Positive-sense RNA viruses often use 3′ tRNA-like structures (TLSs) instead of poly(A) tails to capture the host translation machinery. While TLSs resemble canonical tRNAs and engage host factors, their ability to directly recruit ribosomes has remained unresolved. Here, we present a high-resolution cryo-electron microscopy structure of the Peanut clump virus (PCV) TLS bound to the tobacco 80S ribosome.
Local refinement resolves the PCV2 TLS at 2.37 Å and reveals a bipartite RNA architecture spanning the ribosomal tRNA channel. The tRNA-like domain occupies the E site and forms non-canonical two-base pairing with mRNA, whereas the expanded upstream pseudoknot domain anchors the L1 stalk and extends into the decoding A site, contacting 40S helix h44. This A-to-E-site trajectory closely resembles the ribosome-spanning path of viral IRES elements such as the CrPV IRES, indicating that PCV TLS acts not only as a tRNA mimic but also as an IRES-like ribosome-engagement module. Functional assays show that TLS-containing RNAs directly associate with ribosomal subunits and polysome fractions even under cycloheximide treatment. Reporter assays further show that viral 3′ UTR/TLS-containing mRNAs translate at levels comparable to or greater than poly(A)-tailed controls in wheat germ extract and tobacco BY-2 protoplasts, with more than twofold enhancement in the native plant system.
Together, these findings provide structural and functional evidence that PCV TLS hijacks the ribosomal tRNA channel to support poly(A)-independent translation, and establish a framework for engineering TLS-based 3′ modules for mRNA design.