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Study of the early steps of the Hepatitis B Virus life cycle.

Lu X, Block T - Int J Med Sci (2004)

Bottom Line: Lu et al.Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection.The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.

View Article: PubMed Central - PubMed

Affiliation: Jefferson Center for Biomedical Research and Agricultural Medicine, Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, USA.

ABSTRACT
Hepatitis B virus (HBV) is a human pathogen, causing the serious liver disease. Despite considerable advances in the understanding of the natural history of HBV disease, most of the early steps in the virus life cycle remain unclear. Virus attachment to permissive cells, fusion and penetration through cell membranes and subsequent genome release, are largely a mystery. Current knowledge on the early steps of HBV life cycle has mostly come from molecular cloning, expression of individual genes and studies of the infection of duck hepatitis B virus (DHBV) with duck primary duck hepatocytes. However, considering of the difference of the surface protein of HBV and DHBV both in the composition and sequence, the degree to which information from DHBV applies to human HBV attachment and entry may be limited. A major obstacle to the study HBV infection is the lack of a reliable and sensitive in vitro infection system. We have found that the digestion of HBV and woodchuck hepatitis virus (WHBV) by protease V8 led to the infection of HepG2 cell, a cell line generally is refractory for their infection [Lu et al. J Virol. 1996. 70. 2277-2285 . Lu et al. Virus Research. 2001. 73(1): 27-4].. Further studies showed that a serine protease inhibitor Kazal (SPIK) was over expressed in the HepG2 cells. Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection. The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.

No MeSH data available.


Related in: MedlinePlus

Southern blot detection of HBV DNA in the infected HepG2 cells. 106 HepG2 cells were pre-treated with anti-sense or sense oligo of SPIK or left untreated, then infected by patients' serum. Cells were harvested at 8th day p.i. After removal of nuclei, HBV DNA in cytoplasma was isolated, resolved in 1% agarose gel and detected by southern blot with HBV specific probe.
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Figure 5: Southern blot detection of HBV DNA in the infected HepG2 cells. 106 HepG2 cells were pre-treated with anti-sense or sense oligo of SPIK or left untreated, then infected by patients' serum. Cells were harvested at 8th day p.i. After removal of nuclei, HBV DNA in cytoplasma was isolated, resolved in 1% agarose gel and detected by southern blot with HBV specific probe.

Mentions: HepG2 cells were susceptible to HBV infection after treatment with SPIK specific anti-sense oligo was confirmed by the results from Southern blot. At 8th day post infection, the replicative form of HBV in cytoplasm was detected feebly in infected cells that were pre-treated with anti-sense of SPIK (Figure 5. lane 1). However, no HBV DNA was found in cells treated with SPIK sense oligo or when untreated (Figure 5. lane 2, 3). The examining cccDNA in the nuclear fraction of infected cells was unsuccessful. It is likely that the levels were far below the detection limit.


Study of the early steps of the Hepatitis B Virus life cycle.

Lu X, Block T - Int J Med Sci (2004)

Southern blot detection of HBV DNA in the infected HepG2 cells. 106 HepG2 cells were pre-treated with anti-sense or sense oligo of SPIK or left untreated, then infected by patients' serum. Cells were harvested at 8th day p.i. After removal of nuclei, HBV DNA in cytoplasma was isolated, resolved in 1% agarose gel and detected by southern blot with HBV specific probe.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074507&req=5

Figure 5: Southern blot detection of HBV DNA in the infected HepG2 cells. 106 HepG2 cells were pre-treated with anti-sense or sense oligo of SPIK or left untreated, then infected by patients' serum. Cells were harvested at 8th day p.i. After removal of nuclei, HBV DNA in cytoplasma was isolated, resolved in 1% agarose gel and detected by southern blot with HBV specific probe.
Mentions: HepG2 cells were susceptible to HBV infection after treatment with SPIK specific anti-sense oligo was confirmed by the results from Southern blot. At 8th day post infection, the replicative form of HBV in cytoplasm was detected feebly in infected cells that were pre-treated with anti-sense of SPIK (Figure 5. lane 1). However, no HBV DNA was found in cells treated with SPIK sense oligo or when untreated (Figure 5. lane 2, 3). The examining cccDNA in the nuclear fraction of infected cells was unsuccessful. It is likely that the levels were far below the detection limit.

Bottom Line: Lu et al.Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection.The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.

View Article: PubMed Central - PubMed

Affiliation: Jefferson Center for Biomedical Research and Agricultural Medicine, Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, USA.

ABSTRACT
Hepatitis B virus (HBV) is a human pathogen, causing the serious liver disease. Despite considerable advances in the understanding of the natural history of HBV disease, most of the early steps in the virus life cycle remain unclear. Virus attachment to permissive cells, fusion and penetration through cell membranes and subsequent genome release, are largely a mystery. Current knowledge on the early steps of HBV life cycle has mostly come from molecular cloning, expression of individual genes and studies of the infection of duck hepatitis B virus (DHBV) with duck primary duck hepatocytes. However, considering of the difference of the surface protein of HBV and DHBV both in the composition and sequence, the degree to which information from DHBV applies to human HBV attachment and entry may be limited. A major obstacle to the study HBV infection is the lack of a reliable and sensitive in vitro infection system. We have found that the digestion of HBV and woodchuck hepatitis virus (WHBV) by protease V8 led to the infection of HepG2 cell, a cell line generally is refractory for their infection [Lu et al. J Virol. 1996. 70. 2277-2285 . Lu et al. Virus Research. 2001. 73(1): 27-4].. Further studies showed that a serine protease inhibitor Kazal (SPIK) was over expressed in the HepG2 cells. Therefore, it is possible that to silence the over expressed SPIK and thus to reinstate the activity of indispensable cellular proteases can result in the restoration of the susceptibility of HepG2 cells for HBV infection. The establishing a stable cell line for study of the early steps of HBV life cycle by silencing of SPIK is discussed.

No MeSH data available.


Related in: MedlinePlus