Affiliation:
1. Department of Plant Pathology, University of California, Riverside, California 92521-0122
Abstract
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).
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
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