Hantavirus Maturation

Journal of General Virology

et al. 2010

Here we characterize the interaction between the glycoproteins (Gn and Gc) and the ribonucleoprotein (RNP) of Puumala virus (PUUV; genus Hantavirus, family Bunyaviridae). The interaction was initially established with native proteins by co-immunoprecipitating PUUV nucleocapsid (N) protein with the glycoprotein complex. Mapping of the interaction sites revealed that the N protein has multiple binding sites in the cytoplasmic tail (CT) of Gn and is also able to bind to the predicted CT of Gc. The importance of Gn-and Gc-CTs to the recognition of RNP was further verified in pull-down assays using soluble peptides with binding capacity to both recombinant N protein and the RNPs of PUUV and Tula virus. Additionally, the N protein of PUUV was demonstrated to interact with peptides of Gn and Gc from a variety of hantavirus species, suggesting a conserved RNP-recognition mechanism within the genus. Based on these and our previous results, we suggest that the complete hetero-oligomeric (Gn-Gc) 4 spike complex of hantaviruses mediates the packaging of RNP into virions. INTRODUCTIONHantaviruses comprise a rodent-and insectivore-borne genus in the family Bunyaviridae (Schmaljohn & Hjelle, 1997;Vaheri et al., 2008). Bunyaviruses, presently classified in five genera (Hanta-, Orthobunya-, Nairo-, Phlebo-and Tospovirus), are enveloped, single-stranded RNA viruses with a trisegmented genome of a negative-sense coding strategy (Elliott et al., 2000;Nichol et al., 2005;Schmaljohn & Hjelle, 1997). The three genome segments [small (S), middle (M) and large (L)] encode the nucleocapsid (N) protein, envelope proteins (Gn and Gc) and RNAdependent RNA polymerase (L protein) (Elliott, 1997;Plyusnin & Morzunov, 2001). Unlike other members of the family Bunyaviridae that utilize arthropod vectors, hantaviruses have co-evolved with, and are maintained by, different rodent and insectivore species (Heyman et al., 2009;Klempa, 2009;Vaheri et al., 2008;Vapalahti et al., 2003). Whilst rather benign to the carrier rodents, the transmission of hantaviruses to man is known to cause haemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas (Klempa, 2009;Mertz et al., 2006;Nichol et al., 2005;Pini, 2004;Schonrich et al., 2008;Vaheri et al., 2008;Vapalahti et al., 2003). Enveloped hantavirus particles are described as pleiomorphic or spherical, measuring 80-120 or 120-160 nm in diameter depending on the method of analysis (Huiskonen et al., 2010; Martin et al., 1985;Nichol et al., 2005). The lipid bilayer of the virion encloses the ribonucleoprotein (RNP) in which the genomic RNA segments are encapsidated, possibly as closed circles organized in helical structures with trimers of N protein (Alminaite et al., 2008;Kaukinen et al., 2005;Plyusnin et al., 1996; Wang et al., 2008). Glycoproteins Gn and Gc assemble in virions as extrusions called spikes and arrange to form an unusual fourfold grid-like surface symmetry Huiskonen et al., 2010; Martin et al., 1985). By transmembrane prediction, b...

Section: Discussionmentioning

confidence: 99%

Cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein

Hepojoki

Strandin

Journal of General Virology

et al. 2010

“…During infection, nucleocapsids are trafficked to the cytoplasm (22) to assemble into mature virions (56). Mammalian cells transfected with the N protein alone show a granular pattern of immunofluorescence (32,34).…”

Section: Discussionmentioning

confidence: 99%

“…Immunocytochemistry of N Protein-Hantaviruses are believed to mature intracellularly; specifically, in the Golgi complex (56). During infection, the N protein was shown to localize cytoplasmically in the endoplasmic reticulum-Golgi intermediate compartment, presumably as they traffic from the endoplasmic reticulum to the Golgi (22).…”

Section: Circular Dichroism Spectroscopy Of N 1-74mentioning

confidence: 99%

See 1 more Smart Citation

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

Journal of Biological Chemistry

Boudreaux

Estrada

et al. 2008

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...

“…Gn and Gc are co-translationally cleaved from a precursor and targeted to the cis-Golgi compartment (Shi & Elliott, 2002;Spiropoulou et al, 2003). The Gn glycoprotein consists of an external domain, a transmembrane domain and a Cterminal cytoplasmic domain (Spiropoulou, 2001).…”

Section: Introductionmentioning

confidence: 99%

Degradation and aggresome formation of the Gn tail of the apathogenic Tula hantavirus

Journal of General Virology

Strandin

Hepojoki

et al. 2009

The cytoplasmic tails of envelope glycoprotein Gn of pathogenic hantaviruses but not of the apathogenic Prospect Hill virus (PHV) were recently reported to be proteasomally degraded in simian COS7 cells. Here, we show that the cytoplasmic tails of the glycoproteins of the apathogenic hantaviruses Tula virus (TULV) and PHV are also degraded through the ubiquitinproteasome pathway, both in human HEK-293 and in simian Vero E6 cells. TULV Gn tails formed aggresomes in cells with proteasomal inhibitors. We conclude that degradation upon aggregation of Gn tails, which may represent a general cellular response to misfolded protein used by hantaviruses to control maturation of virions, is unrelated to pathogenicity.