cis -Acting Elements Required for Efficient Packaging of Brome Mosaic Virus RNA3 in Barley Protoplasts

Abstract

Viral RNAs are specifically selected for packaging during viral infection. Specific packaging occurs through an interaction between viral RNAs and structural coat proteins (CPs). The specific recognition of viral RNAs by CPs plays a crucial role in diverse facets of the viral life cycle and in the packaging event. The binding of CP to specific RNA elements is required for viral protein translation during infection initiation in alfalfa mosaic virus (28) and for the regulation of translation and the initiation of RNA packaging in the RNA phages (38). In retroviruses, nucleocapsid protein is thought to stimulate genomic RNA dimerization, which is required for efficient RNA packaging, reverse transcription, and recombination (14, 31). Many plant viruses require RNA packaging for systemic spread and for cell-to-cell movement (3, 10). Therefore, the CP-RNA interaction is a critical event in the viral life cycle. Compared with the characterization of trans-acting factors such as CP in RNA packaging, however, the RNA elements involved in specific packaging have not been well characterized for many viruses. One of the best-characterized RNA elements is "origin of assembly" of tobacco mosaic virus (36, 40). cis-acting sequences for RNA packaging have also been demonstrated in several icosahedral viruses, including flock house virus (39), Sindbis virus (37), hepatitis B virus (17), the human immunodeficiency viruses (2,14,22), and turnip crinkle virus (32).Brome mosaic virus (BMV) is an icosahedral plant RNA virus and is the type member of the genus Bromovirus in the family Bromoviridae in the alphavirus-like superfamily (18). The genome of BMV consists of three species of messenger sense single-stranded RNA (1). RNA1 (3.2 kb) and RNA2 (2.9 kb), which encode the 1a and 2a replicase proteins, respectively (1, 13, 19), are packaged separately into individual particles (21). RNA3 (2.1 kb), which encodes the 3a cell-to-cell movement protein (35), is packaged into a single particle together with subgenomic RNA4 (0.9 kb) (21). RNA4 is synthesized from the minus strand of RNA3 (25) and encodes CP. CP is required for packaging, cell-to-cell movement, and the systemic spread of the virus (29,33,34).A highly conserved N-terminal arginine-rich motif in BMV CP plays an important role in BMV RNA packaging through RNA-CP interactions (4,5,33,34). The crystallographic structure of BMV virions has been determined (23). RNA regions or elements involved in the packaging of BMV RNAs have been assigned to the coding region of BMV RNA1 by UV cross-linking and band-shift assays (11) as well as to the 3Ј-proximal region of the 3a open reading frame (ORF) in RNA3 (9). The tRNA-like structures (TLS) in the 3Ј-untranslated regions of BMV RNAs also play a crucial role in BMV RNA packaging in vitro (6). In the present study, we delimit a nucleotide sequence required for the efficient packaging of BMV RNA3 and show that 69 nucleotides (nt) in the 3Ј-proximal region of the BMV 3a ORF, especially a predicted stem-loop structure (30 nt), is essential fo...

Packaging of Brome Mosaic Virus Subgenomic RNA Is Functionally Coupled to Replication-Dependent Transcription and Translation of Coat Protein

“…Packaging of virus-specific RNAs into stable virions was shown to be mediated through specific recognition of sequences or structures unique to viral nucleic acids, often termed origins of assembly or packaging signals. These signals strongly enhance RNA packaging, and they were identified in several icosaheral RNA viruses infecting animals (40), insects (42), and plants (10,12,26). However, the presence of a packaging signal alone does not guarantee packaging of RNA into virions, since several factors of the packaging signal, such as secondary structure, position with respect to genomic context, and location (cis or trans), also affect packaging efficiency (10,12).…”

“…These signals strongly enhance RNA packaging, and they were identified in several icosaheral RNA viruses infecting animals (40), insects (42), and plants (10,12,26). However, the presence of a packaging signal alone does not guarantee packaging of RNA into virions, since several factors of the packaging signal, such as secondary structure, position with respect to genomic context, and location (cis or trans), also affect packaging efficiency (10,12). This study revealed active replication as being an important and intrinsic criterion for selective and enhanced packaging of BMV sgRNA.…”

Interaction between Brome Mosaic Virus Proteins and RNAs: Effects on RNA Replication, Protein Expression, and RNA Stability

“…Sequences for BMV RNA encapsidation are only beginning to be elucidated (20,22,23). The sequences required for RNA replication and transcription are somewhat better characterized, and the RNA motifs that bind the replicase in vitro and direct RNA synthesis have been mapped (17,19,80,84).…”

Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection

“…In many cases, virus capsid proteins have been found to bind specific high-affinity sequences and/or structures on viral RNA, which helps to preclude encapsidation of host RNAs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In the case of the satellite tobacco necrosis virus (STNV) and MS2 bacteriophage, specific RNA sequences may not exist; rather, the genome may contain numerous distinct but functionally similar packaging sites (e.g., small stem-loops) that are bound simultaneously but independently by multiple coat protein (CP) subunits (4,18,19).…”