In Brome mosaic virus (BMV), genomic RNA1 (gB1) and RNA2 (gB2), encoding the replication factors, are packaged into two separate virions, whereas genomic RNA3 (gB3) and its subgenomic coat protein (CP) mRNA (sgB4) are copackaged into a third virion. In vitro assembly assays performed between a series of deletion variants of sgB4 and wild-type (wt) CP subunits demonstrated that packaging of sgB4 is independent of sequences encoding the CP open reading frame. To confirm these observations in vivo and to unravel the mechanism of sgB4 copackaging, an Agrobacterium-mediated transient in vivo expression system (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005) that effectively uncouples replication from packaging was used. Cultures of agrotransformants, engineered to express sgB4 and CP subunits either transiently (sgB4(Trans) and Cp-Trans) or in replication-dependent transcription and translation when complemented with gB1 and gB2 (sgB4(Rep) and Cp-Rep), were mixed in all four pair-wise combinations and infiltrated to Nicotiana benthamiana leaves to systematically evaluate requirements regulating sgB4 packaging. The data revealed that (i) in the absence of replication, packaging was nonspecific, since transiently expressed CP subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed sgB4 and its deletion variants; (ii) induction of viral replication increased specificity of RNA packaging; and most importantly, (iii) efficient packaging of sgB4, reminiscent of the wt scenario, is functionally coupled not only to its transcription via replication but also to translation of CP from replication-derived mRNA, a mechanism that appears to be conserved among positive-strand RNA viruses of plants (this study), animals (flock house virus), and humans (poliovirus).

Viruses are intracellular parasites that can invade the cells of all known organisms. All viruses contain two components: a DNA or RNA genome and the capsid. Although the essential role of a viral capsid is to protect its genome, it must also release its genome for replication. To meet these two contesting requirements, viral capsids undergo a dynamic and reversible reorganization of biologically relevant surface peptides critical for their infectivity. Plant RNA viruses in the family Bromoviridae have a single-strand, positive-sense RNA genome divided among three genomic RNAs. These RNAs and subgenomic RNA get packaged into three homogeneous or four heterogeneous virion particles. In bromoviruses, the three virions, each carrying unique RNA species, have been physically inseparable by known separation techniques due to their particle homogeneity. Therefore, whether essential structural dynamism existed in these morphologically indistinguishable virions which could be of biological importance was unknown. Chapter 1 of this dissertation focused on investigating the stability and capsid dynamics of the three virions of brome mosaic virus (BMV), the type species of the genus Bromovirus, assembled autonomously in Nicotiana benthamiana using a robust agroinfiltration strategy. As the three virion types in BMV are identical in their morphology and electrophoretic mobility patterns, the use of a thermal denaturation assay and limited proteolysis followed by peptide mass mapping with MALDI-TOF enabled us to identify qualitative differences in capsid dynamics of three virions in BMV. Chapter 2 focused on understanding the comparative capsid dynamics among the three virions of BMV and cowpea chlorotic mottle virus (CCMV), a member of the family Bromoviridae that shares an identical genome organization and packaging scheme as BMV. Chapter 3 analyzed the relative dynamics of BMV and CCMV capsids assembled in the presence of heterologous replicase. Chapter 4 investigated the biological significance of co-packaging of a genetically redundant subgenomic RNA4 in BMV. Chapter 5 characterized Virus-like Particles (VLPs) assembled from the Gag protein of human immunodeficiency virus (HIV-1) in N. benthamiana. Finally, Chapter 6 attempted to silence the NbSKP1 gene in N. benthamiana using virus-induced gene silencing (VIGS) and studied the following effect on cucumber mosaic virus-Q strain accumulation.

Satellite RNA variant-Q associated with cucumber mosaic virus (CMV) is a highly structured single-stranded RNA molecule of 336 nucleotides in length. Satellite RNA is an important subviral pathogen in agriculture because it modulates symptom expression in CMV-infected plants. In the agricultural industry, CMV is economically very important since it exhibits a broad host range comprising over 1200 plant species world-wide. Although satellite RNA has no sequence homology with CMV genome, satellite RNA replication is thought to occur entirely in the cytoplasm and is exclusively dependent upon CMV replicase. Chapter 1 of this dissertation describes an Agrobacterium-mediated transient gene expression system that allows the expression of individual viral and satellite RNA uncoupled from the virus replication in vivo, and by utilizing this system we demonstrate that CMV coat protein expressed independently of replication can enhance the accumulation of an individual CMV RNA. Virions formed by the CMV coat protein in the absence of virus replication encapsidates some host RNAs, and the satellite RNA can accumulate to a detectable level in the absence of the helper CMV. Chapter 2 describes a novel finding that satRNA expressed in the absence of its helper CMV is amplified by the host. By using immunostaining with antibody against double strand RNA, and confocal microscopy to identify the subcellular distribution of double stranded satellite RNA in situ, we have found that in the absence of the helper virus, satRNA can amplify in the nucleus. Furthermore, sequence analysis of satRNA oligomers formed in the absence of CMV replication showed that the junction between two monomeric forms have a unique heptanucleotide sequence GGGAAAA, which is not present in the junction of satRNA oligomers formed in the presence of CMV. Finally in chapter 3, by using agroinfiltration system, we demonstrate that the negative-sense strand of satellite RNA is not amplified by the host cell because it is not recruited into the nucleus. However, consistent with the previously published results, the negative-sense satellite RNA is replicated by the helper CMV but only to a low-level. These novel findings will place the viroids, hepatitis delta virus and satellite RNAs in a closer relationship with each other in terms of their replication mechanisms. Implications of these findings are discussed in Conclusion chapter of this dissertation.

tRNAs, the adapter molecules in protein synthesis, also serve as metabolic cofactors and as primers for viral RNA-directed DNA synthesis. The genomic and subgenomic RNAs of some plant viruses have a 3'-terminal tRNA-like structure (TLS) that can accept a specific amino acid and serve as a site for initiation of replication and as a simple telomere. We report a previously undescribed role for the TLS of brome mosaic virus (BMV), and potentially for cellular tRNA, in mediating the assembly of its icosahedral virions. BMV genomic RNAs and subgenomic RNA lacking the TLS failed to assemble into virions when incubated with purified BMV coat protein. Assembly was restored by addition of a 201-nt RNA containing the BMV TLS. TLSs from two other plant viruses as well as tRNAs from wheat germ and yeast were similarly active in the BMV virion assembly reaction, but ribosomal RNA and polyadenylate did not facilitate assembly. Surprisingly, virions assembled from TLS-less BMV RNA in the presence of tRNAs or TLS-containing short RNA did not incorporate the latter molecules. Consistent with a critical role for the BMV TLS in virion assembly, mutations in the BMV genomic RNAs that were designed to disrupt the folding of the TLS also abolished virion assembly. We discuss the likely roles of the TLS in early stages of virion assembly.