Formation of Vesicular Stomatitis Virus Pseudotypes Bearing Surface Proteins of Hepatitis B Virus

Abstract

Hepatitis B virus (HBV), which belongs to the virus genusHepadnaviridae, is an important etiological agent for acute and chronic hepatitis as well as liver cirrhosis or primary hepatocellular carcinoma (15, 18). The surface proteins of HBV contain three related HBV-encoded glycoprotein species, termed large (L), middle (M), and small (S) proteins (37). As shown in Fig. 1A, all surface proteins are produced from a single open reading frame (ORF) by the use of three different translational start sites that divide this ORF into three domains: preS1, preS2, and S. Translation from the first ATG yields L protein, comprising 390 amino acids (aa), whereas translation from the ATG at aa positions 109 and 164 generates M and S proteins, respectively. The three proteins are posttranslationally modified: each of them exhibits a glycosylation site in the S domain, and an additional glycosylation occurs in the preS2 region of the M protein (Fig. 1A). Either M or S protein, when expressed alone, can be secreted as virus-like particles (25, 45). In contrast, L protein cannot be secreted when expressed alone; instead, it is mainly retained within the endoplasmic reticulum and pre-Golgi regions in the form of intraluminal particles (27,45). It has been reported that the sequence between amino acids 3 and 77 of the preS1 region is necessary for infection of primary human hepatocytes in vitro (21). The preS2 region of M protein has been thought to be involved in polymerized albumin-mediated interaction of HBV (48), but no precise function has yet been definitively assigned to the preS2 region of the M protein. Kann et al. (18) proposed that in association with the preS1 domain of L protein, the preS2 domain of M protein might have a supportive function in viral attachment. Thus, it is still necessary to study the roles of all three proteins in the HBV infection process to clarify the process of entry of HBV into cells.Analysis of HBV infection in vitro has still been difficult due to the lack of a suitable culture system, i.e., lack of susceptible cell lines or of cells producing HBV abundantly. Most in vitro studies have used concentrated, whole virions or human serum samples as virus sources and primary human hepatocytes or human liver cell lines, e.g., HepG2, HuH7, and PH5CH8, as target cells (12,13,21,31,41). However, their infectivities are not so high, and human serum samples and human hepatocytes are not readily available. Despite these difficulties, infection of cells with viral inocula or transfection of cells with HBV DNA has been employed in order to analyze HBV infection in vitro (12,14,46). In vitro infection of human hepatoma (HepG2) cells with hepatitis B virus obtained from the serum of a chronic HBV patient was first reported by Bchini et al. (3).There seems to be only one report that describes an HBV pseudotype: murine leukemia virus (MLV) pseudotype, expected to bear the L and S proteins of HBV (41). The reported MLV(HBV) pseudotype, which was prepared after cotransfection of 293T cells with plasmids expressing...

Assembly of Hepatitis B Virus Envelope Proteins onto a Lentivirus Pseudotype That Infects Primary Human Hepatocytes

“…Two previous studies detected HBsAg at the surface of transfected 293T cells by flow cytometry and immunocytochemistry analyses (25,30). Our results by flow cytometry (Fig.…”

“…The three envelope proteins of HBV, abbreviated here LMS, are cotranslationally inserted into the endoplasmic reticulum (ER) and, subsequently, within the ER or ER-Golgi intermediate compartment, facilitate HBV and HDV particle assembly and release (3,15,22,31). However, others have reported experimental situations where at least some of the HBsAg can actually be detected at the cell surface (25,30). Therefore, we undertook to confirm and extend such reports, with the ultimate aim of achieving the assembly of HIV vectors pseudotyped with LMS.…”

Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins

“…The pseudotype viruses were thus expected to show similar behaviors to RV during the process of virus entry, and the subsequent processes of gene expression are dependent on the VSV replication machinery. In the initial experiment, only RV envelope E2 and E1 proteins were provided in trans to generate the GFP gene-and FLuc gene-encoding pseudotype viruses, VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1, respectively, like other VSV pseudotype viruses 13,[19][20][21][22][23][24][25][26][27][28][29][30] . The infectivity titers for VSV GFP -RV/E2E1 and VSV FLuc -RV/E2E1 were 10-fold higher than those of the counterpart control viruses, VSV GFP -∆G and VSV FLuc -∆G, respectively, which lack envelope glycoproteins, in Vero cells ( Fig.…”

Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

“…These rVSV-ΔG pseudotypes, when plated on susceptible cells, undergo a single cycle of infection and the entry of the pseudotypes is dictated by the cell tropism and entry properties of the heterologous glycoprotein(s). Since the first description of this system in 1997 (Takada et al, 1997) rVSV-ΔG pseudotypes have been used in numerous studies examining virus entry (Abe et al, 2007;Fukushi et al, 2005;Glende et al, 2008;Hanika et al, 2005;Ito et al, 1999;Kaimori et al, 2004;Matsuura et al, 2001;Okuma et al, 2001;Perez et al, 2001;Saha et al, 2005;Tamura et al, 2005;Tatsuo et al, 2000a), for identification of novel host cell receptors (Tatsuo et al, 2000b), for screening antiviral libraries and development of neutralizing antibody tests (Fukushi et al, 2008;Ge et al, 2006;Ogino et al, 2003;Porotto et al, 2007), and as vaccine vectors (Klas et al, 2002;Lee et al, 2006;Miller et al, 2004;Publicover et al, 2005).…”