Truncated yet functional viral protein produced via RNA polymerase slippage implies underestimated coding capacity of RNA viruses

Abstract

RNA viruses use various strategies to condense their genetic information into small genomes. Potyviruses not only use the polyprotein strategy, but also embed an open reading frame, pipo, in the P3 cistron in the –1 reading frame. PIPO is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO) via transcriptional slippage of viral RNA-dependent RNA polymerase (RdRp). We herein show that clover yellow vein virus (ClYVV) produces a previously unidentified factor, P3N-ALT, in the +1 reading frame via transcriptional slippage at a conserved G1–2A6–7 motif, as is the case for P3N-PIPO. The translation of P3N-ALT terminates soon, and it is considered to be a C-terminal truncated form of P3. In planta experiments indicate that P3N-ALT functions in cell-to-cell movement along with P3N-PIPO. Hence, all three reading frames are used to produce functional proteins. Deep sequencing of ClYVV RNA from infected plants endorses the slippage by viral RdRp. Our findings unveil a virus strategy that optimizes the coding capacity.

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas

“…Previously, we reported that in the susceptible pea line PI 250438 infected with Cl-90-1 Br2 or ClNo.30, P3N-PIPO of Cl-90-1 Br2 could be detected, but the amount of P3N-PIPO of Cl-No.30 was below the level of detection when tested by using an antibody against the PIPO peptide (7). Furthermore, we showed that the amount of P3N-PIPO produced from the P3 cistron of Cl-No.30 is significantly smaller than that of Cl-RB in an in vitro translation system using MM2 dL (an extract derived from A. thaliana MM2d cells) and wheat germ extract (7,13). In this study, we showed evidence that the P3 cistron of ClNo.30 produced less P3N-PIPO protein than did Cl-RB in vivo in agroinfiltrated N. benthamiana leaves (Fig.…”

“…We previously showed that P3N-PIPO was detected in Cl-RB-infected plants but was below the level of detection in Cl-No.30-infected plants of a susceptible pea cultivar, PI 250438, indicating that the level of P3N-PIPO from Cl-No.30 is significantly lower than that of P3N-PIPO from Cl-90-1 Br2 (7). The same difference was observed when P3N-PIPO was produced from the P3 cistron of each virus by using an in vitro translation system with an A. thaliana cell-free system and wheat germ extract (7,13). In this study, we compared the accumulation of Cl-No.30 P3N-PIPO with that of Cl-RB P3N-PIPO in a transient-expression system, agroinfiltration in N. benthamiana leaf tissues.…”

“…The pTA/RB-P3(PIPO:FLAG Ϫ1 ), pTA/No.30-P3(PIPO:FLAG Ϫ1 ), pTA/RB-P3N-PIPO:FLAG mk , and pTA/No.30-P3N-PIPO:FLAG mk constructs were previously described (13).…”

“…As a control experiment, we compared the protein/mRNA ratios of P3N-PIPO:FLAG mk , which has mutations that enable the production of P3N-PIPO in the zero frame but does not produce P3 frame product, between Cl-RB and Cl-No.30 ( Fig. 5A) (13). Western blot analysis indicated that there were no visible differences in protein accumulation between Cl-RB and Cl-No.30 (Fig.…”

“…P3N-PIPO has an essential role in virus cell-to-cell movement (10). Three independent groups, including our own, recently showed that P3N-PIPO is produced mainly by transcriptional slippage within the P3 gene (11)(12)(13).…”