The Hantavirus Nucleocapsid Protein Recognizes Specific Features of the Viral RNA Panhandle and Is Altered in Conformation upon RNA Binding

Abstract

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.The members of the Hantavirus genus of the family Bunyaviridae are spherical, enveloped viruses containing tripartite negative-sense RNA as their genome (37). The three genomic RNA segments, designated L, M, and S, encode an RNAdependent RNA polymerase, envelope glycoproteins (G1 and G2), and nucleocapsid (N) protein, respectively. Hantavirus infections can cause two serious and often fatal human diseases, hemorrhagic fever with renal syndrome and hantaviral pulmonary syndrome, characterized by lung damage and cardiac dysfunction (36). Humans are infected with hantaviruses from rodent reservoirs that are persistently infected without signs of disease (38).The initial steps in hantavirus infection require binding of the G1 and/or G2 protein to ␤3 integrin (9) or other cell surface receptors, followed by virus entry and uncoating. After entry, L-protein mediates transcription of minus-strand RNA, resulting in plus-stranded mRNA in the cytoplasm, apparently with an orthomyxovirus-like cap-snatching mechanism (7,8,16). Following viral mRNA translation, transcription shifts from mRNA to plus-strand complementary RNA and de novo minus-strand viral RNA synthesis concomitant with the formation of ribonucleoprotein structures (7,16,37). The ribonucleoproteins appear to be composed of viral RNA, N protein, and presumably viral polymerase and accumulate on the cytoplasmic side of intracellular me...

Characterization of the Interaction between Hantavirus Nucleocapsid Protein (N) and Ribosomal Protein S19 (RPS19)

“…Similar quenching studies were carried out with 1,5-IAEDANS-labeled RPS19 that was preincubated with 500 nM N. The data points in a plot of F 0 /F versus [Q] fit to a straight line, indicating a single species of 1,5-IAEDANS moieties that are equally accessible to the neutral quencher. The Stern-Volmer quenching constant (K sv ) was calculated from the slope of the straight line fitted to the data points as reported previously (14,58). The K sv values calculated from three independent experiments were used for the calculation of standard deviation.…”

Hantavirus Nucleocapsid Protein Has Distinct m7G Cap- and RNA-binding Sites

“…However, if the data points in the plot show a biphasic nature that indicates two species of tryptophan residues that are differentially accessible to the quencher. Stern-Volmer quenching constant (K sv ) was calculated from the slope of the straight line fitted to the data points, as reported previously (13,53).…”

The bunyavirus nucleocapsid protein is an RNA chaperone: Possible roles in viral RNA panhandle formation and genome replication

“…Trimeric N protein specifically interacts with the bunyavirus panhandle, and this specific interaction could have a role in either viral RNA encapsidation or initiation of genome replication or in both processes (Mir and Panganiban 2004). The latter process would be expected to require at least transient panhandle dissociation during replication initiation.…”

“…N exhibits high-affinity binding to the vRNA panhandle in vitro and has therefore been proposed to recognize the panhandle structure during the encapsidation process (Mir and Panganiban 2004). N also recognizes the cRNA panhandle but with lower affinity (Mir and Panganiban 2005).…”