NMR Structure of the N-terminal Coiled Coil Domain of the Andes Hantavirus Nucleocapsid Protein

Abstract

The hantaviruses are emerging infectious viruses that in humans can cause a cardiopulmonary syndrome or a hemorrhagic fever with renal syndrome. The nucleocapsid (N) is the most abundant viral protein, and during viral assembly, the N protein forms trimers and packages the viral RNA genome. Here, we report the NMR structure of the N-terminal domain (residues 1-74, called N 1-74 ) of the Andes hantavirus N protein. N 1-74 forms two long helices (␣ 1 and ␣ 2 ) that intertwine into a coiled coil domain. The conserved hydrophobic residues at the helix ␣ 1 -␣ 2 interface stabilize the coiled coil; however, there are many conserved surface residues whose function is not known. Site-directed mutagenesis, CD spectroscopy, and immunocytochemistry reveal that a point mutation in the conserved basic surface formed by Arg 22 or Lys 26 lead to antibody recognition based on the subcellular localization of the N protein. Thus, Arg 22 and Lys 26 are likely involved in a conformational change or molecular recognition when the N protein is trafficked from the cytoplasm to the Golgi, the site of viral assembly and maturation.Hantaviruses can cause two emerging infectious diseases known as the hantavirus cardiopulmonary syndrome (HCPS) 3 and the hantavirus hemorrhagic fever with renal syndrome (1). Annually, there are over 150,000 cases of hantaviral infections reported world wide (2). Rodents are the primary reservoir of hantaviruses, and humans are normally infected by inhalation of aerosol contaminated with the excreta of infected rodents. The first reported cases of HCPS in North America (3) was caused by a novel hantaviral species (4, 5), the Sin Nombre virus, and had an initial mortality rate of 78%. HCPS has since been reported throughout the United States with a current mortality rate of 35% when correctly diagnosed (6). The major cause of HCPS in South America is the Andes virus, and person-to-person transmission of the Andes virus was reported in Argentina and Chile (7). Hantaviruses are known to invade and replicate primarily in endothelial cells, including the endothelium of vascular tissues lining the heart (8 -10).The genome of hantaviruses consists of three negativestranded RNAs, which encode the nucleocapsid (N) protein, two integral membrane glycoproteins (G1 and G2), and an RNAdependent RNA polymerase (L protein). The N protein is highly immunogenic (11, 12) and elicits a strong immune response, which confers protection in mice (13-15). It is highly conserved and is the most abundant viral protein, and it plays important roles in viral encapsidation, RNA packaging, and host-pathogen interaction (16). The N protein binds to viral proteins (16), host proteins (17-23), and viral RNA (24 -28). The self-association of the N protein into trimers was shown by gradient fractionation and chemical cross-linking (29). Deletion mapping identified that regions at the N and C termini are important in N-N interaction (29 -31), and a model of trimerization was proposed based on the head-to-head and tail-to-tail association of the...

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

“…Previous results revealed that hantavirus NP undergoes trimerization (34,35) and that NP trimers specifically recognize a conserved panhandle structure that is formed by the complementary base pairing of the 5= and 3= ends of the hantaviral genome (36,37). Further results showed that the N-terminal domains of the ANDV (38) and SNV NPs (34) possess a coiled-coil architecture that is comprised of two antiparallel helices stabilized by conserved hydrophobic residues, while the C-terminal domain (residues 370 to 429) forms two helices; both domains are essential for NP oligomerization (35).…”

Identification of Two Functional Domains within the Arenavirus Nucleoprotein

“…Coiled coils have been shown to be important for self-interaction of hantavirus nucleoprotein (2)(3)(4)63). Nevertheless, the contribution of additional sequences that stabilize homotypic interactions has also been reported (28,29).…”

Characterization of Two Substrains of Puumala Virus That Show Phenotypes That Are Different from Each Other and from the Original Strain

“…This shows that the N protein is involved in, and therefore potentially can interfere with, several cellular functions. The secondary structure of the first 70 amino acids in the N protein has been solved (3,6,52). From these reports, it has been suggested that PUUV N-protein D27 is located within ␣-helix 1, while D35 is on the apex formed between ␣-helix 1 and ␣-helix 2.…”