A. L. N. Rao

Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. In viruses, conformational changes are essential for establishing infection and evolution.

The tripartite genome of Cowpea chlorotic mottle virus is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1 ^V and C2 ^V , package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4 ^V , co-packages genomic RNA3 and its sub-genomic RNA (RNA4). In this study, each virion type was separately assembled in planta using a previously established agroinfiltration approach. The three virion types are indistinguishable under the EM and by electrophoretic mobility. However, the stability and the capsid dynamics evaluated respectively by the differential scanning fluorimetry and MALDI-TOF analysis following trypsin digestion distinguished the three virions type. Proteolytic analysis revealed that, while retaining the structural integrity, C1 ^V and C2 ^V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4 ^V virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. In addition, we also evaluated the effect of the two host organisms on the capsid dynamics. The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from N. benthamiana remained resistant. Finally, the MALDI-TOF evaluated the relative dynamics of C3+4 ^V and B3+4 ^V virions assembled under the control of the homologous vs. heterologous replicase. The significance of the viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4 ^V and C3+4 ^V virions assembled under the homologous vs. heterologous replicase. The significance of these results in relation to viral biology is discussed. IMPORTANCE: Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. Cowpea chlorotic mottle virus (CCMV) and Brome mosaic virus (BMV), RNA viruses pathogenic to plants, contain a segmented genome distributed among three physically and structurally homogenous icosahedral virion types. This study examines the role of the host organism and the heterologous viral replicase in modulating the thermal stability and capsid dynamics in CCMV and BMV. The results accentuate that alteration of the capsid dynamics by the host type and heterologous replicase are likely to affect the biology of the virus.

Protein–protein interactions (PPI) are vital in regulating the biological and physiological functions in a given cell or organism. Proteomics, in conjunction with bioinformatic tools, represents the study involving the characterization of the protein content of the genome of a given biological system. Like PPI, an interaction between either coding or noncoding RNA and a complex set of host proteins protein plays an essential role in gene expression at translational, posttranscriptional, and epigenetic level. Although a wide range of techniques such as shotgun proteomics, MuDPIT, etc. are available for characterizing PII, those for characterizing RNA–protein interactions are infancy. Given the significance of the long noncoding RNAs (lnc-RNA) in plant biology, it is imperative to isolate and characterize the functionality of the host proteome interacting with RNA. In this context, riboproteomics approach becomes a valuable tool to study these interactions. Here, using a noncoding plant pathogenic satellite-RNA (Sat-RNA) of Cucumber mosaic virus (CMV) as an RNA source, we describe a stepwise protocol for identifying the host proteome interacting specifically with the Sat-RNA. This protocol streamlines steps starting from in vitro transcription of RNA, preparation of RNA affinity column, preparation of cell lysate from Nicotiana benthamiana leaves infected with the Sat-RNA followed by the Co-IP and preparation of samples for LC-MS/MS. We believe this approach is applicable to a wide range of RNAs of any nature associated with eukaryotic and prokaryotic organisms.

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.

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. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium -mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

A hallmark feature of (+)-strand RNA viruses of eukaryotic cells is that progeny (+)-strands are accumulated 100-fold over (−)-strands. Previous experimental evidence suggests that, in Brome mosaic virus (BMV), a plant-infecting member of the alphavirus-like superfamily, the addition of RNA3 and, specifically, translation of the wild-type (WT) coat protein (CP) gene contributes to increased accumulation of (+)-strands. It is unclear whether this stimulation of (+)-strand accumulation by CP is due to direct regulation of viral RNA replication or RNA stabilization via encapsidation. Analysis of BMV progeny RNA in Nicotiana benthamiana plants revealed that expression of RNA3 variants that did not express WT CP led to a severe defect in BMV (+)-strand accumulation. The (+)-strand accumulation could be rescued when CP was complemented in trans. To verify whether stimulation of (+)-strand accumulation is coupled with encapsidation, two independent mutations were engineered into CP open reading frames. An N-terminal deletion that prevented CP binding to the viral RNAs resulted in a severe reduction of BMV (+)-strand accumulation but stimulated (−)-strand accumulation over the WT. On the other hand, a C-terminal mutation affecting CP dimerization caused a significant decrease in (+)-strand accumulation but had no detectable effect on (−)-strand accumulation. Nucleotide sequences in the movement protein-coding region were also found to contribute to (+)-strand accumulation, in part by providing packaging signals for efficient RNA3 encapsidation. Overall, these results show that RNA encapsidation is a significant determinant of BMV RNA intracellular accumulation.

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replicated three-molecule RNA progeny of Brome mosaic virus (BMV) are packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasi-symmetry. Type 1 (B1v) and type 2 (B2v) virions respectively package genomic RNA1 or RNA2, while type 3 (B3+4v) co-packages genomic RNA3 (B3) and its sub-genomic RNA4 (B4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta . Physical and biochemical approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types, so protease-based mapping experiments were used to analyze the conformational dynamics of the individual virions. The crystallographic structure of the BMV capsid shows four trypsin-cleavage sites (K ₆₅ , R ₁₀₃ , K ₁₁₁ and K ₁₆₅ on the A, B and C subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1v and B2v released only two peptides involving amino acids 2-8 and 16-22 from the N-proximal arginine-rich RNA binding motif. In contrast, B3+4v capsids are unstable to trypsin, releasing several peptides in addition to the four sites predicted to be exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids. IMPORTANCE: 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. The three genomic RNAs (RNAs 1, 2 and 3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the capsid dynamics by MALDI-TOF analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium- mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNAs1 and 2 are more stable and dynamically distinct from those co-packaging RNA3 and 4, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

Pathogenic or non-pathogenic small (17 to 30 nt) and long (>200 nt) non-coding RNAs (ncRNAs) have been implicated in the regulation of gene expression at transcriptional, post-transcriptional and epigenetic level by interacting with host proteins. However, lack of suitable experimental system precludes the identification and evaluation of the functional significance of host proteins interacting with ncRNAs. In this study, we present a first report on the application of riboproteomics to identify host proteins interacting with small, highly pathogenic, noncoding satellite RNA (sat-RNA) associated with Cucumber mosaic virus, the helper virus (HV). RNA affinity beads containing sat-RNA transcripts of (+) or (-)-sense covalently coupled to cyanogen bromide activated sepharose beads were incubated with total protein extracts from either healthy or HV-infected Nicotiana benthamiana leaves. RNA-protein complexes bound to the beads were eluted and subjected to MudPIT analysis. Bioinformatics programs PANTHER classification and WoLF-PSORT were used to further classify the identified host proteins in each case based on their functionality and subcellular distribution. Finally, we observed that the host protein network interacting with plus and minus-strand transcripts of sat-RNA, in the presence or absence of HV is distinct, and the global interactome of host proteins interacting with satRNA in either of the orientations is very different.

Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3' terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.