Three-dimensional structure of baculovirus-expressed Norwalk virus capsids

Abstract

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T=3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T=3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (Pl), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 nm. It is

A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles

“…There are limitations in NoV VLP production in terms of inadequate yield and quality of the VLPs [1,3,9,20]. Both sucrose and CsCl gradients ultracentrifugation have been used for purification of NoV VLPs [1,7,9,17], even though studies on rotavirus-like particles demonstrated a low yield and impurities resulting from CsCl gradient purification [16].…”

“…In the present study, we compared commonly used methods for NoV GII-4 VLP purification [1,14,17] and concentration [6,19], considering the purity, yield, morphological integrity, antigenicity and functionality of the purified VLPs.…”

“…VLPs in the supernatant were concentrated by ultracentrifugation (L8-60M ultracentrifuge, Beckman SW-32.1 Ti rotor) at 100,0009g for 2 h at 4°C, and pellets were resuspended in 0.2 M Tris-HCl, pH 7.3. VLPs were loaded onto a 10-60% discontinuous sucrose gradient and ultracentrifugated at 100,0009g for 1 h at 4°C as described before [17]. Fractions were collected by bottom puncture.…”

“…NoV VLPs have been used extensively to study protein interactions [8], and virus assembly [17], and have been used as a tool in diagnostic serological assays [7]. Clinical trials have been performed with the NoV VLPs used as a vaccine [2].…”

Nonculturable agents of viral gastroenteritis

“…The name calicivirus is derived from the Latin calyx, meaning "cup" or "goblet," and refers to the 6)]. These depressions are frequently not clearly visible (e.g., for NV), although structural studies have demonstrated their presence (7). Because of these structural differences and the previous use of IEM to detect these viruses, many of the HuCVs were initially referred to as small, roundstructured viruses (SRSVs).…”