skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Superinfection Exclusion by p28 of Turnip Crinkle Virus Is Separable from Its Replication Function
We recently reported that the p28 auxiliary replication protein encoded by turnip crinkle virus (TCV) is also responsible for eliciting superinfection exclusion (SIE) against superinfecting TCV. However, it remains unresolved whether the replication function of p28 could be separated from its ability to elicit SIE. Here, we report the identification of two single amino acid mutations that decouple these two functions. Using an Agrobacterium infiltration-based delivery system, we transiently expressed a series of p28 deletion and point mutants, and tested their ability to elicit SIE against a cointroduced TCV replicon. We found that substituting alanine (A) for valine (V) and phenylalanine (F) at p28 positions 181 and 182, respectively, modestly compromised SIE in transiently expressed p28 derivatives. Upon incorporation into TCV replicons, V181A and F182A decoupled TCV replication and SIE diametrically. Although V181A impaired SIE without detectably compromising replication, F182A abolished TCV replication but had no effect on SIE once the replication of the defective replicon was restored through complementation. Both mutations diminished accumulation of p28 protein, suggesting that p28 must reach a concentration threshold in order to elicit a strong SIE. Importantly, the severe reduction of F182A protein levels correlated with a dramatic loss in the number of intracellular p28 foci formed by p28–p28 interactions. Together, these findings not only decouple the replication and SIE functions of p28 but also unveil a concentration dependence for p28 coalescence and SIE elicitation. These data further highlight the role of p28 multimerization in driving the exclusion of secondary TCV infections.  more » « less
Award ID(s):
1758912
PAR ID:
10479655
Author(s) / Creator(s):
; ; ; ; ; ; ;
Publisher / Repository:
APS Press
Date Published:
Journal Name:
Molecular Plant-Microbe Interactions®
Volume:
33
Issue:
2
ISSN:
0894-0282
Page Range / eLocation ID:
364 to 375
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; (, Virology)
    We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wildtype p28 (Zhang et al., 2017). Here we show that p88, the TCV-encoded RNA-dependent RNA polymerase, likewise trans-complemented a p88-defective TCV replicon, but repressed one encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans-complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans-complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a novel class of small TCV RNAs. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance. 
    more » « less
  2. ; ; ; ; (, Virology)
    We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wildtype p28 (Zhang et al., 2017). Here we show that p88, the TCV-encoded RNA-dependent RNA polymerase, likewise trans-complemented a p88-defective TCV replicon, but repressed one encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans-complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans-complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a novel class of small TCV RNAs. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance. 
    more » « less
  3. ; ; ; (, Viruses)
    RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication. 
    more » « less
  4. ; ; (, Viruses)
    Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus. 
    more » « less
  5. ; ; ; ; ; (, Journal of Virology)
    Simon, Anne E. (Ed.)
    ABSTRACT Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5′-to-3′ direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (−) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans . Consistent with its replicational origin, ttsgR accumulation required a 5′ terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5′ CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis . Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5′-to-3′ degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email