Replication-Dependent Biogenesis of Turnip Crinkle Virus Long Noncoding RNAs

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, 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 wildtype 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. 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 over-accumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR itself are readily replicated by TCV replication proteins (p28 and RdRp) provided from non-viral 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.

A cowpea severe mosaic virus-based vector bolsters virus-induced gene silencing and foreign protein expression in soybean

“…However, to develop a CPSMV-based VIGS and gene expression vector, we rst needed to generate infectious clones for both genomic RNAs of CPSMV. To this end, we chose to clone the RNA1 and RNA2 cDNAs into pAIDE, a binary vector modi ed from pCambia1300 by our lab [20][21][22][23]. The modi ed pAIDE plasmid no longer has the cassette that confers hygromycin resistance in transgenic plants, but acquired a new empty cassette with the duplicated 35S promoter (2X35S) and the 35S terminator (T35S), both originated from cauli ower mosaic virus, anking a multiple cloning site (Fig.…”

Single-cell mutation rate of turnip crinkle virus (-)-strand replication intermediates

“…Constructs. Both TCVdMP_sg2R and RTdMP_sg2R are binary constructs modified from TCV_sg2R and RT_sg2R (21,36), respectively, by creating a 92-nt deletion (positions 2,425-2,516) within the p8/p9 coding region. This deletion was previously shown to abolish TCV cell-to-cell movement without affecting the production of sgRNAs (14).…”

“…The RNA extraction procedure included a DNase treatment step that removed DNA contamination. The RNA was then quantified with NanoDrop and subjected to Northern blotting as described (21,22). proteins p28, p88, p8, p9, and p38.…”

Single-cell mutation rate of turnip crinkle virus (-)-strand replication intermediates

“…Both TCVdMP_sg2R and RTdMP_sg2R are binary constructs modified from TCV_sg2R and RT_sg2R [ 21 , 37 ], respectively, by creating a 92-nt deletion (positions 2,425–2,516) within the p8/p9 coding region. This deletion was previously shown to abolish TCV cell-to-cell movement without affecting the production of sgRNAs [ 14 ].…”