an eventual of understanding how capsid dynamics play an important biological role in the viral life cycle and pathogenesis.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although viral capsids appear to be highly uniform, recent measurements suggest that virions are highly dynamic in nature. Do capsid dynamics functionally regulate pathogenicity? And to what extent does the packaged genomic RNA affect capsid dynamics? We propose to answer these questions via a multidisciplinary approach involving genetics and molecular biology, biochemistry, physical chemistry, and nanotechnology. To this end, we exploit as a model system the genetically and structurally well-characterized Brome mosaic virus (BMV), pathogenic to plants. The three BMV genomic RNAs (RNAs 1, 2, and 3) and a single subgenomic RNA (RNA4) are distributed among three virions that cannot be separated - purified from one another - by physical methods. Using the Agrobacterium-mediated transient expression approach, we propose to assemble in planta the three virion particles of BMV one at a time, i.e., capsids containing only RNA1 (type 1: B1 capsids) or RNA (type 2: B2 capsids) or RNA3+4 (type 3: B3+4 capsids). The central objectives of this proposal are to: (i) extend to in vitro reconstituted virions and to in vivo hybrid virus-like particles - in different hosts - the kind of proteolysis MALDI-TOF analyses we have recently carried out with in vivo pure virions; (ii) extend to type 1 and 2 virions the kind of high-resolution 3D cyroEM reconstruction we have performed on type-3 virions and to enhance the analysis by employing single-particle tomography (SPT), looking for differences not only in RNA organization but also in the structure of the capsid itself; and finallyObjective: 1 Determine the role of packaged RNA and the host in controlling capsid dynamics and pathogenesisObjective: 2 Determine the atomic structure of independently assembled pure virions of the BMV and employ single-particle cryoelectron tomography (STP) to characterize differences in the capsid structure
Project Methods
(1) Mutational analysis of the surface-exposed trypsin sites of B1V or B2V or B3+4V: To evaluate the effect of altering specific trypsin sites on the dynamics and subsequently on pathogenicity of B1V, B2V and B3+4V, we propose to introduce a series of CP mutations involving the trypsin-accessible sites located on the different virion types.(2) Assembly of BMV and CCMV hybrid VLPs: We propose: (i) to construct six hybrid VLPs using our established in vitro assembly procedures (Annamalai and Rao, 2005; 2008) mixing purified CP of each virus with one or the other RNAs from its own genome or the other viral genome; (ii) to assemble BMV VLPs packaging non-viral RNAs of different lengths varying between 140-5300 nt.. The desired RNA templates will be generated by in vitro transcription of linearized plasmids using T7 RNA polymerase. Dissociated CP subunits amenable to in vitro assembly will be obtained from purified virions using the CaCl2 method.(3) Contribution of sub-genomic (sg) RNA4 packaging to capsid dynamics: We propose to perform both in vitro and in vivo experiments to assemble virions containing only B3- containing VLPs. In vitro assembly of only B3-containing VLPs involves our standardized procedure of mixing transcripts of full-length B3 RNA with CP at a ratio of 4.5:1 (w/w) followed by dialysis for 24h at 4°C. VLPs co-packaging B3+4 will be used as controls. For in vivo assembly experiments, the strategy used to assemble B3+4V(Chakravarthy et al., 2020), will be used except for wild-type B3 plasmid being replaced by a mutant plasmid of B3, referred to as B3SGP, engineered to be defective in the synthesis of sgB4 due to a mutation in the sgRNA initiation site [30]. Consequently, following infiltration, B3SGP will replicate to produce B3 RNA but not sgB4. Transiently co-expressed CP subunits after their interaction with replicase proteins would package replicated B3 resulting in the accumulation of VLPs having only B3. Virions B3+4V will be used as positive controls. Dynamics of the B3 VLPs and the B3+4V virions, assembled in vitro and in vivo, will be evaluated by digesting each sample with trypsin at various time points and identifying released peptide fragments by MALDI-TOF analysis.(4) Functional relationship between capsid dynamics and pathogenesis: BMV and CCMV are readily distinguished by the symptom phenotypes they induce in barley, cowpea and C. quinoa. For example, barley is susceptible to BMV but not to CCMV, and cowpea is susceptible to CCMV but not to BMV. C. quinoa is a commonly shared host between BMV and CCMV, but in this host BMV induces chlorotic local lesions followed by systemic mottling whereas CCMV induces necrotic local lesions without systemic infection. We previously have demonstrated that a mutation engineered into the N-ARM region of the BMV CP modified the symptom phenotype. To evaluate the contribution of the capsid dynamics to regulating the host range and pathogenesis we propose to perform experiments involving the exchange of coat protein genes between BMV and CCMV.(5) Role of replicase-CP interaction in controlling capsid dynamics:In BMV and other viruses it has been conclusively shown that interaction of replicase with CP dictates packaging specificity. Therefore, we hypothesize that the replicase-CP interaction controls capsid dynamics. To substantiate this hypothesis, we propose to exploit the inherent compatibility of replicase and capsid proteins by assembling a series of hybrid viruses between BMV and CCMV.(6) Symptom phenotypes: Symptom expression will be monitored for 5-15 days and virions purified from the symptomatic plants will be subjected to trypsin digestion followed by MALDI-TOF analysis. Since the BMV and CCMV CPs show such strong homology at the amino acid level, the contribution of otherwise conserved amino-acid sequences to capsid dynamics will be tested by assembling a set of CP chimeras. Each full-length chimera construct will be co-infiltrated with p1a and p2a and the resulting virions assembled from chimeric CP will be subjected to trypsin digestion followed by MALDI-TOF analysis and compared to control virions of BMV and CCMV containing, respectively, RNA3 and sgRNA. Because barley plants are not amenable to agroinfiltration we will mechanically inoculate barley, cowpea and C. quinoa plants with in vitro synthesized transcripts in desired combinations, in order to analyze the symptom phenotypes induced by each chimera. Plants inoculated with wild-type BMV and CCMV will serve as controls.(7) Cryo EM and tomography: We propose to use cryoEM and asymmetric reconstruction methods to build atomic resolution density maps of B1V and B2V, and to compare these to our recent study of B3+4V (Beren et al., 2020). By building density maps for each pure virion type we can examine structural differences, differences in structural heterogeneity, and differences between protein-RNA contacts in the three virion types. cryoelectron tomography (SPT), which reconstructs virus particles one-by-one rather than by sorting into 3D classes; the trade-off is that without averaging over many particles, the resolution obtained decreases from several A? to several nm. However, the resolution obtained for the B3+4V RNA genome using averaging and classification is already as low as several nm, so that the theoretical limitations related to doing tomography may be irrelevant for imaging the RNA inside of viral capsids. We propose to use this tomographic reconstruction to generate many 10s of density maps for each of the three BMV virion types, and to compare both the capsid and RNA structures generated within and across particle types. In this way we will directly compare both structural differences and differences in structural heterogeneity associated with the differences in dynamics associated with the virion types. An advantage of the tomographic studies is that they can be applied to follow the evolution of structural features after a change in conditions such as pH, temperature or protease digestion. By taking images of samples at different time points (i.e., matching the time course used in the mass spectrometric analyses), we can follow the evolution of the density maps for each particle type after subjecting the virions to protease. Using this technique, we hope to resolve portions of both the RNA and CP that become accessible at various time points during the proteolysis. The technique can also potentially be useful for resolving an RNA end fluctuating out of the capsid. To enhance our ability to distinguish between CP and RNA that has fluctuated outside the capsid we can carry out the studies in the presence of a translation initiation factor such as eIF4F, which will bind to the emerging RNA.