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

Abstract

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).Packaging of viral genomes by structural protein components leading to the assembly of infectious progeny virions is an essential step in the life cycle of plus-strand RNA viruses pathogenic to humans, animals, insects, and plants (28,33). The assembly of infectious virions is a carefully orchestrated process that maintains a high degree of precision and specificity, requiring protein subunits interacting with each other and with viral nucleic acids (5). The majority of RNA viruses infecting eukaryotic cells are assembled in the cytoplasm, and therefore an opportunity exists to copackage cellular RNA. However, remarkably mature virions exclusively contain viral RNA, with the exception of a few viruses, such as human immunodeficiency virus, whose virions contain cellular tRNAs. The mechanism and factors that regulate this specificity are not fully understood. However, a high degree of specificity between sequence-and/or structure-dependent interactions of RNA and coat protein (CP) operates during the assembly process (28, 33). Thus, knowledge of detailed mechanisms by which viruses assemble and package their genomes into structurally stable virions is an important prerequisite for understanding overall biology of eukaryotic RNA viru...

In Vivo Packaging of Brome Mosaic Virus RNA3, but Not RNAs 1 and 2, Is Dependent on a cis -Acting 3′ tRNA-Like Structure

“…5, lane 1). Similar observations were reported previously with nonreplicating or inefficiently replicating BMV RNA3 variants (1,4). By contrast, efficient accumulation and detection of sgB4 in samples complemented with either TLS 200 or UTR 336 suggested that the 3Ј end of the B3⌬TLS progeny was restored, permitting B3 to enter replication.…”

Self-Assembly of Viral Capsid Protein and RNA Molecules of Different Sizes: Requirement for a Specific High Protein/RNA Mass Ratio

“…More explicitly, CCMV virions purified from infected leaf tissue display a remarkable homogeneity in size and physical appearance: RNA1 (3,171 nt) and RNA2 (2,774 nt) are each packaged independently into Tϭ3 capsids, whereas RNA3 (2,173 nt) and RNA 4 (824 nt) are copackaged into a third virion. It has been shown that in vivo assembly of nucleocapsids in positive-strand RNA viruses pathogenic to humans (32), animals (4,46), and plants (3) is functionally coupled to replication-dependent transcription and translation, i.e., the assembly is mediated by capsid protein that is translated from replication-derived mRNA (5). Consequently, macromolecular interactions, such as the interaction between CP and viral replicase (RNA1 and RNA2 gene products), resulting from commonly shared subcellular localization sites (6), might play an important role, ensuring that only one kind of stable virion population with Tϭ3 symmetry is assembled and maintained.…”

Replication-Coupled Packaging Mechanism in Positive-Strand RNA Viruses: Synchronized Coexpression of Functional Multigenome RNA Components of an Animal and a Plant Virus in Nicotiana benthamiana Cells by Agroinfiltration

“…The mechanism by which the CP regulates this balanced distribution of four BMV RNAs into three individual virions is still unknown. More recent studies using experimental systems that are competent to effectively uncouple replication from packaging revealed that efficient packaging of viral RNA is functionally coupled to replication-dependent transcription and translation in FHV and BMV (5,58). However, it is not known whether such functional coupling between replication-dependent transcription and translation requires homologous replicase machinery.…”