Limits...
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

Northern blot detects SPIK. 10μg RNA from Huh7 (1), HepG2 (2) and human liver cells purchased from CloneTech (3) were resolved in the 1% non-denatured agarose gel, then, transferred to nylon membrane. The membrane was hybridized with 32p labeled SPIK specific probe. The image of ethidium bromide (EB) staining was taken from gel before transference. The image of northern blot was determined by phosphateImager.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1074507&req=5

Figure 3: Northern blot detects SPIK. 10μg RNA from Huh7 (1), HepG2 (2) and human liver cells purchased from CloneTech (3) were resolved in the 1% non-denatured agarose gel, then, transferred to nylon membrane. The membrane was hybridized with 32p labeled SPIK specific probe. The image of ethidium bromide (EB) staining was taken from gel before transference. The image of northern blot was determined by phosphateImager.

Mentions: The over-expression of the SPIK gene in HepG2 cells has also been confirmed by Northern blot analysis. Ten micrograms RNA from HepG2 cells and human liver cells were resolved on a 1% non-denatured agarose gel, subsequently transferred to a nylon membrane, and then hybridized with a 32P labeled SPIK specific probe derived from the SPIK gene by PCR. In agreement with the results from RT-PCR, figure 3 shows that the SPIK mRNA can only be detected in the HepG2 cells. The signal in the human liver cells was invisible (Figure 3, Northern blot, lane 2 & lane 3). The undetectable signal from human liver cells was not as a result of the unequal loading. The equality of RNAs were applied on to the gel as evidenced by the equivalent ethidium bromide (EB) staining of ribosomal RNA in both samples (Figure 3, EB stain lane 2 and lane 3. See 18S and 26S ribosome RNA). Similar increase of the SPIK expression was observed in yet another hepatoma cell line Huh7, which is also refractory to HBV infection similarly to HepG2 (Figure 3. Northern blot, lane 1). Taken together, these results demonstrate that SPIK gene is over-expressed in cells that are not susceptible to HBV infection, such as HepG2 and Huh7 cells. In contrast, the expression of SPIK maintains at undetectable level in the HBV infectible cells such as the normal human liver cells. Although, this needs to be further investigated, it is also worth noting that if over expression of SIPK is common to hepatoblastoma and other cancer cells, it could be a useful biomarker for early detection of cancer (Lu, in progress).


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

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

Northern blot detects SPIK. 10μg RNA from Huh7 (1), HepG2 (2) and human liver cells purchased from CloneTech (3) were resolved in the 1% non-denatured agarose gel, then, transferred to nylon membrane. The membrane was hybridized with 32p labeled SPIK specific probe. The image of ethidium bromide (EB) staining was taken from gel before transference. The image of northern blot was determined by phosphateImager.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Northern blot detects SPIK. 10μg RNA from Huh7 (1), HepG2 (2) and human liver cells purchased from CloneTech (3) were resolved in the 1% non-denatured agarose gel, then, transferred to nylon membrane. The membrane was hybridized with 32p labeled SPIK specific probe. The image of ethidium bromide (EB) staining was taken from gel before transference. The image of northern blot was determined by phosphateImager.
Mentions: The over-expression of the SPIK gene in HepG2 cells has also been confirmed by Northern blot analysis. Ten micrograms RNA from HepG2 cells and human liver cells were resolved on a 1% non-denatured agarose gel, subsequently transferred to a nylon membrane, and then hybridized with a 32P labeled SPIK specific probe derived from the SPIK gene by PCR. In agreement with the results from RT-PCR, figure 3 shows that the SPIK mRNA can only be detected in the HepG2 cells. The signal in the human liver cells was invisible (Figure 3, Northern blot, lane 2 & lane 3). The undetectable signal from human liver cells was not as a result of the unequal loading. The equality of RNAs were applied on to the gel as evidenced by the equivalent ethidium bromide (EB) staining of ribosomal RNA in both samples (Figure 3, EB stain lane 2 and lane 3. See 18S and 26S ribosome RNA). Similar increase of the SPIK expression was observed in yet another hepatoma cell line Huh7, which is also refractory to HBV infection similarly to HepG2 (Figure 3. Northern blot, lane 1). Taken together, these results demonstrate that SPIK gene is over-expressed in cells that are not susceptible to HBV infection, such as HepG2 and Huh7 cells. In contrast, the expression of SPIK maintains at undetectable level in the HBV infectible cells such as the normal human liver cells. Although, this needs to be further investigated, it is also worth noting that if over expression of SIPK is common to hepatoblastoma and other cancer cells, it could be a useful biomarker for early detection of cancer (Lu, in progress).

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