Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: A functional role for viral replicase in RNA packaging

Abstract

To begin elucidation of the relationship between Brome mosaic virus (BMV) 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 genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (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' tRNA-like structure (3' TLS). Co-expression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing 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.

Selective Repression of Translation by the Brome Mosaic Virus 1a RNA Replication Protein

“…Recently, an Agrobacterium-mediated T-DNA gene delivery system was established to study BMV replication, gene expression, and RNA packaging in Nicotiana benthamiana (4,14). The agroinfiltration system provides robust transient expression of viral proteins and allows careful dissection of the cisand trans-acting requirements for BMV RNA replication in plants.…”

Reverse Genetic Analysis of Ourmiaviruses Reveals the Nucleolar Localization of the Coat Protein in Nicotiana benthamiana and Unusual Requirements for Virion Formation

“…Furthermore, a specific mutant of the N-terminal part of the CP can assemble in vivo, but not in vitro, and an interaction between the CP and the replicase was hypothesized (11). Nevertheless, for BMV, in vivo production of BMV CP and RNA3 indeed results in virion formation (5), whereas in the ourmiavirus model, virion formation itself occurs only with active replication of RNA3. In ourmiaviruses, active replication is therefore also required for the structural aspects of capsid assembly, which to our knowledge has not yet been described in nonenveloped plus-strand RNA viruses.…”

“…Other virus models where the virus is able to self-assemble in vitro include the Brome mosaic virus (BMV), Turnip crinkle virus (TCV), Alfalfa mosaic virus (AMV), and Cowpea mosaic virus (CPMV) (22). Studies of virion formation in vivo are more recent, and new insights into virion in vivo assembly and packaging have been gained with the use of agroinfiltration (5). Within this context, very few model systems are under investigation, and detailed descriptions of plus-stranded isometric viruses have only recently been reviewed (52).…”

Replication-Independent Long-Distance Trafficking by Viral RNAs in Nicotiana benthamiana

“…RNA gel blot analysis revealed the presence of R3CPKOII and subgenomic RNA4 in leaf 4 after 2 d, indicating that CP is not required for RNA3 trafficking ( Figure 3B). The accumulation of R3CPKOII was lower than that of wild-type RNA3, but this is to be expected since the CP contributes to increased accumulation of (þ)-strand BMV RNAs (Annamalai and Rao, 2005;Gopinath et al, 2005).…”