Maturation of Papillomavirus Capsids

Abstract

The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1-and L2-specific antibodies.Papillomaviruses, which are etiologically implicated in the development of warts, cervical cancer, and other epithelial tumors, replicate in the stratified squamous epithelium of the skin or mucous membranes (reviewed in reference 18). The papillomavirus life cycle is closely tied to the epithelial differentiation program, which takes many days to complete. The eventual release of the nonenveloped virion is thought to depend primarily on the normal cellular disintegration typically seen near the surface of squamous epithelia.The complexity of mimicking stratified epithelial differentiation in culture has made it challenging to study the papillomaviral life cycle in vitro. As a result, much of what is known about papillomavirus morphogenesis has been learned by studying recombinant versions of the two viral structural proteins, L1 and L2. The major capsid protein, L1, can spontaneously self-assemble into 72-pentamer virus-like particles (VLPs) that closely resemble native papillomavirus virions (20). The minor capsid protein, L2, is important for papillomavirus infectivity (38), but its arrangement in the virion and its role in encapsidation of the 8-kb double-stranded circular viral genomic DNA remain unclear.We have recently described a system for generating hightiter papillomavirus-based gene transfer vectors (also known as...

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…HPV16 pseudovirion (PsV) particles were produced as described previously (22)(23)(24). Briefly, 293T cells were transfected with pXULL, an human papillomavirus type 16 (HPV16) L1/L2 capsid protein expression plasmid (a gift of J. Schiller, NIH) and a "pseudogenome" reporter plasmid encoding a histone H2B-GFP fusion (a gift of H. Kimura, Tokyo Medical and Dental University) (25).…”

Replication and Assembly of Human Papillomaviruses

“…Assembly within the reducing environment of the cytosol/lysates of these cells is insufficient to form the much more extensive disulfide bonding network that is formed in native virions (Sapp et al, 1998). In fact, overnight incubation of PsV allows for the formation of additional disulfide bonds in a redox-dependent and cellular lysate-dependent fashion (Buck et al, 2005). Similarly, stabilization of VLPs has been reported through the removal of reducing agents, lowering of pH, and increasing of ionic strength (Mach et al, 2006).…”

“…Similarly, the L1 and L2 capsid proteins are expressed only in terminally differentiated keratinocytes (Barksdale and Baker, 1993). Studies with both papillomaviruses and polyomaviruses suggest that, once expressed, capsid proteins assemble into icosahedral capsids via assistance from chaperone proteins (Buck et al, 2005;Chromy et al, 2003Chromy et al, , 2006Bird et al, 2008). It is unknown if encapsidation of the viral genome takes place during capsid assembly or after; however, encapsidation is assisted by L2 and may be facilitated by E2 proteins (Heino et al, 2000;Gu et al, 2004;Holmgren et al, 2005).…”

“…Each particle is composed of 72 pentamers of the L1 capsid protein. L1 pentamers are thought to form a network of intra-and interpentameric disulfide interactions which serve to stabilize the capsid (Sapp et al, 1998;Fligge et al, 2001;Buck et al, 2005;Kondo et al, 2007). In addition to L1, an unknown amount of the minor capsid protein, L2, exists within the virion.…”

“…While many protocols exist, with various degrees of efficacy, to produce PsV, the most efficient PsV production protocol utilizes transfection of codon-optimized, L1-and L2-expression plasmids, in addition to reporter-gene-expressing plasmids into 293T or 293TT cells, which allows for efficient intracellular production of papillomavirus gene transfer vectors (Buck et al, 2005;Gambhira et al, 2007) (Fig. 3).…”