CEPCEB Members

A. L.N. Rao Professor Department of Plant Pathology 3264 A Webber Hall University of California Riverside, CA 92521 Phone: (951) 827-3810 Fax: (951) 827-4294 Areas of Expertise Molecular Biology RNA Packaging Virus Assembly
Background Mechanism of RNA Packaging in BMV Current Laboratory Personnel Selected Recent Publications (Bibliography page)

Background

After getting B.SC (Ag) and M.Sc (Plant Pathology) degrees from India, I went Waite Agricultural Institute, University of Adelaide, South Australia to get Ph.D in Plant Virology). I joined UCR in 1993 after postdoctoral studies at Texas A&M University. The long term goal of my research is to elucidate the functional role of viral coat protein gene in pathogenesis, the movement and encapsidation. To fulfill these objectives my laboratory is using brome mosaic virus (BMV), a multicomponent RNA virus pathogenic to monocotyledonous hosts, as a model system (Rao, 2001). The three genomic and a single subgenomic RNA (mRNA for coat protein synthesis Fig. 1A) of BMV are packaged by a single coat protein of 180 subunits into three morphologically indistinguishable icosahedral virions (Fig. 1B). Initial efforts characterized the functional significance of the viral coat protein, specifically the N-terminal arginine rich RNA binding motif conserved among BMV coat protein, HIV Tat and Rev proteins, bacterial antiterminators and ribosomal proteins. It was observed that N-ARM contained crucial amino acids that specifically dictate packaging of coat protein subgenomic RNA4 of BMV (Choi and Rao, 2000). I presented an invited lecture on RNA packaging at a bromovirus symposium organized by the American Society of Virology.

Fig. 1. (A) Schematic diagram of the tripartite bromovirus genome and (B) proposed packaging scheme for four BMV RNAs into three icosahedral virions of identical size and shape.

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Mechanism of RNA Packaging in BMV

Genome packaging is considered to be a highly specific process since the majority of purified virions contain exclusively viral RNAs. Assembly of infectious virions has long been recognized to be an important phase in the life cycle of a given virus pathogenic to plants, humans and animals. However the mechanism by which viruses assemble and package their genomes (RNA or DNA) into stable virions is poorly understood. Since BMV is amenable for in vitro reconstruction of infectious virions from dissociated coat protein subunits and RNA, we initiated a project to unravel the mechanism of RNA packaging in BMV. Our recent studies indicated that assembly of infectious BMV virions requires highly conserved 3' co-terminal tRNA-like structures (TLS). The transient yet critical involvement of the TLS or host tRNAs in BMV assembly suggest a role in the nucleation of coat protein dimmers that serve as intermediates in the encapsidation pathway (Choi et al., 2002).

In BMV, 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 (Fig. 1B). Our results demonstrated 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. 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. A schematic model for sequential packaging of BMV RNA3 and 4 into a single virion is shown in Fig. 2.

Fig. 2. Sequential packaging model. Panel A: (Step 1) Prior packaging of BMV RNA3 (B3) occurs with wt CP subunits positioning the N-ARM on the surface of the virion. (Step 2) A specific interaction between BMV RNA4 (B4) and arginine residues of N-ARM occurs. (Step 3) This results in B3 and B4 co-packaging into the same virion. Panel B: (Step 1) As in panel A, B3 is packaged into the virions by mutant CP subunits. (Step 2) However, mutation of a crucial arginine residue disrupts B4 interaction with the N-ARM. (Step 3) Consequently the virion contains only B3 as exemplified by previous in vivo analysis (Choi and Rao, 2000).

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Current Laboratory Personnel

Dr. Annamalai Postdoctoral Fellow	Shauni Killien Graduate Student	George L. Grantham Research Technician

Selected Publications (Bibliography page)

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