Phleboviruses encapsidate their genomes by sequestering RNA bases

Abstract

Rift Valley fever and Toscana viruses are human pathogens for which no effective therapeutics exist. These and other phleboviruses have segmented negative-sense RNA genomes that are sequestered by a nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes of irregular, asymmetric structure, previously uncharacterized at high resolution. N binds nonspecifically to single-stranded RNA with nanomolar affinity. Crystal structures of Rift Valley fever virus N-RNA complexes reconstituted with defined RNAs of different length capture tetrameric, pentameric and hexameric N-RNA multimers. All N-N subunit contacts are mediated by a highly flexible α-helical arm. Arm movement gives rise to the three multimers in the crystal structures and also explains the asymmetric architecture of the RNP. Despite the flexible association of subunits, the crystal structures reveal an invariant, monomeric RNP building block, consisting of the core of one N subunit, the arm of a neighboring N, and four RNA nucleotides with the flanking phosphates. Up to three additional RNA nucleotides bind between subunits. The monomeric building block is matched in size to the repeating unit in viral RNP, as visualized by electron microscopy. N sequesters four RNA bases in a narrow hydrophobic binding slot and has polar contacts only with the sugar-phosphate backbone, which faces the solvent. All RNA bases, whether in the binding slot or in the subunit interface, face the protein in a manner that is incompatible with base pairing or with "reading" by the viral polymerase. transmitted by arthropod vectors and cause a variety of severe diseases worldwide. The Rift Valley fever virus is a highly infectious, mosquito-borne pathogen endemic to sub-Saharan Africa. RVFV infects livestock and humans and generally causes a flu-like illness; however, 1% of cases result in hemorrhagic fever disease, which has a 50% case-fatality rate (1). The closely related Toscana virus (TOSV) is endemic to the Mediterranean basin, is transmitted by infected phlebotomine sandflies and causes neurological dysfunction in humans (2). The membrane envelope of bunyaviruses encloses a three-segment, negative-sense RNA genome that is encapsidated by a nucleocapsid protein (N), forming the ribonucleoprotein (RNP) (3). However, the size and sequence of N vary extensively among the five genera of the Bunyaviridae family. Phlebovirus N are highly similar, but they appear unrelated to N of the other four bunyavirus genera.We showed previously that phleboviruses have a unique genome-packaging strategy and an RNP that lacks the helical symmetry observed in some other negative-sense RNA viruses (NSVs) (4, 5). However, the detailed interactions between phlebovirus N and the viral RNA genome are unknown. The crystal structure of a RVFV N monomer revealed a compact helical fold with two lobes (5). The structure of an RVFV N hexamer demonstrated conformational flexibility in N and showed a putative RNA binding site on the inner surface of the hexameric ring (6). An α-helical arm, which is...

Structure of the Leanyer orthobunyavirus nucleoprotein–RNA complex reveals unique architecture for RNA encapsidation

“…So far, structures of only five binary complexes of N with RNA are known. These include N proteins from rabies virus (RV) (4), vesicular stomatitis virus (VSV) (5), respiratory syncytial virus (RSV) (6), Lassa virus (1, 7) and Rift Valley fever virus (RVFV) (8). The structures reveal a ring-like oligomeric assembly of N with the RNA threading the cavity running along the inner or outer edge of the ring such that it cannot be accessed by the host's defense machinery.…”

“…The interaction sites have been mapped principally on the N and C terminals (14). The N protein of RVFV, a member of the Phlebovirus genus, has been proposed to oligomerize into a tetrameric, pentameric, or hexameric ring-like ribonucleoprotein (RNP) complex (8,15). In contrast, CCHFV N (Nairovirus genus) exists as a monomer when expressed as a recombinant protein (3,16,17).…”

Helical capsids of plant viruses: architecture with structural lability

“…Surprisingly, the tertiary structure of potexvirus CP is similar to that of the nucleoprotein (NP protein) encoded by viruses of the genus Phlebovirus (family Bunyaviridae) (Agirrezabala et al, 2015). The NP protein binds single-stranded phlebovirus RNA to form ribonucleoproteins (RNPs) representing sinuous loose helices with a variable pitch (Raymond et al, 2012;Zhou et al, 2013). Taking into account the distant evolutionary relationship of mammal-infecting phleboviruses and plant potexviruses, the evolution of these virus groups is suggested to involve an event of horizontal gene transfer of a CP-NP progenitor (Agirrezabala et al, 2015).…”

Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein ofTomato spotted wilt virus

“…Recently, the structures of N proteins from three animal virus genera of the Bunyaviridae family have been reported and several encapsidation mechanisms of viral genome RNAs have been suggested based on the formation of different oligomers of N proteins (Raymond et al, 2010(Raymond et al, , 2012Ferron et al, 2011;Guo et al, 2012;Carter et al, 2012;Wang et al, 2012;Dong et al, 2013;Niu et al, 2013;Li et al, 2013;Reguera et al, 2013). Interestingly, the sequence and structure of N proteins are highly conserved within a genus but differ between genera.…”