The membrane glycoproteins Gn and Gc of Hantaan virus (HTNV) (family (HTNV) is the prototype from the genus in the family and is a causative agent of hemorrhagic fever with renal syndrome. theme WAASA (21) and adult Gn and Gc possess obvious molecular weights of 68 0 and 55 0 respectively (35). (Gn and Gc are specified according with their location in accordance with the N and C termini of GPC and had been previously known as G1 and G2.) Both Gn and Gc are type I integral transmembrane proteins with their own signal sequences (33). Our recent work indicated that the Gc signal sequence is an integral part of the Gn cytoplasmic tail and is required for Golgi targeting of both proteins (38). Similar to most other viruses in the family WYE-125132 HTNV glycoproteins Gn and Gc form a heterodimer and accumulate in the Golgi complex where viruses mature and assemble (reviewed in reference 39). It is accepted that the heterodimerization of Gn and Gc is essential for their correct folding and transport to the Golgi complex (32 38 40 HNTV Gn and Gc proteins are both modified by N-linked glycosylation. They possess six potential N-linked glycosylation sites (Fig. ?(Fig.1) 1 five on Gn (at N residues 134 235 347 399 and 609) and one on Gc (N928) (37). The N glycosylation sites are highly conserved among all hantaviruses suggesting that N glycosylation is crucial for the conformation and functions of the proteins. HTNV glycoproteins are sensitive to endoglycosidase H (endo H) treatment indicating that the N-glycan chains are predominantly of the high-mannose type (1 36 However there is disagreement as to whether the glycans are completely of the high-mannose type (1) or whether WYE-125132 some of WYE-125132 them have acquired endo H resistance (32 36 FIG. 1. Schematic of HTNV GPC and potential N-linked glycosylation sites. The GPC is represented by the bar with signal sequences (SS) and transmembrane domains (TMD) shown as open WYE-125132 boxes and filled boxes respectively. Lollipops indicate the locations of the … N-linked glycosylation is important not only for correct protein folding but also for glycoprotein function (16 18 27 CGB 44 Enveloped viruses may contain one or more types of integral membrane proteins and the majority of them are modified by the addition of N-linked carbohydrate chains (7). N-linked glycosylation is associated with the diverse functions of viral glycoproteins such as receptor binding mediation of membrane fusion and penetration into cells directing virus morphogenesis at the budding site and working as antigens to elicit a protective immune response (3 7 In this study we reexamined the properties of the N-linked oligosaccharides on HTNV glycoproteins and confirmed that the N-glycans are completely endo H sensitive. To determine the utilization of the potential N glycosylation sites and study their role in protein folding and intracellular trafficking of the glycoproteins we constructed 10 mutants bearing either single or double glycosylation site mutations. WYE-125132 Our data show that the individual sites of N glycosylation have different effects on protein folding and intracellular trafficking of the glycoproteins. Two endoplasmic reticulum (ER) chaperones calnexin (CNX) and calreticulin (CRT) were found associated with the glycoproteins and presumably play a role in HTNV glycoprotein folding. MATERIALS AND METHODS Cells and viruses. HeLaT4+cells and Vero E6 cells (ATCC C1008) were grown in Dulbecco’s modified Eagles’s medium containing 10% fetal bovine serum (FBS). BSR-T7 cells which stably express T7 RNA polymerase (4) were kindly provided by K. K. Conzelmann (Max-von-Pettenkofer Institut Munich Germany) and were grown in Glasgow modified Eagle’s medium containing 10% FBS. vTF7-3 a recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase (9) was a gift from B. Moss National Institutes of Health Bethesda Md. WYE-125132 vT-HTN M a recombinant vaccinia virus that expresses HTNV glycoproteins (Gn and Gc) was constructed in this laboratory (unpublished data). Antibodies. Monoclonal antibodies (MAbs) recognizing HTNV glycoproteins were kindly provided by C. S. Schmaljohn (Virology Department U.S. Military Medical Analysis Institute for Infectious Illnesses Frederick Md.). MAbs 8B6 300000 1600 and 6D4 are particular for Gn and MAbs 11E10 80000000000 16000000 and HCO2 are particular for Gc (2). A rabbit polyclonal antibody against GM130 a DNA polymerase (Stratagene). To create the N glycosylation site mutants one (for one mutation) or two (for dual mutations) from the asparagine (N) residues on the potential sites on Gn and Gc had been replaced with.