Modulation of Capsid Dynamics in Bromoviruses by the Host and Heterologous Viral Replicase.

The three genomic and a single subgenomic RNA of Cowpea chlorotic mottle virus (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1V and C2V, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4V, copackages genomic RNA3 and its subgenomic RNA (RNA4). In this study, we sought to evaluate how the alteration of native capsid dynamics by the host and viral replicase modulate the general biology of the virus. The application of a series of biochemical, molecular, and biological assays revealed the following. (i) Proteolytic analysis of the three virion types of CCMV assembled individually in planta revealed that, while retaining the structural integrity, C1V and C2V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4V virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. (ii) The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from Nicotiana benthamiana remained resistant, and (iii) finally, the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis evaluated the relative dynamics of C3+4V and B3+4V virions assembled under the control of the homologous versus heterologous replicase. The role of viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4V and C3+4V virions assembled under the homologous versus heterologous replicase. Our results collectively conclude that constant modulation of capsid dynamics by the host and viral replicase is obligatory for successful infection. IMPORTANCE 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. 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. The primary focus of this study is to address how capsid dynamics of plant-pathogenic RNA viruses, namely, Cowpea chlorotic mottle (CCMV) and Brome mosaic virus (BMV), are modulated by the host and viral replicase. The results presented have improved and transformed our understanding of the functional relationship between capsid dynamics and the general biology of the virus. They are likely to provide stimulus to extend similar studies to viruses pathogenic to eukaryotic organisms.

Studying RNA-Protein Interaction Using Riboproteomics.

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.

Newly emerged resistance-breaking variants of cucumber mosaic virus represent ongoing host-interactive evolution of an RNA virus.

Understanding the evolutionary history of a virus and the mechanisms influencing the direction of its evolution is essential for the development of more durable strategies to control the virus in crop fields. While the deployment of host resistance in crops is the most efficient means to control various viruses, host resistance itself can act as strong selective pressure and thus play a critical role in the evolution of virus virulence. Cucumber mosaic virus (CMV), a plant RNA virus with high evolutionary capacity, has caused endemic disease in various crops worldwide, including pepper (Capsicum annuum L.), because of frequent emergence of resistance-breaking variants. In this study, we examined the molecular and evolutionary characteristics of recently emerged, resistance-breaking CMV variants infecting pepper. Our population genetics analysis revealed that the high divergence capacity of CMV RNA1 might have played an essential role in the host-interactive evolution of CMV and in shaping the CMV population structure in pepper. We also demonstrated that nonsynonymous mutations in RNA1 encoding the 1a protein enabled CMV to overcome the deployed resistance in pepper. Our findings suggest that resistance-driven selective pressures on RNA1 might have contributed in shaping the unique evolutionary pattern of CMV in pepper. Therefore, deployment of a single resistance gene may reduce resistance durability against CMV and more integrated approaches are warranted for successful control of CMV in pepper.

Genome organization and interaction with capsid protein in a multipartite RNA virus.

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 Qß 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.

Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy.

Atomic Force Microscopy was utilized to study the morphology of Gag, ΨRNA, and their binding complexes with lipids in a solution environment with 0.1Å vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). The morphology of specific complexes Gag-ΨRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-ΨRNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific ΨRNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than ΨRNA. When both ΨRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.

Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo.

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites 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 (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.

Regulation of Positive-Strand Accumulation by Capsid Protein During Brome mosaic virus Infection In Planta.

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.

Riboproteomics: A versatile approach for the identification of host protein interaction network in plant pathogenic noncoding RNAs.

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.

Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging.

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.

Functionality of host proteins in Cucumber mosaic virus replication: GAPDH is obligatory to promote interaction between replication-associated proteins.

Here, we evaluated the role of two host proteins, a Bromo domain containing RNA binding protein (BRP1) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in the replication of Cucumber mosaic virus (CMV). LC-MS/MS analysis of host/viral proteins pull down against BRP1 from CMV-infected plants co-infiltrated with BRP1-FLAG agroconstruct identified that BRP1 specifically interacts with a ten amino acid motif (843-SPQDVVPLVR-852) encompassing the helicase domain of replicase protein p1a. The interaction between BRP1 and p1a was subsequently confirmed using a BiFC assay. Among fourteen other host proteins identified to interact with BRP1 during CMV infection, six were found to block accumulation of viral progeny in Arabidopsis thaliana lines defective in each of these host proteins. Additional BiFC assays followed by trans-complementation assays identified that plant lines defective in the expression of GAPDH blocked CMV replication by interfering with p1a:p2a interaction. Distinct roles of BRP1 and GAPDH in the replication of CMV are discussed.

A shift in plant proteome profile for a Bromodomain containing RNA binding Protein (BRP1) in plants infected with Cucumber mosaic virus and its satellite RNA.

Host proteins are the integral part of a successful infection caused by a given RNA virus pathogenic to plants. Therefore, identification of crucial host proteins playing an important role in establishing the infection process is likely to help in devising approaches to curbing disease spread. Cucumber mosaic virus (Q-CMV) and its satellite RNA (QsatRNA) are important pathogens of many economically important crop plants worldwide. In a previous study, we demonstrated the biological significance of a Bromodomain containing RNA-binding Protein (BRP1) in the infection cycle of QsatRNA, making BRP1 an important host protein to study. To further shed a light on the mechanistic role of BRP1 in the replication of Q-CMV and QsatRNA, we analyzed the Nicotiana benthamiana host protein interactomes either for BRP1 alone or in the presence of Q-CMV or QsatRNA. Co-immunoprecipitation, followed by LC-MS/MS analysis of BRP1-FLAG on challenging with Q-CMV or QsatRNA has led us to observe a shift in the host protein interactome of BRP1. We discuss the significance of these results in relation to Q-CMV and its QsatRNA infection cycle. BIOLOGICAL SIGNIFICANCE: Host proteins play an important role in replication and infection of eukaryotic cells by a wide-range of RNA viruses pathogenic to humans, animals and plants. Since a given eukaryotic cell typically contains ~30,000 different proteins, recent advances made in proteomics and bioinformatics approaches allowed the identification of host proteins critical for viral replication and pathogenesis. Although Cucumber mosaic virus (CMV) and its satRNA are well characterized at molecular level, information concerning the network of host factors involved in their replication and pathogenesis is still on its infancy. We have recently observed that a Bromodomain containing host protein (BRP1) is obligatory to transport satRNA to the nucleus. Consequently, it is imperative to apply proteomics and bioinformatics approaches in deciphering how host interactome network regulates the replication of CMV and its satRNA. In this study, first we established the importance of BRP1 in CMV replication. Then, application of co-immunoprecipitation in conjunction with LC-MS/MS allowed the identification of a wide range of host proteins that are associated with the replication of CMV and its satRNA. Interestingly, a shift in the plant proteome was observed when plants infected with CMV were challenged with its satRNA.

Infectious long non-coding RNAs.

Long non protein coding RNAs (lncRNAs) constitute a large category of the RNA world, able to regulate different biological processes. In this review we are focusing on infectious lncRNAs, their classification, pathogenesis and impact on the infected organisms. Here they are presented in two separate groups: 'dependent lncRNAs' (comprising satellites RNA, Hepatitis D virus and lncRNAs of viral origin) which need a helper virus and 'independent lncRNAs' (viroids) that can self-replicate. Even though these lncRNA do not encode any protein, their structure and/or sequence comprise all the necessary information to drive specific interactions with host factors and regulate several cellular functions. These new data that have emerged during the last few years concerning lncRNAs modify the way we understand molecular biology's 'central dogma' and give new perspectives for applications and potential therapeutic strategies.

Properties of satellite tobacco mosaic virus phenotypes expressed in the presence and absence of helper virus.

In this study, we assembled an Agrobacterium-based transient expression system for the ectopic expression of Satellite tobacco mosaic virus (STMV) (+) or (-) transcripts and their biological activity was confirmed when Nicotiana benthamiana plants were co-expressed with helper Tobacco mosaic virus replicase. Characterization of STMV in the presence and absence of its HV revealed: (i) HV-dependent expression of STMV (+) in N. benthamiana, but not in N. tabacum, generated a replication-deficient but translation and encapsidation competent variant lacking the highly conserved 3' 150 nucleotides (nt) (STMVΔ150); (ii) mutational analysis demonstrated that a conserved 3' stem-loop structure in wild type and STMVΔ150 located between nt 874 and 897 is essential for translation of CP; (iii) helper virus-independent expression of CP from wt STMV was competent for the assembly of empty aberrant virion-like particles; whereas, CP translated from STMVΔ150 resulted in disorganized CP aggregates suggesting a role for the 3'tRNA-like structure in STMV assembly.

Virus-associated small satellite RNAs and viroids display similarities in their replication strategies.

Since the discovery of non-coding, small, highly structured, satellite RNAs (satRNAs) and viroids as subviral pathogens of plants , have been of great interest to molecular biologists as possible living fossils of pre-cellular evolution in an RNA world. Despite extensive studies performed in the last four decades, there is still mystery surrounding the origin and evolutionary relationship between these subviral pathogens. Recent technical advances revealed some commonly shared replication features between these two subviral pathogens. In this review, we discuss our current perception of replication and evolutionary origin of these petite RNA pathogens.

The brome mosaic virus 3' untranslated sequence regulates RNA replication, recombination, and virion assembly.

The 3' untranslated region in each of the three genomic RNAs of Brome mosaic virus (BMV) is highly homologous and contains a sequence that folds into a tRNA-like structure (TLS). Experiments performed over the past four decades revealed that the BMV 3' TLS regulates many important steps in BMV infection. This review summarizes in vitro and in vivo studies of the roles of the BMV 3' TLS functioning as a minus-strand promoter, in RNA recombination, and to nucleate virion assembly.

Heterologous replicase driven 3' end repair of Cucumber mosaic virus satellite RNA.

To investigate the extent of the 3' end repair in a satellite RNA of Cucumber mosaic virus (CMV) strain Q (Q(sat)) by a heterologous Tomato aspermy virus (TAV), a set of biologically active agrotransformants corresponding to the three genomic RNAs of TAV was developed. Analysis of Nicotiana benthamiana plants agroinfiltrated with TAV and either wild type or each of the six 3' deletion mutants of Q(sat) revealed that (i) heterologous replicase failed to generate Q(sat) multimers, a hallmark feature of homologous replicase dependent replication of Qsat; (ii) manifestation of severe symptom phenotypes and progeny analysis suggested that heterologous replicase was competent to repair Q(sat) deletion mutants lacking up to 3'13 nucleotides (nt) but not beyond and (iii) comparative in silico analysis indicated that the 3' secondary structural features of the repaired Q(sat) progeny from heterologous vs homologous driven replicases are remarkably very similar. The significance of these observations is discussed.

A novel minimal in vitro system for analyzing HIV-1 Gag-mediated budding.

A biomimetic minimalist model membrane was used to study the mechanism and kinetics of cell-free in vitro HIV-1 Gag budding from a giant unilamellar vesicle (GUV). Real-time interaction of Gag, RNA, and lipid, leading to the formation of mini-vesicles, was measured using confocal microscopy. Gag forms resolution-limited punctae on the GUV lipid membrane. Introduction of the Gag and urea to a GUV solution containing RNA led to the budding of mini-vesicles on the inside surface of the GUV. The GUV diameter showed a linear decrease in time due to bud formation. Both bud formation and decrease in GUV size were proportional to Gag concentration. In the absence of RNA, addition of urea to GUVs incubated with Gag also resulted in subvesicle formation. These observations suggest the possibility that clustering of GAG proteins leads to membrane invagination even in the absence of host cell proteins. The method presented here is promising, and allows for systematic study of the dynamics of assembly of immature HIV and help classify the hierarchy of factors that impact the Gag protein initiated assembly of retroviruses such as HIV.

Integration of replication and assembly of infectious virions in plant RNA viruses.

For all plant pathogenic viruses with positive-strand RNA genomes, the assembly of infectious virions is a carefully orchestrated process. The mature virions of such viruses exhibit a remarkable degree of packaging specificity, despite the opportunity that exists to package cellular RNAs. Recent technical developments in the fields of molecular and cellular biology have revealed that the processes regulating genome replication and virion assembly are integrated. The main focus of this review is to (i) apprise readers of the technical breakthroughs that have facilitated the dissection of replication from virion assembly and genome packaging in vivo and (ii) describe the critical factors that have been shown to be involved in the regulation and integration of these processes.

Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: implications for replication and genome packaging.

In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein-protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed.