Structural Investigation of RNA Silencing Supression by Plant Viruses

RNA silencing is a conserved eukaryotic gene regulation mechanism that provides sequence-specific antiviral defense in higher plants and a few taxa of invertebrates. Double-stranded RNA (dsRNA) molecules trigger Dicer-like (DCL) Class-III RNases in the host cytoplasm that result in hydrolysis of dsRNAs into small 21-24 nt siRNA duplexes, typically with 2-nt 3′ overhangs. Guided by the siRNA, RISC complex surveils the cytoplasm for complementary viral RNA targets and cleaves them by PIWI-domain mediated hydrolysis, rendering antiviral defense through post-transcriptional gene silencing (PTGS).

High plains wheat mosaic virus (HPWMoV) is an important wheat and maize pathogen that belongs to the newly discovered Emaravirus genus (Family: Fimoviridae, Order: Bunyavirales). Recently, the complete genome sequence of various isolates of HPWMoV revealed eight segments, of which two encode siRNA suppression proteins termed P7 and P8. While P7 shows binding to long dsRNA fragments (180 nt), P8 seems to bind preferably to 21 nd dsRNA, implying different siRNA suppression mechanisms.

We expressed P8 and purified it to homogeneity and found it to oligomerizes in solution to a very stable tetramer. We had success in crystallizing P8 and determined its crystal structure to 1.9 Å resolution. The structure reveals new class of short RNA binding protein. Using our structure we biochemically explored the mode of binding of short RNA duplex to P8. Our structural and biochemical investigations provide new insights into viral evasion mechanisms to win in the tug-of-war between the virus and its host-cell.