Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target audience includes research scientists, graduate students and audience at the annual meetings. In addition, results obtained from this project are disseminated to students through classroom instruction and to provide advanced training to graduate and postdoctoral fellows. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? During this project, we developed several new approaches for testing the functional significance host-protein interactions as well as protein-protein interactions leading to evaluate how plant viruses replicate and cause diseases. These technical advancements allowed graduate students and postdoctoral fellows to get acquainted with state-of-the-art technologies and compete in securing desired positions in academia and industry. How have the results been disseminated to communities of interest? Results obtained from this project were disseminated to audience at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? We continue to develop and apply novel approaches in unraveling the molecular mechanisms involved in packaging viral genomes, identify how heterologous virus replicase repairs the 3' ends of a satellite RNA associated with cucumber mosaic virus.
Impacts
What was accomplished under these goals?
We accomplished the following: (i) We demonstrated that the nuclear importation of Q-satRNA is mediated by a bromodomain-containing host protein (BRP1), which is also apparently involved in the nuclear localization of PSTVd. A comparative analysis of nuclear and cytoplasmic fractions from Nicotiana benthamiana plants coinfected with Q-satRNA and its HV confirmed the association of Q-satRNA but not HV with the nuclear compartment. A combination of the MS2-capsid protein-based RNA tagging assay and confocal microscopy demonstrated that the nuclear localization of Q-satRNA was completely blocked in transgenic lines of Nicotiana benthamiana (ph5.2nb) that are defective in BRP1 expression. This defect, however, was restored when the ph5.2nb lines of N. benthamiana were trans-complemented by ectopically expressed BRP1. The binding specificity of BRP1 with Q-satRNA was confirmed in vivo and in vitro by coimmunoprecipitation and electrophoretic mobility shift assays, respectively. Finally, infectivity assays involving coexpression of Q-satRNA and its HV in wild-type and ph5.2nb lines of N. benthamiana accentuated a biological role for BRP1 in the Q-satRNA infection cycle. (ii) InBrome 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-expressionin plantaof 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. (iii) A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Gopal, A.J., Egecioglu, D., Yoffe, A, Ben-Shaul.A., Rao, A., Knobler,C., and Gelbart, W (2014). Viral RNAs are unusually Compact. PLOS ONE 9 (9) e105875.
Sonali Chaturvedi and A.L.N. Rao (2014) Live Cell Imaging of Interactions Between Replicase and Capsid Protein of Brome Mosaic Virus using Bimolecular Fluorescence Complementation: Implications for Replication and Genome Packaging. Virology 464-465: 67-75.
Sonali Chaturvedi, Kriton Kalantidis and A. L. N. Rao (2014). A bromodomain containing host protein mediates the nuclear import of a satellite RNA of cucumber mosaic virus. Journal of Virology 88(4) 1890-1896.
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Progress 10/01/09 to 09/30/14
Outputs
Target Audience:The target audience includes fellow scientists, graduate students, and postdoctoral fellows. Results obtained from the proposed studies are presented at the annual meetings of American Society for Virology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project offered training to graduate and undergraduate students in molecular, biochemical and cell biology areas. How have the results been disseminated to communities of interest?Results were disseminatedto scientific communities of interest via publications in scientificjournals and presentations at annual meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
? 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 likely to help in devising approaches to curbing disease spread. Cucumber mosaic virus (CMV) and its satellite RNA (satRNA) 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 satRNA, making BRP1 an important host protein to study. To further shed a light on the mechanistic role of BRP1 in the replication of CMV and satRNA, we analyzed the Nicotiana benthamiana host protein interactomes either for BRP1 alone or in the presence of CMV or satRNA. Co-immunoprecipitation, followed by LC-MS/MS analysis of BRP1-FLAG on challenging with CMV or satRNA has led us observe a shift in the host protein interactome of BRP1. p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 7.0px Times} span.s1 {font: 7.0px Helvetica}
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Sonali Chaturvedi and A. L. N. Rao (2015). 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. Jornal of Proteomics 131: 1-7.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Target audience include fellow scientists, graduate students and postdoctoiral fellow and results obtained from the proposed studies are presented at the annual meetings of Amrican Society for Virology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project offered training to graduate and undergraduate students in molecular, biochemical and cell biology areas. How have the results been disseminated to communities of interest? Results were disseminated to communities of interest via publication in scientific journals. What do you plan to do during the next reporting period to accomplish the goals? We are continuing on appalying cell biology and genomc apparoches to identify the critical host protein involved in the nuclear import of satellite RNA assocaited with cucumber mosaic virus.
Impacts
What was accomplished under these goals?
By testing the role of CP, we demonstrated that CP has an inherent property to rearrange cellular membranes into vesicle like structures. We also showed that this membrane modifying property is linked to encapsidation competence of CP. Keeping these observations in perspective, we envision two likely roles for CP induced vesicles. The first role is to promote virion assembly. A second hypothesized role for CP-mediated vesicles is to promote cell-to-cell movement. Consequently, results of this study provide a previously un-recognized cytopathology-based evidence to explain the mechanism of cell-to-cell movement of BMV. Apart from replication and assembly, the major differential phenotype exhibited by wt BMV in N. benthamiana and N. clevelandii is cell-to-cell movement. This defective cell-to-cell spread of BMV in N. clevelandii could be attributed to the defective MP resulting in subliminal infection. Alternatively, since cell-to-cell movement of BMV requires encapsidation competent-CP, compelling evidence for the conjecture that cell-to-cell movement of BMV correlates with CP-mediated vesicle induction. Replication of Q-sat is cytoplasmic since the replication of its helper virus is confined to the same cellular compartment. However, using cell biology based approaches we recently demonstrated Q-sat has a propensity to localize in the nucleus for a template independent addition of a hepta nucleotide motif at the monomer junctions to form multimers that subsequently serve as efficient templates for helper virus-dependent replication. Since none of the six 3’ end deletion mutants are repaired by the cellular polymerase, we hypothesize that 3’ end repair, specifically addition of -CCCOH, can only be mediated by the helper virus-encoded replicase and whereas nuclear encoded polymerases would add hepta nucleotide motif only to those Q-sat transcripts that terminated with-CCCOH. Consequently, following transient expression in the nucleus, all 3’ terminal Q-sat mutant transcripts exit the cytoplasm to get repaired by the helper virus-replicase, generating RNA transcripts terminating in -CCCOH and re-enter the nucleus for the addition of hepta nucleotide motif and multimer formation as described above. Therefore it would be significant not only to identify the host factors involved in nuclear import of Q-sat but also the Q-sat sequences that play an important role in this active process.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
1. Bamunusinghe, D., Sonali Chaturvedi, Jang-Kyun Seo and A. L. N. Rao (2013) Mutations in the Capsid Protein of Brome Mosaic Virus Affecting Encapsidation Eliminate Vesicle Induction in planta: Implications for Virus Cell-to-Cell Spread. Journal of Virology 87: 8982-8992.
2. Sun-Jung Kwon, Sonali Chaturvedi and A. L. N. Rao (2014). Repair of the 3' proximal and internal deletions of a satellite RNA associated with Cucumber mosaic virus is directed toward restoring structural integrity. Virology 450-451: 222-232
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Genome packaging in the plant-infecting Brome mosaic virus (BMV), a member of the alphavirus-like superfamily as well as in other positive strand RNA viruses pathogenic to humans and animals, is functionally coupled to replication. The onset of a given viral disease and its progression relies on coordinated strategies on the host cell infrastructure and metabolism. Positive-strand RNA viruses pathogenic to humans, animals and plants rearrange internal cellular membranes to create an environment facilitating virus replication. Genetic analysis of viral genes involved in cellular modifications has revealed that membrane rearrangement into vesicles could be attributed to a particular viral gene product, usually a non-structural protein. Although the subcellular localization site of BMV replication has been identified, that of the capsid protein (CP) has remained elusive. The application of immunofluorescence confocal microscopy to Nicotiana benthamiana leaves expressing replication-derived BMV CP as a GFP fusion, in conjunction with antibodies to the CP and double-stranded RNA, a presumed marker of RNA replication, revealed that the subcellular localization sites of replication and CP overlap. Our temporal analysis by transmission electron microscopy of ultrastructural modifications induced in BMV infectedN. benthamiana leaves revealed a reticulovesicular network of modified endoplasmic reticulum (ER) incorporating large assemblies of vesicles derived from ER accumulated in the cytoplasm during BMV infection. Additionally, for the first time, we have found by ectopic expression experiments that BMV CP it self has the intrinsic property of modifying ER to induce vesicles similar to those present in BMV infections. A number of Sat-RNAs have been shown to modulate the course of disease development in plants incited by their helper viruses (HVs), and hence are of importance to practical agriculture. Consequently, a majority of studies have focused on characterizing various strains of Sat-RNAs, their relationship to HVs, symptom expression and origin. Since the size and structural features of Sat-RNA are similar to those of viroids, studies conducted in the 1980's attempted to find similarities in the replication mechanisms of Sat-RNAs and viroids. In 1977, a Sat-RNA associated with Cucumber mosaic virus (CMV) has been shown to the casual agent responsible for an epidemic of lethal necrosis of tomato in France. Since then many variants of Sat-RNA associated with CMV have been characterized. All analyzed CMV Sat-RNAs have a 5'-terminal cap and a 3'-terminal CCCOH, the latter of which cannot be aminoacylated. These Sat-RNAs are completely dependent on CMV (i.e. HV) for their replication and are efficiently encapsidated by HV CP. CMV Sat-RNAs are shown to either attenuate or intensify phenotypic symptoms induced by the HV in certain host plants although the effect of Sat-RNA on CMV disease severity depends on the Sat-RNA strain. However, since Sat-RNAs are dependent on their HV for replication, the biological properties exhibited by Sat-RNAs are likely to be due to their direct interaction with components of HV. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We have discovered that multimers of a CMV Sat-RNA generated in the absence of its HV are characterized by the addition of a hepta nucleotide motif (HNM) at the monomer junctions. We evaluated the functional significance of HNM in HV-dependent replication by ectopically expressing wild type and mutant forms of satRNA multimers in planta either in (+) or (-)-strand polarity. Comparative replication profiles revealed that (-)-strand multimers with complementary HNM (cHNM) are the preferred initial templates for HV-dependent replication than (-)-strand monomers and multimers lacking the cHNM. Further mutational analyses of the HNM accentuate that preservation of the sequence and native length of HNM is obligatory for efficient replication of Sat-RNA. Positive-strand RNA viruses are known to rearrange the endomembrane network to make it more conducive for replication, maturation or egress. Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus; FHV) and plants (Brome mosaic virus, BMV). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular Fluorescence Complementation (BiFC) in conjunction with fluorescent cellular protein markers and co-immunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and this interaction requires either the N-proximal 1-31 or 32-50 amino acid regions of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to re-localize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.
Publications
- Jang-Kyun Seo, Sun-Jung Kwon, Sonali Chaturvedi and A.L.N. Rao (2013). Functional significance of a hepta nucleotide motif present at the junction of cucumber mosaic virus satellite RNA multimers in helper-virus-dependent replication. Virology 435: 214-219.
- Jang-Kyun Seo, Sun-Jung Kwon and A.L.N. Rao (2012). Molecular dissection of Flock house virus protein B2 reveals that electrostatic interactions between N-terminal domains of B2 monomers are critical for dimerization. Virology 432: 296-305.
- Jang-Kyun Seo, Sun-Jung Kwon and A.L.N. Rao (2012). A physical interaction between viral replicase and capsid protein is required for genome packaging specificity in an RNA virus. Journal of Virology 86(11): 6210-6221.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Subviral pathogens are the smallest known infectious molecules that manipulate the cellular systems of much higher organisms, including animals and plants, to replicate themselves. Plant subviral pathogens can be divided into two major groups based on their replicability: Helper virus-dependent or independent subviral pathogens. The former includes plant satellite RNAs (satRNAs) while the latter includes viroids. satRNAs associated with Cucumber mosaic virus (CMV), the type member of the genus Cucumovirus, are among the earliest found and well studied subviral pathogens. Since the discovery of satRNAs, these petite subviral molecules remain of interest to molecular biologists as relatively simple models for studying interactions between macromolecules in plant cells. A number of satRNAs have been shown to modulate the course of disease development in plants incited by their HVs, and hence are of importance to practical agriculture. In addition, CMV satRNA has been shown to survive up to 25 days without its HV. However, the molecular basis for this abnormal long-term survival of CMV satRNA still remains obscure although their high secondary structure was envisioned to contribute to this HV-independent survival. Consequently, a majority of studies have focused on characterizing various strains of satRNAs, their relationship to HVs, symptom expression and origin. Because of the inherent dependency on HV, most research on satRNA replication to date has been performed in the presence of HV using mechanical inoculation of either virion RNA or in vitro transcripts. Therefore, the major purpose of this work is to define in molecular terms the basis for HV-independent survival of satRNA. Consequently, in this study, we sought to examine the subcellular localization and the biological activities of a satRNA in the absence of its HV. The Agrobacterium tumefaciens-mediated transient expression (agroinfiltration) not only facilitates synchronized delivery of multiple DNA-based transgene expression but also results in high-level expression of mRNAs independent of replication. Thus, a T-DNA based Q-satRNA construct (pQ-satRNA) was engineered to initiate transcription by the Cauliflower mosaic virus 35S promoter precisely at the authentic 5' end of Q-satRNA and terminate with the natural 3'CCCOH through a self-cleaving Hepatitis delta virus (HDV) ribozyme. Agroinfiltration in conjunction with a series of molecular, biochemical and cell-biology based approaches were used to investigate the above-mentioned research problem. Despite overwhelming interest on the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Thus, the role of the polarity of inoculum RNA in RNA recombination was examined. Once again, we exploited the salient features of agroinfiltration in examining the extent to which RNA polarity would contribute to recombination. Northern blot analysis of the progeny RNAs followed by sequencing of products of RT-PCR were employed to characterize the recombinants emerged from expressing either plus-or minus sense RNAs. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). When expressed autonomously in the absence of HV, a variant of satellite RNA (Q-satRNA) associated with Cucumber mosaic virus has a propensity to localize in the nucleus and transcribe, generating genomic and anti-genomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA tagging and dsRNA labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent hepta-nucleotide motif (HNM) at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus. By agro-infiltrating Nicotiana benthamiana leaves, we showed that Brome mosaic virus (BMV) replicase is competent to initiate (+)-strand synthesis on an ectopically expressed RNA3 (-)-strand and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-)-polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+)-strand, the recombinant pool accumulated during early phases of replication was a mixture of non-homologous recombinants. These are longer than the inoculum template length and a nascent 3' un-translated region (UTR) of wild type (wt) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-)-strand, the progeny contained a pool of native length homologous recombinants generated by template switching of BMV replicase with nascent UTR from wt RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+)-strand. These results contribute to explain the functional role of RNA polarity in recombination mediated by copy-choice mechanisms.
Publications
- Soon Ho Choi, Jang-Kyun Seo, Sun-Jung Kwon and A. L. N. Rao (2012). Helper virus independent transcription and multimerization of a satellite RNA associated with cucumber mosaic virus. Journal of Virology (In Press) (doi:10.1128/JVI.00018-12).
- Sun-Jung Kwon and A. L.N. Rao (2012). Emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum RNA. Journal of Virology (In Press) (doi:10.1128/JVI.00351-12).
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Undoubtedly viruses are important plant pathogens that continue to cause serious economic losses in a variety of crops in California and worldwide. In RNA viruses, replication of the genome followed by its encapsidation by viral capsid protein (CP) into stable virions is considered to be important during establishment of a successful infection in susceptible host plants. Despite significant advances made in understanding viral replication, the mechanism of genome packaging by viral capsid protein is poorly understood. In brome mosaic virus (BMV), two important phases in the viral life cycle implicate the existence of an intimate relationship between CP and replication. First, CP is involved in the up-regulation of plus-strand synthesis over minus-strands. Second, CP translated from a replication-derived mRNA exclusively encapsidates the progeny RNA into stable infectious virions, a process commonly referred to as replication coupled packaging that is highly conserved among many positive-strand RNA viruses. Although the subcellular localization site of BMV replication has been delineated to ER-derived spherules that of the CP is not known. Therefore, immuno fluorescence confocal microscopy (IFCM) was used to explore the subcellular localization sites of BMV CP synthesis. In addition, transmission electron microscopy (TEM) of whole plants infected either with wild type BMV or expressing CP ectopically was employed to evaluate the cytopathological changes induced in Nicotiana benthamiana. To this end, we employed agroinfiltration for ectopic expression of viral genes followed by fixation of leaf material for TEM analysis. The application of immunofluorescence confocal microscopy to Nicotiana benthamiana leaves expressing replication-derived BMV CP as a GFP fusion, in conjunction with antibodies to the CP and double-stranded RNA, a presumed marker of RNA replication, revealed that the subcellular localization sites of replication and CP overlap. Our temporal analysis by transmission electron microscopy of ultrastructural modifications induced in BMV infected N. benthamiana leaves revealed a reticulovesicular network of modified endoplasmic reticulum (ER) incorporating large assemblies of vesicles accumulated in the cytoplasm during BMV infection. Additionally, for the first time, we have found by ectopic expression experiments that BMV CP it self has the intrinsic property of modifying ER to induce vesicles similar to those present in BMV infections. These observations offer a new perspective toward elucidation of CP-organized viral functions that are intimately linked to replication-coupled RNA packaging. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One of the ultimate goals of biological sciences, and certainly one with a high impact on society, is to improve our understanding of the processes and events related to diseases. Molecular biologists, who traditionally study the structure and function of individual proteins and genes, have gained insight and introduced several discoveries that have ultimately reached the bedside. However, biological processes are not realized by a single molecule, but rather by the complex interaction of proteins with their environment, including nucleic acids, ions, lipids, membranes and, of course, other proteins. Thus, while the analysis of the structure and function of individual proteins is crucial for the understanding of their role in biological processes, it has a limited capability to explain the processes themselves. Defining protein and ribonucleoprotein complexes is critical to virtually all aspects of cell biology, because stable protein complexes regulate many cellular processes and their identification often provides insights into their function. In viruses, protein-protein interactions regulate various functions essential to establish a successful infection in a given susceptible host. Many proteins mediate their function through the formation of both stable or transient protein complexes and networks. Patterns of protein accumulation in various cell types are regulated spatially and temporally and often the same protein may interact with various partners in response to different stimuli or at different developmental stages. One such scenario was found in brome mosaic virus (BMV) where an interaction between capsid protein (CP) and viral replicase appears to regulate packaging specificity. The reticulovesicular network of ER modified by the wild type BMV infection along with that induced by the ectopic expression of CP places the intrinsic involvement of CP in up regulating plus-strand synthesis and promoting replication-coupled packaging in a new perspective. These results together with on going studies in evaluating protein-protein interactions using bimolecular fluorescence complementation assay collectively impact in accurately defining the subcellular localization sites critical for virus propagation. Such information is valuable in understanding the overall infection process of RNA viruses pathogenic to crop plants of agricultural importance.
Publications
- Devinka Bamunusinghe, Jang-Kyun Seo and A. L. N. Rao (2011) Subcellular localization and rearrangement of endoplasmic reticulum by brome mosaic virus capsid protein. Journal of Virology 85(6) 2953-2963.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Viruses are important plant pathogens that continue to cause major economic damage to a variety of crops in California and worldwide. One of the important phases in the cycle of RNA viruses is efficient replication and encapsidation of the infectious genome into stable virions required for dissemination to healthy hosts by insect vectors. A hallmark feature of (+)-strand RNA viruses of eukaryotic cells is that progeny (+)-strands are accumulated 100-fold over (-)-strands. Analysis of brome mosaic virus (BMV) progeny RNA in Nicotiana benthamiana plants revealed that (+)-strand accumulation was severely debilitated when expression of wild type capsid protein (CP) was ceased by either a knockout mutation or by inhibiting subgenomic RNA synthesis. Plus strand accumulation was rescued when CP was complemented in trans. GFP expressed in transgenic 16c N. benthamiana is targeted to ER. In order to monitor sites of BMV accumulation, 16c plants were infiltrated with wild type BMV RNAs 1and 2 (B1 and B2) and either wild type RNA3 (B3) or a RNA3 mutant defective in virus assembly (184A). After 5 days, mock and virus infiltrated leaves were subjected to confocal laser scanning microscopy. As expected in mock infiltrated leaves GFP was localized on ER. In BMV infected leaves, large mass of GFP perhaps representing replication complex was also found to accumulate on ER. In addition, we also found that GFP accumulated at multiple foci on perinuclear ER. These multiple foci represent coat protein aggregates and perhaps virus assembly sites since they were absent in leaves infiltrated with virus assembly defective mutant 184A. In a parallel experiment, wild type N. benthamiana plants were inoculated with B1+B2+B3/CP-GFP. In this CP is expressed as a GFP fusion following replication of B3. In order to identify the subcellular localization of CP, after 4 dpi, infected leaves were excised, fixed and treated with a enzyme solution (cellulase and pectolyase) to remove cell wall. The fixed samples were then transferred on to microscopic slides and incubated with KDEL monoclonal antibody (specific for ER-resident proteins) followed by alexa fluor 633 conjugated secondary antiserum. Confocal laser scanning microscope was used to analyze the slides. In this, CP-GFP showed a distinct subcellular localization and appeared as punctate bodies on ER. As expected, counter staining with antibody specific to ER-resident proteins containing KDEL motifs showed that CP signal closely followed ER network. These results demonstrate that CP localizes on ER, which happen to be sites of viral replication. Taken together results of these studies suggest that replicase and CP subunits co-localize on ER supporting the mechanism of replication-coupled packaging. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One of the ultimate goals of biological sciences, and certainly one with a high impact on society, is to improve our understanding of the processes and events related to diseases of plants. Molecular biologists, who traditionally study the structure and function of individual proteins and genes, have gained insight and introduced several discoveries that have ultimately reached the bedside. However, biological processes are not realized by a single molecule, but rather by the complex interaction of proteins with their environment, including nucleic acids, ions, lipids, membranes and, of course, other proteins. In the context of Virology, one such scenario that hallmark eukaryotic RNA viruses with icosahedral symmetry pathogenic to humans (eg. Polio and HIV), animals (eg. foot and mouth), insects (eg. flock house virus, FHV) and plants (eg. brome mosaic virus, BMV) is the assembly of infectious virions by the capsid protein. Although several biochemical approaches have been used for evaluating protein-protein interactions, they do not monitor the dynamics of interaction and localization in vivo in real time. This information is indispensable to understand protein function at the cellular, tissue and organism level. Thus, a study involving analysis of membrane rearrangements to given viral infection at ultra structural level and application of Bimolecular Fluorescence Complementation (BiFC) analysis that permits simultaneous determination of protein-protein interaction and sub-cellular localization in a wide variety of cell types is underway. The results obtained from these studies collectively impact in accurately defining not only protein:protein interactions but also precisely identifies the subcellular location where each of these interaction occurs. Such information is valuable in understanding the overall infection process of RNA viruses pathogenic to humans, animals, insects and plants.
Publications
- de Wispelaere, V. Sivanandam and A. L. N. Rao (2010) Effect of coat protein and movement protein deletions on brome mosaic virus replication in plants. (In Preparation for Virus Research).
- Bahunasighe, B and A. L. N. Rao (2010) Ultrastructure and origin of vesicles induced by brome mosaic virus capsid protein: Implications for replication coupled packaging mechanism. (In Preparation for Journal of Virology).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The major focus of this study is to evaluate the mechanism responsible for the production of RNA5 in cucumber mosaic virus (CMV). In addition to three genomic and two known subgenomic RNAs, CMV strains of subgroup II (eg. Q-CMV), but not subgroup I (eg. Fny-CMV), produce and package a genetically redundant RNA species, referred to as RNA5. Previous studies revealed that RNA5 in Q-CMV is contiguous with the 3' 304-307 nucleotides (nt) of genomic RNAs 2 (Q2) and 3 (Q3) but not RNA1 (Q1). The mechanism regulating RNA5 production is unknown. In this study, wild-type and mutant genomic Q-CMV RNAs defective in RNA5 production were expressed in Nicotiana benthamiana plants using agroinfiltration. Efficient accumulation of RNA5 in N. benthamiana leaves infiltrated autonomously with Q2 or Q3 suggested that its production is independent of replication. Deletion mutants of Q-CMV RNAs, characterized by lacking a highly conserved region (RNA5-box) adjacent to the 5' end of RNA5, completely blocked RNA5 production but had no effect on viral replication. Mutational analysis of nt 5' to the conserved RNA5-box further identified UC as being the preferred nt pair for efficient production of RNA5. Although mutant Q-CMV inoculum defective in RNA5 production (Q1Δ+Q2Δ+Q3Δ) affected accumulation of viral progeny in N. benthamiana, it failed to exhibit discernable effects on either progeny encapsidation or virion stability. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Many viruses with single-stranded, positive sense RNA genomes cause serious diseases of humans (eg. AIDS, hepatitis), animals (eg. foot and mouth), insects (eg. flock house virus) and plants (eg. cucumber mosaic virus). Egress from infected host followed by dissemination to susceptible new hosts is contingent on efficient packaging of infectious genome into stable virions by the coat protein (CP). Information gleaned from recent studies with eukaryotic RNA viruses pathogenic to humans and animals (eg. Polio and Venezuelan equine encephalitis viruses, insects (eg. flock house virus, FHV) and plants (eg. brome mosaic virus, BMV) revealed that the mechanism of genome packaging in these taxonomically distinct viral systems is commonly shared and is functionally coupled to replication. Thus, results of this study collectively address the molecular interactions between macromolecules at increasingly complex biological levels with the promise of being able to apply what is learned about packaging of viral RNA genomes to understanding the overall infection process of RNA viruses pathogenic to humans, animals, insects and plants.
Publications
- de Wispelaere, M and A. L. N. Rao. (2009). Production of cucumber mosaic virus RNA5 and its role in recombination. Virology 384: 179-191.
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Progress 01/01/07 to 12/31/07
Outputs
The major focus of our research is to investigate the mechanism controlling cucumber mosaic virus (CMV) satellite RNA (Sat-RNA) packaging followed by determination of the constellation of CMV virions required for efficient transmission of the virus by aphid vectors to healthy hosts. A well characterized Agrobacterium-mediated transient expression system (agroinfiltration), developed in our lab, will be employed to fulfill the proposed research quest. AIM 1: Determine whether packaging of CMV sat-RNA is replication contingent. Proposed experiments will also evaluate the distribution of Sat-RNA among CMV virions. AIM 2: Investigate the required form of CMV coat protein (CP), produced in either replication dependent or independent mode, for efficient assembly. Each of this virion form will be tested for acquisition and transmission to healthy hosts by aphid vectors. During the past year, we made good progress on AIM 1 and 2. We expressed CMV Sat-RNA and CP independent of
replication. Assembled virions were purified and their RNA content analyzed. We are in the process of evaluating factors regulating efficient Sat-RNA packaging. We are raring healthy aphids on plants for transmission experiments. Virions packaging Sat-RNA expressed in either replication-dependent or independent mode will be tested for their transmission competence to healthy host plants by aphid vectors. An agrotransformant that will be used to express multiple copies of CMV Sat-RNA is being constructed. We also characterized the mechanism regulating CMV RNA5 production and shown that its synthesis is independent of viral replication. We are now testing a series of CMV mutant RNAs defective in RNA5 synthesis to evaluate its role in packaging, cell-to-cell and long distance movement, symptom expression, insect transmission and host range.
Impacts
We have submitted a proposal to USDA focusing on the mechanism of CMV virion conformation modulating host-pathogen interactions and aphid transmission.
Publications
- Calhoun, S.L. and A. L. N. Rao. 2007. Functional analysis of brome mosaic virus coat protein-RNA interacting domains. Archives of Virology 153:235-241.
- Calhoun, S. L., J. A. Speir, and A. L. Rao. 2007. In vivo particle polymorphism results from deletion of a N-terminal peptide molecular switch in brome mosaic virus capsid protein. Virology 364: 407-21.
- Annamalai, P., and A. L. Rao. 2007. In vivo packaging of brome mosaic virus RNA3, but not RNAs 1 and 2, is dependent on a cis-acting 3' tRNA-like structure. Journal of Virology 81:173-81.
- Annamalai, P. Fady Rofail, D.A. Demason and A.L.N. Rao, 2007 Replication coupled packaging mechanism in positive-strand RNA viruses; Synchronized co-expression of functional multigenome components of an animal and plant virus in Nicotiana benthamiana cells by agroinfiltration. Journal of Virology 82: 1484-1495.
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Progress 01/01/06 to 12/31/06
Outputs
In 2005 my laboratory has developed a novel agrobacterium-mediated transient expression system for RNA viruses. We extended this approach for evaluating three independent aspects related to RNA packaging. The first study evaluated the mechanism that regulates specificity of RNA packaging in eukaryotic viruses that are pathogenic to many crops of agricultural importance. Using brome mosaic virus, pathogenic to monocotyledonous hosts, as a model system we observed that (i) in the absence of replication, packaging was non-specific since transiently expressed coat protein subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed coat protein mRNA and its deletion variants; (ii) induction of viral replication increased the specificity of RNA packaging and most importantly (iii) efficient packaging of coat protein mRNA, reminiscent of wild type scenario, is functionally coupled not only to its transcription via replication but also to
translation of coat protein from replication-derived mRNA. This replication-coupled packaging was demonstrated by our lab for the first time for plant RNA viruses which appears to be conserved among positive strand RNA viruses of animals (flock house virus) and humans (poliovirus) as well. The second study evaluated the critical role played by the highly conserved 3' tRNA-like structure of the four encapsidated BMV RNAs. Coexpression of TLS-less constructs of BMV RNA 1 or RNA2 or RNA3 and coat protein mRNAs in Nicotiana benthamiana leaves resulted in packaging of TLS-less RNA1 and RNA2 but not RNA3, suggesting that packaging of RNA3 requires the TLS in cis. This conjecture was confirmed by the efficient packaging of a RNA3 chimera in which the viral TLS was substituted with a cellular tRNA. This intrinsic cis-requirement of TLS in promoting RNA3 packaging was further confirmed by additional in vivo experiments. Collectively the data suggested that the requirement of a cis-acting TLS
is distinct for RNA3 compared with RNA1 or RNA2. The third study examined the interaction between BMV coat protein and viral RNA leading to formation of homogeneous population of infectious virions with T=3 symmetry. To this end, we engineered a series of mutations into BMV coat protein and one mutant characterized by deletion of amino acid residues 41-47 from the N-proximal region was chosen for additional in vivo studies. Purified virion preparations of this mutant from symptomatic leaves remain non-infectious and Northern blot analysis of virion RNA displayed packaging defects. Biochemical characterization of variant coat protein by circular dichroism and MALDI-TOF, respectively, revealed that the engineered deletion affected the protein structure and capsid dynamics. Most significantly, coat protein subunits dissociated from polymorphic virions are incompetent for in vitro reassembly. Based on these observations, we propose a chaperon mediated mechanism for the assembly of variant
coat protein in vivo and also hypothesize that 41KAIKAIA47 N-proximal peptide functions as a molecular switch in regulating T= 3 virion symmetry.
Impacts
Many viruses with single-stranded, positive sense RNA genomes cause serious diseases of humans (eg. AIDS, hepatitis), animals (eg. foot and mouth), insects (eg. flock house virus) and plants (eg. cucumber mosaic virus). Among many events that take place during the life-cycle of eukaryotic RNA viruses, the assembly of infectious genome (packaging) into stable virions is an important phase since viruses that infect animals, humans and insects are disseminated only in the assembled form. Likewise, in several plant viruses assembly into infectious virions is obligatory for cell-to-cell and/or long distance spread as well as for acquisition and dissemination to new hosts by a variety of insect vectors. Information gleaned from recent studies with eukaryotic RNA viruses pathogenic to humans and animals (eg. Polio and Venezuelan equine encephalitis viruses), insects (eg. flock house virus, FHV) and plants (eg. brome mosaic virus, BMV) revealed that the mechanism of genome
packaging in these taxonomically distinct viral systems is commonly shared and is functionally coupled to replication. Thus, knowledge of the detailed mechanism by which viruses assemble and package their genomes into structurally stable virions is an important prerequisite for understanding the overall biology of viruses and pathogenesis. Consequently results obtained from our studies will have major impact in providing basic foundation to understand virus assembly mechanisms.
Publications
- Rao, A. L. N. 2006. Sensitivity of brome mosaic virus replication to 3 prime mutations in RNA1 implies a requirement for sustained synthesis of 1a replicase protein. Archives of Virology 151: 721-733.
- Annamalai, P and A. L. N. Rao. 2006. Packaging of brome mosaic virus subgenomic RNA is functionally coupled to replication-dependent transcription and translation of coat protein. J. Virol. 80: 10096-10108.
- Annamalai, P and A. L. N. Rao. 2007. In vivo packaging of brome mosaic virus genomic RNA3, but not RNAs 1 and 2, is dependent on a cis- acting 3 prime tRNA-like structure. J. Virol. 81: 173-181.
- Calhoun, S., Speir, J. A and A. L.N. Rao. 2007. In vivo particle polymorphism results from deletion of a N-terminal peptide molecular switch in brome mosaic virus capsid protein. Virology (in Press).
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Progress 01/01/05 to 12/31/05
Outputs
Cowpea chlorotic mottle virus (CCMV) and Brome mosaic virus (BMV) are the member viruses of the genus Bromovirus of the family Bromoviridae. The N-proximal region of CCMV capsid protein (CP) contains an arginine rich RNA binding motif (ARM) that are also found in the CPs of other members of Bromoviridae, and other RNA binding proteins such as Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/delta 919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/delta919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer assisted three-dimensional reconstruction of wt and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of
virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs: however the packaging effect was more pronounced on genomic RNAs 1 and 2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNAs1 (53%) and 2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimera, constructed by exchanging CPs having the delta 919 deletion, exemplified that the role of the conserved N-ARM in recognizing and packaging specific RNA is distinct for each virus. To further elucidate the relationship between bromovirus replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to
express either individual or desired pairs of the three BMV genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed BMV CP was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1+RNA3 or RNA2+RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNA1 and RNA2 were constructed by deleting the 3 prime TRNA-like structure (TLS). Coexpression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves coexpressing TLS-less RNA1 and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent
whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging.
Impacts
One of the important events in the life cycle of any eukaryotic RNA virus is packaging of the newly synthesized genome components into stable and infectious virions by the capsid protein. Absence of cellular RNA in progeny virions of plant viruses suggests that packaging is a highly coordinated process involving specific sequence and/or structure dependent interactions between coat protein and viral RNAs. Alternatively specificity of RNA packaging in plant viruses could be coupled to replication as proposed for poliovirus and other non-plant viruses. Our present study involving the analyses of packaging of replication competent and replication-defective BMV RNAs by transiently expressed CP demonstrated that, as in other non-plant viral systems, specificity of BMV RNA packaging is coupled to expression of viral replicase. Thus the results of this study reveal that mechanisms that regulate genome packaging among RNA viruses that are infectious to plant, animal and
humans are highly conserved
Publications
- Annamalai, P., Apte, S., Wilkens, S., Rao, A.L.N., 2005. Deletion of highly conserved arginine rich RNA binding motif in cowpea chlorotic mottle virus capsid protein results in virion structural alterations and RNA packaging constraints. J. Virol. 79, 3277-3288.
- Annamalai, P and Rao, A. L . N. 2005. Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: A functional role for viral replicase in RNA packaging. Virology 338: 96-111.
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Progress 01/01/04 to 12/31/04
Outputs
The 3 prime ends of three genomic RNAs (gRNAs) of cowpea chlorotic mottle virus (CCMV) terminate in a highly conserved tRNA-like structure (TLS). To examine the intrinsic role played the TLS in packaging, the competence of each gRNA lacking the TLS to interact with dissociated coat protein (CP) subunits and form virions was assayed in vitro. In contrast to the well established requirement for the participation of either viral TLS or host-tRNAs in the assembly of RNA-containing virions in brome mosaic virus (BMV), CCMV CP does not require the presence of viral TLS in cis or in trans. Similar in vitro assembly assays showed that CCMV CP subunits also packaged BMV RNAs lacking TLS as well as two other non-bromoviral RNAs although with lesser efficiency. To characterize sequences of CCMV RNA3 (C3) required for packaging, a series deletions were engineered into C3 and their effect on virus assembly was examined. It was observed that, unlike BMV RNA3 whose packaging
requires a bipartite signal, packaging of C3 is independent of either movement protein (MP) ORF or CP ORF or 3 prime non-coding regions. Based on the differential prerequisites identified in this study for the assembly of BMV and CCMV, we hypothesize that the adaptive condition for movement in monocotyledonous host has made packaging a necessary co-requirement for BMV.
Impacts
The majority of plant viruses are disseminated to healthy plants in virion forms acquired by insect vectors; therefore, it is important to understand the mechanism of genome packaging into infectious virions and the mechanism of virus assembly. Results obtained from these studies are important in developing novel strategies of virus disease control.
Publications
- Annamalai, P and Rao, A. L. N. (2005). Dispensability of 3 prime tRNA-like sequence for packaging cowpea chlorotic mottle virus genomic RNAs. Virology 332: 650-658.
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Progress 01/01/03 to 12/31/03
Outputs
The three genomic and a single subgenomic RNA of brome mosaic virus (BMV), an RNA virus infecting plants, are packaged by a single coat protein (CP) into three morphologically indistinguishable icosahedral virions with T=3 quasi-symmetry. Genomic RNAs 1 and 2 are packaged individually into separate particles whereas genomic RNA3 and subgenomic RNA4 (coat protein mRNA) are co-packaged into a single particle. We report here that packaging of dicistronic RNA3 requires a bipartite signal. A highly conserved 3' tRNA-like structure postulated to function as a nucleating element (NE) for CP subunits and a cis-acting, position-dependent packaging element (PE) of 187nt present in the non structural movement protein gene are the integral components of the packaging core. Efficient incorporation into BMV virions of non viral RNA chimeras containing NE and the PE provides confirmatory evidence that these two elements are sufficient to direct packaging. Analysis of virion RNA
profiles obtained from barley protoplasts transfected with a RNA3 variant lacking the PE provides the first genetic evidence that de novo synthesized RNA4 is incompetent for autonomous assembly whereas prior packaging of RNA3 is a prerequisite for RNA4 to co-package.
Impacts
The majority of plant viruses are disseminated to healthy plants in virion forms acquired by insect vectors; therefore, it is important to understand the mechanism of genome packaging into infectious virions and the mechanism of virus assembly. Results obtained from these studies are important in developing novel strategies of virus disease control.
Publications
- Choi, Y. G and A. L. N. Rao (2003). Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal. Journal of Virology 77: 9750-9757.
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Progress 01/01/02 to 12/31/02
Outputs
The three genomic and a single subgenomic RNA of brome mosaic virus (BMV), an RNA virus infecting plants, are packaged by a single coat protein (CP) into three morphologically indistinguishable icosahedral virions with T equals 3 quasi-symmetry. Genomic RNAs 1 and 2 are packaged individually into separate particles whereas genomic RNA3 and subgenomic CP mRNA are co-packaged into a single particle. This distribution of genomic content suggests that each of the four RNAs interact with specific CP domains during the encapsidation process. To identify these specific CP interacting sites, we engineered mutations into a putative N-proximal RNA binding domain (amino acids 40-46) of BMV CP and analyzed their competence to mediate assembly of virions in vivo. Deletion of a region encompassing all seven amino acids rendered the resulting variant incompetent for virion assembly and it remained non-infectious to susceptible host plants. Whereas simultaneous mutation of amino
acids located at positions 32, 40 and 43 resulted in an infectious variant sequence but it exhibited defects in encapsidation. One variant characterized by a deletion of bases specifying isoleucine located at position 42 was infectious like wild type but exclusively defective in packaging genomic RNA1.
Impacts
Majority of plant viruses are transported between cells of initially infected leaf and to upper leaves in assembled form. Therefore it is imperative to understand the regions of coat protein interacting with viral RNAs during encapsidation process. Results obtained from these studies are important in developing novel strategies of virus disease control.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Tobacco mosaic virus (TMV), a rod shaped plant virus and brome mosaic virus (BMV), an isometric plant virus share similarities in replication mechanisms. However, they significantly differ in their host range and most importantly in packaging genomic and subgenomic RNAs. In brome mosaic virus, the three genomic and the single subgenomic coat protein mRNA are encapsidated into three separate particles of identical shape and size with T=3 icosahedral symmetry: genomic RNAs 1 and 2 are packaged individually into separate particles whereas RNAs 3 and 4 are thought to be co-packaged into a single particle. In Nicotiana benthamiana plants, TMV induces a severe necrosis whereas BMV remains symptomless. We engineered a TMV expression vector to express BMV coat protein in addition to that of TMV. Infection of this hybrid to whole plants is expected to synthesize one genomic and four subgenomic RNAs. As expected tobacco plants inoculated with transcripts of the hybrid resulted
in the production of symptoms different from either of the parental virus. Molecular characterization of the progeny recovered from symptomatic plants revealed the presence of rods as well as icosahedral virions. The two types of virions were separated on sucrose density gradient and their RNA content analyzed. Comparative Northern blot analysis of total and virion RNA revealed that rod shaped virions packaged only the genomic RNA of TMV hybrid. No other RNAs were found. By contrast, only the subgenomic RNAs were found to be packaged by two differently sized icosahedral virus-like particles. Interestingly, a subgenomic RNA capable of synthesizing TMV coat protein was not found in any virions. We are currently examining the mechanism of subgenomic RNA packaging by BMV coat protein.
Impacts
Viruses exhibit a high degree of specificity in packaging their genomes. Packaging of viral genomic RNAs by heterologous coat protein, a mechanism often referred to as heteroencapsidation, is likely to yield novel pathogens. Therefore, dissecting the events leading to tobacco mosaic virus RNA packaging by brome mosaic virus coat protein is likely to shed more light in to mechanism of RNA packaging.
Publications
- No publications reported this period
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Progress 01/02/00 to 12/31/00
Outputs
Despite similarities in replication mechanisms, tobacco mosaic virus and brome mosaic virus differ significantly in their host range and most importantly in packaging genomic and subgenomic RNAs. In brome mosaic virus, the three genomic and the single subgenomic coat protein mRNA are encapsidated into three separate particles of identical shape and size with T=3 icosahedral symmetry: genomic RNAs 1 and 2 are packaged individually into separate particles whereas RNAs3 and 4 are thought to be co-packaged into a single particle. The majority of tobamoviruses having the origin of assembly sequence in the movement protein cistron package detectable amounts of genomic RNA but only the movement protein subgenomic RNA is detected upon enrichment of the high speed supernatant. Sunn-hemp mosaic virus having origin of assembly in the coat protein cistron has been shown to package genomic RNA into normal size rods and detectable levels of subgenomic RNAs of movement protein and
coat protein into shorter rods. The coat protein of icosahedral brome mosaic virus was expressed from a genetically engineered rod-shaped Tobacco mosaic virus. Molecular characterization of the progeny recovered from symptomatic plants revealed that brome mosaic virus coat protein selectively packaged the three subgenomic RNAs of the hybrid virus into two differently sized icosahedral virus-like particles. The smaller virus-like particles packaged only the two smaller subgenomic RNAs. Additional in vitro reassembly assays with brome mosaic virus coat protein subunits and transcripts of hybrid subgenomic RNAs further demonstrated that the ability of brome mosaic virus capsids to display polymorphism is not dependent on the RNA size alone and appears to be controlled by some other features of the genetically engineered RNA.
Impacts
Viruses exhibit a high degree of specificity in packaging their genomes. Packaging of viral genomic RNAs by heterologous coat protein, a mechanism often referred to as heteroencapsidation, is likely to yield novel pathogens. Therefore, dissecting the events leading to tobacco mosaic virus RNA packaging by brome mosaic virus coat protein is likely to shed more light in to mechanism of RNA packaging.
Publications
- Choi, Y. G., and Rao, A. L. N. (2000). Packaging of Tobacco Mosaic Virus Subgenomic RNAs by Brome Mosaic Virus Coat Protein Exhibits RNA Controlled Polymorphism. Virology275, 249-257.
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Progress 01/01/99 to 12/31/99
Outputs
In order to elucidate the function of the C-terminal region of cowpea chlorotic mottle bromovirus (CCMV) movement protein (MP) in cell-to-cell movement, a set of deletions ranging from 10 to 80 amino acids (DMP10, DMP20, DMP33, DMP43, DMP60 and DMP80) was engineered into the MPgene encoded by the biologically active clone C3/DCP-EGFP, a variant of CCMV RNA3 that contained wild-type (wt) MP and the enhanced green fluorescent protein (EGFP) gene in place of the coat protein (CP). The effect of each MP deletion on cell-to-cell movement was examined in three susceptible host plants: Chenopodium quinoa, Nicotiana benthamiana and cowpea (VIGNA SINENSIS cv. Black Eye). The results indicate that, except for mutant DMP43, infections resulting from the deletion mutants remained subliminal. Interestingly, infections resulting from inoculating mutant DMP43, which lacked the 43 most C-terminal amino acids, spread rapidly between cells and the number of infected cells expressing
EGFP approached that of control inoculations made with C3/DCP-EGFP. To verify whether the presence of wt CP altered the movement behavior of these mutants, each MP deletion was also incorporated into the genetic background of wt CCMV RNA3 (pCC3) and inoculated independently to all three hosts. These accentuate that the overall movement process exhibited by each MP mutant is influenced profoundly by the presence of CP and the particular host plant tested.
Impacts
Above results shed more light into understanding how plant viruses are transported between cells and the role of viral genes in this process. This information is valuable in devising novel strategies for virus disease control.
Publications
- Osman, F., Schmitz, I. and Rao, A. L. N. 1999. Effect of C-terminal deletions in the movement protein of cowpae chlorotic mottle virus on cell-to-cell and long distance movement. Journal of General Virology 80: 1357-1365.
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Progress 01/01/98 to 12/01/98
Outputs
Brome mosaic bromovirus (BMV) and cucumber mosaic cucumovirus (CMV) are structurally and genetically very similar. The specificity of the BMV and CMV coat proteins (CPs) during in vivo encapsulation was studied using two RNA3 chimera in which the respective CP genes were exchanged. The replicative competence of each chimera was analyzed in NICOTIANA BENTHAMIANA protoplasts and their ability to cause infections was examined in two common permissive hosts, CHENOPODIUM QUINOA and N. BENTHAMIANA. Each RNA3 chimera replicated to near wild type (wt) levels and synthesized CPs of expected parental origin, when co-inoculated with their respective genomic wt RNAs 1 and 2. However, inoculum containing each chimera was non-infectious in the common permissive hosts tested. Encapsidation assays in N. BENTHAMIANA protoplasts revealed that CMV CP expressed from chimeric BMV RNA3 was capable of packaging heterologous BMV RNA, however, at a lower efficiency than parental BMV CP. By
contrast, BMV CP expressed from chimeric CMV RNA3 was unable to package heterologous CMV RNA. These observations demonstrate that BMV CP, but not CMV CP, exhibits a high degree of specificity during in vivo packaging. The reasons for the non-infectious nature of each chimera in the host plants tested and factors likely to affect encapsulation in vivo are discussed.
Impacts
(N/A)
Publications
- OSMAN, F., GRANTHAM, G. L. and RAO, A. L. N. 1998. Molecular studies on bromovirus capsid protein V. Evidence for the specificity of brome mosaic virus encapsulation using RNA3 chimera of brome mosaic and cucumber mosaic viruses expressing.
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Progress 01/01/97 to 12/01/97
Outputs
To analyze whether the replication competence and cell-to-cell movement of dicot adapted cowpea mottle bromovirus (CCMV) is distinct from that of monocot adapted brome mosaic bromovirus (BMV), two reporter genes, b-glucuronidase (GUS) or enhanced green fluorescent protein (EGFP) were substituted for the CP in a biologically active clone of CCMV RNA3 (C3). Primary leaves of NICOTIANA BENTHAMIANA, CHENOPODIUM QUINOA and cowpea were coinoculated with wild type (wt) CCMV RNAs 1 and 2 and either C3/DCP-GUS or C3/DCP-EGFP and analyzed for GUS activity or presence of green fluorescence. The visual appearance of infections caused by GUS or EGFP variants indicated that, in CCMV, epidermal cell-to-cell movement can occur without a functional CP. By contrast, inoculation of MP defective variants of C3/DCP-GUS or C3/DCP-EGFP resulted in subliminal infections. Additional experiments examining the infectivity of wt BMV RNAs 1+2 and a BMV RNA3 variant bearing the EGFP in the place
of CP (B3/DCP-EGFP) confirmed previous observations that, unlike CCMV, epidermal cell-to-cell movement of BMV is dependent on the expression of a functional CP. Taken together, the results demonstrate that BMV and CCMV use different mechanisms for initial epidermal cell-to-cell spread.
Impacts
(N/A)
Publications
- RAO, A.L.N. 1997. Molecular studies on bromovirus capsid protein, III. Analysis of cell-to-cell movement competence of coat protein defective variants of cowpea chlorotic mottle virus. Virology
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Progress 01/01/95 to 12/30/95
Outputs
My laboratory is investigating the role of viral coat protein in pathogenesis and movement, using brome mosaic virus-CHENOPODIUM as a model system. Inoculation of six brome mosaic virus (BMV) RNA3 transcripts with defined deletions in the coat protein (CP) gene to three CHENOPODIUM spp demonstrated that synthesis of a functional, encapsidation-competent CP is required for the induction of local lesions. The BMV CP ORF contains two in-frame AUG codons separated by 7 amino acids resulting in the synthesis of two CPs (CP1 and CP2). To elucidate the biological significance of the N-terminal basic region of BMV CP, RNA3 variants capable of producing either CP1 or CP2 but not both were constructed. Infection phenotypes elicited on three CHENOPODIUM spp by each RNA3 variant revealed that amino terminal residues 1 to 7 are required to establish chlorotic local lesions and systemic infection in C. QUINOA. Deletion of this region has no effect on infection in barley plants but
resulted in the induction of the hypersensitive response on the inoculated leaves of C. QUINOA and blocked systemic spread. Analysis of seven additional RNA3 variant transcripts, each having a six base deletion (two amino acids) in the sequence encoding the N-terminal 7 residues, indicated that variants that share a common deletion of positively charged lysine rendered the CP encapsidation-incompetent and failed to establish infection.
Impacts
(N/A)
Publications
- RAO, A. L. N., and GRANTHAM, G. (1995). Biological significance of the first seven amino-terminal basic residues of brome mosaic virus coat protein. Virology 211, 42-52.
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