Open Access
Poole, Brian Douglas
Graduate Program:
Integrative Biosciences
Doctor of Philosophy
Document Type:
Date of Defense:
June 09, 2004
Committee Members:
  • Stanley J Naides, Committee Chair
  • Robert Harold Bonneau, Committee Member
  • Sarah Bronson, Committee Member
  • Michael J Chorney, Committee Member
  • Mark Kester, Committee Member
  • parvovirus B19
  • Apoptosis
  • Acute fulminant liver failure
  • DNA damage
ABSTRACT Parvovirus B19 persists in multiple tissues and has been implicated in a variety of diseases including acute fulminant liver failure (AFLF). Despite multiple reports demonstrating the presence of B19 DNA in liver tissues from patients with AFLF, the causal relationship of B19 infection to liver failure is not yet proven. In addition, the mechanism by which B19 infection induces liver failure remains unknown. Although hepatocytes express the B19 receptor, globoside, these cells are non-permissive for B19 replication. B19, although unable to replicate productively in liver cells, may establish a limited infection that kills these cells. B19 cytotoxicity is likely mediated through the viral nonstructural protein, NS1. The NS1 protein has the potential to damage cellular DNA through DNA nicking, covalent DNA binding, and helicase activities. We hypothesize that DNA damage induced by these activities leads to apoptosis through PARP and ATR-mediated pathways. To investigate whether B19 is able to establish infection of hepatocytes, both primary liver cells and the hepatocellular carcinoma cell line Hep G2 were inoculated with parvovirus B19 and examined for the presence of RNA transcripts of viral genes. RT-PCR analysis demonstrated that B19 was able to infect both primary and transformed hepatocytes and produce RNA for NS1. No transcripts correlating to the structural capsid proteins VP1 or VP2 were detected. Immunofluorescence-based studies confirmed the presence of NS1 in infected cells. These experiments demonstrate that B19 enters liver cells, and that the NS1 gene is actively transcribed and the mRNA is translated in infected hepatocytes. The ability of B19 to infect hepatocytes allows for the possibility that B19 infection of these cells is cytotoxic. Pathological studies of tissue from patients with AFLF are characterized by non-inflammatory hepatocellular death. Non-inflammatory disappearance of hepatocytes suggests apoptosis as the mechanism for B19-induced cell death in AFLF. To investigate whether B19 induces apoptosis in liver cells, hepatocytes and Hep G2 cells were infected and assayed for apoptosis by PARP cleavage assays and annexin-V staining. Infection with B19 led to PARP cleavage and induced apoptosis, with a mean of 28% of infected Hep G2 cells and 24% of infected primary hepatocytes undergoing apoptosis as determined by annexin-V staining, compared to 7% apoptosis in mock-infected cells. Several lines of evidence suggest that NS1 is the molecule responsible for the B19-induced apoptosis of liver cells. Irradiated virions, which are incapable of having their genes transcribed, do not induce apoptosis, suggesting that transcription is requisite for B19-induced apoptosis. Since NS1 is the only known transcript produced by B19 infection of hepatocytes, it is likely that NS1 is the apoptosis-inducing factor. Apoptosis can proceed through many different mechanisms. These pathways are regulated by the involvement of different caspases, cysteine proteases that mediate apoptosis. Caspase 9 transduces apoptotic signals originating within the cell, such as responses to DNA damage. Caspase 8 transduces apoptotic signals that are initiated by ligation of TNF superfamily receptors. Caspase 3 is a target of caspases 8 and 9, and acts to carry out the final activities of apoptosis. Caspase inhibition studies demonstrated that caspases 3 and 9, but not caspase 8, are required for B19-induced apoptosis. The requirement for caspase 9 in B19-induced apoptosis suggests that apoptosis is initiated by an internal signal, such as DNA damage. The dispensability of caspase 8 suggests that fas and TNF-á are not involved in B19-induced apoptosis. The DNA modifying activities of NS1 could allow NS1 to induce apoptosis by damaging cellular DNA. To directly examine the apoptosis-inducing effects of NS1, the NS1 gene was cloned and fused to the gene for eGFP in an inducible vector. This vector was transfected into Hep G2 cells. Expression of the NS1 fusion protein was sufficient to cause apoptosis in a caspase-3 and–9-dependent manner, similar to the apoptosis induced by infection with whole virions. Immunoprecipitation experiments revealed that NS1 covalently binds to cellular DNA, a process which likely leads to activation of DNA damage response pathways. Bulky adducts induce apoptosis through a pathway requiring a DNA damage repair molecule, ATR. The single-stranded DNA binding protein RPA initiates the ATR-dependent pathway by binding complexes containing ATR, leading to ATR phosphorylation. Immunofluorescence staining demonstrated that the DNA damage-sensing molecule RPA colocalizes with NS1 in transfected cells. Inhibition of ATR with caffeine decreases apoptosis in NS1-transfected cells by 70%, decreasing the percentage of apoptotic transfected cells from 37% to 12%, and implicating the DNA adduct repair pathway in NS1 induced apoptosis. The ATR-mediated pathway for DNA damage-induced apoptosis is not the only potential DNA repair pathway induced by NS1. NS1 nicks viral genomes, causing single-strand breaks. The cellular pathway that repairs single-strand breaks can also lead to apoptosis. This pathway is initiated by the single-strand nick binding protein PARP. Upon activation, PARP poly(ADPribose)ylates neighboring proteins. Although the mechanisms of PARP-mediated DNA repair and apoptosis are not well understood, PARP activation can lead to apoptosis through changes in mitochondrial membrane permeability, leading to activation of caspase 9. To investigate whether NS1 activates PARP, NS1 was immunoprecipitated from transfected cells and found to be poly(ADPribose)ylated. Poly(ADPribose)ylation of NS1 confirmed that PARP is active in NS1-transfected cells, and in contact with NS1 protein. When PARP was inhibited by 5-aminoisoquinolinone, NS1-induced apoptosis decreased by 57%. PARP activation in the presence of NS1 demonstrates the presence of DNA single strand nicks in the immediate vicinity of NS1. These experiments demonstrate the cytotoxic nature of B19 infection of hepatocytes, and elucidate the mechanisms of B19-induced apoptosis of liver cells. The evidence shows that NS1 binds to DNA in a covalent manner and that NS1 expression induces apoptosis through the single-strand nick and adduct DNA damage repair pathways. A strong association between B19 and AFLF allows treatments for AFLF to be targeted to B19. Since B19 can be neutralized with intravenous immune globulin (IVIG), it is possible that administration of IVIG could abrogate liver injury in cases of AFLF. The knowledge that B19 induces apoptosis through NS1-induced DNA damage also suggests potential therapies. PARP inhibitors have been proven useful in combating apoptosis-related injury due to ischemia, and it is possible that they would also prevent cell death induced by B19 infection. These therapies have the potential to limit the liver damage during B19-associated AFLF, perhaps eliminating the need for transplantations. In addition, many other diseases are associated with B19 infection, and these diseases may also benefit from anti-viral or anti-apoptotic therapies.