Hantavirus Nucleocapsid Protein Oligomerization

Abstract

Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome. The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein. The N protein is a 420-to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood. We have undertaken a biochemical and genetic analysis of N protein oligomerization. Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers. Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer. We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions. Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion. These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils. Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates. Hantaviruses, such as the Sin Nombre hantavirus (SNV) andProspect Hill virus (PHV), are members of the bunyavirus class of viruses (3,20,24). They are enveloped, negative-strand RNA viruses and carry three genomic RNA segments: the L segment, which encodes an RNA-dependent RNA polymerase; the M segment, which encodes envelope glycoproteins G1 and G2; and the S segment, which encodes the viral nucleocapsid (N) protein (24). Hantaviruses are of medical importance, because many of them cause either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome, which is characterized by lung damage and cardiac dysfunction (25).Models for hantavirus replication at the cellular level have been based on direct experiments and by inference from work on other bunyaviruses. The general replication cycle starts with G1 and G2 binding to B3 integrins (7,8) or other cell surface receptors, followed by virus entry and uncoating. After entry, L protein-mediated primary transcription of mRNAs occurs in the cytoplasm, apparently using an orthomyxovirus-like capsnatching mechanism (4, 14). Following mRNA translation, transcription shifts from mRNA to cRNA and viral RNA synthesis, and ribonucleoprotein (RNP) structures are formed (4, 14, 24). The RNPs appear to be composed of viral RNAs, N proteins, and presumably L proteins and accumulate on the cytoplasmic sides of cellular membranes, possibly through interactions with the G1 and G2 proteins (9, 21). Evidence suggests that RNPs use microfilaments for transport to ...

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes

“…This is remarkable because, in the case of THOV, we have previously found the MxA recognizes nucleocapsids rather than free nucleoprotein (23,31,37). Given the tendency of bunyavirus N proteins to associate into multimers (32,33), it is conceivable that N of LACV spontaneously forms nucleocapsid-like structures that are preferentially sequestered by MxA into the perinuclear complexes described here. In the absence of MxA, comparable structures are not detectable, indicating that MxA and N have to cooperate to form these highly ordered structures.…”

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

“…We have previously reported that a single molecule of N interacts with the mRNA 5Ј cap. In addition, N forms homodimers and homotrimers in cells (8). It is possible that dimerization of N molecules individually bound to the mRNA cap and RPS19 mediates the loading of 40 S subunit on capped mRNA during N-mediated translation initiation (Fig.…”

“…The K app (apparent equilibrium constant ϭ 1/K d ) was calculated at four different temperatures (8,15,25, and 30°C) by fluorometric titration of 1,5-IAEDANSlabeled RPS19 with N. ⌬H and ⌬S were calculated from the slope and intercept of a plot of ln K app against 1/T (Equation 4). ⌬G was calculated from Equation 5 after incorporation of the values for ⌬H and ⌬S obtained from Equation 4.…”

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

“…Although the N protein essential for the propagation of the virus adopts a highly conserved structure within a genus, N proteins from different genera differ in their primary sequences and 3D architecture markedly. For example, the Hantaan virus N protein is reported to form trimeric structures using homotypic N-N protein interactions (11)(12)(13). The interaction sites have been mapped principally on the N and C terminals (14).…”