Nat. single-stranded RNA (ssRNA) segments that encode four structural proteins: the Gn and Gc glycoproteins, nucleocapsid protein (N), and RNA-dependent RNA polymerase. As other bunyaviruses, hantaviruses do not have a matrix protein that mediates assembly and budding; hence, a role for the cytoplasmic tails of glycoproteins has been proposed (6,C8). Previous studies of orthobunyavirus mutant glycoproteins showed that this endodomains of both glycoproteins are required for virus-like particle (VLP) and virus assembly (9). Further, studies around the Uukuniemi phlebovirus Gn tail showed that this Gn endodomain plays a crucial role in genome packaging into virus particles (10). Bunyavirus glycoproteins originate from a single glycoprotein precursor (GPC) through cotranslational cleavage in the endoplasmic reticulum (11,C14). Hantaviruses are believed to bud at internal membranes, most probably derived from the Golgi apparatus, and exit cells via exocytosis; alternatively, they may bud directly from the plasma membrane (11, 15, 16). To study viral assembly and budding processes, individual or isolated viral components are expressed in cells to test their release into the culture medium as VLPs corresponding to membrane-containing viral structures (17). Previously, it has been reported that hantavirus VLPs are produced when Gn, Gc, and N proteins are coexpressed (18, 19). However, not all of these proteins may be necessary for VLP production. This notion is usually supported by the observation that animals elicit high neutralizing antibody responses after DNA vaccination solely using a hantavirus Gn and Gc coding plasmid (20,C23), which may be indicative for VLP formation = 3; data not shown). To further characterize the VLPs in terms of density, sucrose gradient sedimentation was performed by ultracentrifugation for 16 h at 38,000 rpm using an SW55 rotor. The refractive index of each fraction was analyzed at 20C, and the presence of VLPs was examined by Western blotting using MAb anti-Gc 2H4/F6. VLPs derived from ANDV glycoprotein expression peaked in fraction 8, corresponding to a buoyant density of 1 1.15 g/ml (Fig. 2I) that coincides Hbegf with the density range of 1.15 to 1 1.18 g/ml, which has been reported for infectious hantaviruses and other bunyaviruses (34, 37). The size range of particles generated by glycoprotein expression was next determined by dynamic light scattering (Zetasizer Nano ZS; Malvern Instruments). The size of Mcl1-IN-11 over 90% of VLPs varied within the range of 90 to 255 nm (Fig. 2J). When VLPs were incubated with 0.1% SDS, their size diminished below 20 nm, confirming their membranous composition (Fig. 2J). Taken together, these data indicate that the release of glycoproteins into cell supernatants in the form of virus-like structures does not require the participation of the viral N protein. Further, the hantavirus glycoproteins are the only viral components required for the assembly and release of VLPs. The hantavirus VLPs were further characterized in terms of their antigenicity using the sera of hantavirus patients. Mcl1-IN-11 For this purpose, ANDV or PUUV VLPs contained in concentrated supernatants were immobilized on enzyme-linked immunosorbent assay (ELISA) plates by incubation for 1 h at room temperature (RT). Subsequently, wells were blocked with 4% casein-sucrose for 2 h at RT, and human sera were then added at a dilution of 1 1:250. After 1.5 h of incubation at RT, the wells were washed 5 times with 0.05% Tween 20Cphosphate-buffered saline (PBS). Next, wells were incubated for 1 h with anti-human immunoglobulins G, A, and M conjugated to peroxidase and finally revealed with a tetramethylbenzidine peroxidase substrate (KPL). The reaction was stopped within 10 min by the addition of 1 M phosphoric acid, and the absorbance was read at 450 nm. As seen Mcl1-IN-11 in Fig. 3, ANDV VLPs reacted with sera derived from Chilean patients. Weak cross-reactivity with ANDV VLPs was detected with the sera.