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1.  Short ORF-Dependent Ribosome Shunting Operates in an RNA Picorna-Like Virus and a DNA Pararetrovirus that Cause Rice Tungro Disease 
PLoS Pathogens  2012;8(3):e1002568.
Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence.
Author Summary
Ribosome shunting, first discovered in plant pararetroviruses, is a translation initiation mechanism that combines 5′ end-dependent scanning and internal initiation and allows a bypass of highly-structured leaders of certain viral and cellular mRNAs. Here we demonstrate that a similar shunt mechanism has been developed by the RNA picorna-like virus RTSV and the DNA pararetrovirus RTBV that form a disease complex in rice. Leader sequences of the RTSV genomic RNA and the RTBV pregenomic RNA possess a conserved shunt configuration with a 5′-proximal short ORF (sORF1) terminating in front of a large stem-loop structure. Like in RTBV and a related pararetrovirus Cauliflower mosaic virus, shunt-mediated translation downstream of the RTSV leader depends on initiation and proper termination of sORF1 translation and on formation of the basal helix of the downstream secondary structure. Given that RTBV-like shunt elements with identical sequence motifs are present in all RTSV isolates but absent in related picorna-like viruses, it is likely that RTSV could have acquired these elements after its encounter with RTBV. Alternatively, the RTSV shunt elements could have evolved independently to adapt to the rice translation machinery. Our study highlights on-going genetic exchange and co-adaptation to the host in emerging viral disease complexes.
PMCID: PMC3291615  PMID: 22396650
2.  Intracellular Targeting of a Hordeiviral Membrane-Spanning Movement Protein: Sequence Requirements and Involvement of an Unconventional Mechanism▿  
Journal of Virology  2007;82(3):1284-1293.
The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.
PMCID: PMC2224415  PMID: 18032484

Results 1-2 (2)