Hepatitis B virus infection and replication in a new cell culture system established by fusing HepG2 cells with primary human hepatocytes.

BACKGROUND: Hepatitis B virus (HBV) infection is strictly species and tissue specific, therefore none of the cell models established previously can reproduce the natural infection process of HBV in vitro. The aim of this study was to establish a new cell line that is susceptible to HBV and can support the replication of HBV. METHODS: A hybrid cell line was established by fusing primary human hepatocytes with HepG2 cells. The hybrid cells were incubated with HBV-positive serum for 12 hours. HBV DNA was detected by quantitative fluorescence polymerase chain reaction (QF-PCR). HBsAg (surface antigen) and HBeAg (extracellular form of core antigen) were observed by electrochemiluminescence (ECL). HBcAg (core antigen) was detected by the indirect immunofluorescence technique. HBV covalently closed circular DNA (cccDNA) was analyzed by Southern blot hybridization and quantified using real-time PCR. RESULTS: A new cell line was established and named HepCHLine-7. The extracellular HBV DNA was observed from Day 2 and the levels ranged from 9.80 (± 0.32) × 10(2) copies/mL to 3.12 (± 0.03) × 10(4) copies/mL. Intracellular HBV DNA was detected at Day 2 after infection and the levels ranged from 7.92 (± 1.08) × 10(3) copies/mL to 5.63 (± 0.11) × 10(5) copies/mL. HBsAg in the culture medium was detected from Day 4 to Day 20. HBeAg secretion was positive from Day 5 to Day 20. HBcAg constantly showed positive signals in approximately 20% (± 0.82%) of hybrid cells. Intracellular HBV cccDNA could be detected as early as 2 days postinfection and the highest level was 15.76 (± 0.26) copies/cell. CONCLUSION: HepCHLine-7 cells were susceptible to HBV and supported the replication of HBV. They are therefore suitable for studying the complete life cycle of HBV.

Generation and characterization of JCV permissive hybrid cell lines.

JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host-cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents.

Isolation and characterization of mouse-human microcell hybrid cell clones permissive for infectious HIV particle release.

Mouse cells are non-permissive to human immunodeficiency virus type 1 (HIV) in that there is a pronounced post-integration block to viral replication. We have recently demonstrated that mouse-human somatic cell hybrids that contain human chromosome 2 increase both HIV Capsid (CA) production and infectious virus release. Here we report on the isolation of three mouse-human microcell hybrids (MCHs) that behave similarly, starting from a pool of 500 MCH clones. Release of virus was specific to HIV and cell revertants that no longer contained any human chromosome fragments did not release CA or infectious virus. Two of the three cell clones were identical as judged by PCR STS content and fluorescence in situ hybridization (FISH) and contained a single 2-12 human chromosome chimera. The third cell clone only contained human chromosome 12, as determined by PCR, FISH, and microarray analyses. There were no consistent differences in Gag protein and spliced/unspliced viral RNA levels between mouse cell lines. CMV promoter-driven, codon-optimized gag-pol had no effect on infectious HIV release from these mouse cells, despite allowing Gag targeting and increasing CA production. These permissive mouse-human MCHs and their corresponding non-permissive revertants may prove useful for mechanistic studies and also for identifying the responsible gene(s) or factor(s) involved in the production of HIV.

Herpes simplex virus regulatory proteins VP16 and ICP0 counteract an innate intranuclear barrier to viral gene expression.

HSV regulatory proteins VP16 and ICP0 play key roles in launching the lytic program of viral gene expression in most cell types. However, these activation functions are dispensable in U2OS osteosarcoma cells, suggesting that this cell line either expresses an endogenous activator of HSV gene expression or lacks inhibitory mechanisms that are inactivated by VP16 and ICP0 in other cells. To distinguish between these possibilities, we examined the phenotypes of somatic cell hybrids formed between U2OS cells and highly restrictive HEL fibroblasts. The U2OS-HEL heterokarya were as non-permissive as HEL cells, a phenotype that could be overcome by providing either VP16 or ICP0 in trans. Our data indicate that human fibroblasts contain one or more inhibitory factors that act within the nucleus to limit HSV gene expression and argue that VP16 and ICP0 stimulate viral gene expression at least in part by counteracting this innate antiviral defence mechanism.

Naltrexone inhibits alcohol-mediated enhancement of HIV infection of T lymphocytes.

Acute and chronic alcohol abuse impairs various functions of the immune system and thus, has been implicated as a cofactor in the immunopathogenesis of human immunodeficiency virus (HIV) disease progression. We determined whether naltrexone, an opioid receptor antagonist widely used in the treatment of alcoholism, inhibits alcohol-mediated enhancement of HIV infection of T cells. Alcohol enhanced HIV infection of peripheral blood lymphocytes (PBL) and a human lymphoid cell line (CEMX174). Alcohol increased HIV X4 envelope (Env), not murine leukemia virus Env-pseudotyped infection of CEMX174 cells. Naltrexone antagonized the enhancing effect of alcohol on HIV infection of PBL and CEMX174 cells. The specific mu-opioid receptor antagonist, Cys2, Tyr3, Arg5, Pen7 (CTAP) amide, also blocked the enhancing effect of alcohol on HIV infection. Investigation of the underlying mechanism for the alcohol action showed that alcohol significantly increased endogenous beta-endorphin production and induced mu-opioid receptor mRNA expression in PBL and CEMX174 cells. The role of beta-endorphin in alcohol-mediated enhancement of HIV infection was indicated by the observations that naltrexone and CTAP antagonized ether alcohol- or exogenous beta-endorphin-mediated enhancement of HIV infection. These findings suggest a biological mechanism for the potential therapeutic benefit of naltrexone in treating HIV-infected alcoholics.

Involvement of caspase-3 pathway in anti-apoptotic action of methionine enkephalin on CEM x 174 cells in prolonged infection with simian immunodeficiency virus in vitro.

The roles of methionine enkephalin, as an immunomodulator, on immunodeficiency virus-induced apoptosis of lymphocytes during prolonged infection are still unclear. In the present study, we evaluated the effects of methionine enkephalin on the viability, the profile of cell cycle and apoptosis, as well as the expression of apoptosis-related genes in CEM x 174 cells infected with simian immunodeficiency virus for 72 h. Our data demonstrated that methionine enkephalin maintains the viability of cells during the period of prolonged infection. Following co-incubation with the virus, CEM x 174 cells were arrested at S phase, with increased mortality as a result of apoptosis. Methionine enkephalin could abolish virus-induced over-expression of caspase-3. Taken together all findings, we conclude that methionine enkephalin may maintain the viability of SIV-infected cells via suppressing the expression of caspase-3, which may have clinical implications in opioid peptide therapy for AIDS.

A notable phenomenon recapitulated. A fusion product of a murine lymphoma cell and a leukemia virus-neutralizing antibody-producer host plasma cell formed spontaneously and secreting the specific antibody continuously.

In the mid-1960s the #620 cell passage line of a murine lymphoma-leukemia was developed at the Section of Clinical Tumor Virology and Immunology, Department of Medicine, The University of Texas M.D. Anderson Hospital in Houston, TX. The diploid lymphoma cells released a retrovirus and were antigenic in young adult Swiss (YAS) mice. Small lymphoma cell inocula were rejected with immunity acquired against large inocula of lymphoma cells. Tissue sections revealed the "starry sky" configurations. In one of the tissue cultures set up from lymphoma #620, a cell line consisting of large round poly- or tetraploid cells arose and was referred to as lymphoma cell line #818. The #818 cells grew in suspension cultures and in the form of large, lethal ascitic tumors in YAS mice. Diploid #620 lymphoma cells stained for retroviral antigens; #818 cells stained both for retroviral antigens and immunoglobulins. Fluids withdrawn from #818 cultures neutralized the leukemia virus in spleen focus assays. Immunoglobulin precipitated from #818 suspension culture fluids strongly and specifically neutralized the leukemia virus. The growth of #620 or #818 cells in YAS mice treated with rabbit anti-lymphoma cell immune sera was strongly inhibited but culture fluids of #818 cells showed weak and insignificant inhibition against leukemia-lymphoma #620 (in one experiment, unpublished). In two experiments #620 lymphoma cells were co-inoculated with immune spleen cells into the peritoneal cavities of YAS mice. The immune spleen cells derived from mice that rejected #620 cell inocula or were actively immunized with a photodynamically inactivated mouse leukemia virus vaccine. In the peritoneal cavities of mice co-inoculated with #620 cells and immune spleen cells, clones of large round cells emerged with tetra- or polyploid chromosomal modes. These cells stained for leukemia viral antigens and immunoglobulins. When passaged in YAS mice these cells induced lethal ascites tumors. It was concluded as early as in 1968-69 that an immune plasma cell can fuse with a lymphoma cell, if the lymphoma cell expresses retroviral antigens against which the plasma cell is producing a specific antibody. Some human lymphoma-leukemia cells express retroviral antigens and/or budding retroviral particles, whether due to the acquisition of new env sequences by incomplete resident endogenous retroviral genomes or due to the entry of exogenous retroviruses into lymphopoietic stem cells. In the Discussion illustrations are provided for the human cell line #778 established from a patient with "lymphosarcoma cell leukemia" in 1966. The malignant cells released unidentified retrovirus-like particles and fused with one another and with reactive lymphoid cells of the host. It should be investigated further if human lymphoma-leukemia cells could fuse with an immune plasma cell of the host and thus alter the clinical course of the disease.

Spontaneous fusion of cells between species yields transdifferentiation and retroviral transfer in vivo.

Human cells can fuse with damaged or diseased somatic cells in vivo. Whether human cells fuse in vivo in the absence of disease and with cells of disparate species is unknown. Such a question is of current interest because blood exchanges between species through direct physical contact, via insect vectors or parasitism, are thought to underlie the transmission of zoonotic agents. In a model of human-pig chimerism, we show that some human hematopoietic stem cells engrafted in pigs contain both human and porcine chromosomal DNA. These hybrid cells divide, express human and porcine proteins, and contribute to porcine nonhematopoietic tissues. In addition, the hybrid cells contain porcine endogenous retroviral DNA sequences and are able to transmit this virus to uninfected human cells in vitro. Thus, spontaneous fusion can occur in vivo between the cells of disparate species and in the absence of disease. The ability of these cell hybrids to acquire and transmit retroviral elements together with their ability to integrate into tissues could explain genetic recombination and generation of novel pathogens. * differentiation * fusion * retrovirus

Restriction of measles virus RNA synthesis by a mouse host cell line: trans-complementation by polymerase components or a human cellular factor(s).

The mouse epithelial MODE-K cell line expressing human CD46 or CD150 cellular receptors was found to be nonpermissive for measles virus (MV) replication. The virus binding and membrane fusion steps were unimpaired, but only very limited amounts of virus protein and RNA synthesized were detected after the infection. In a minigenome chloramphenicol acetyltransferase assay, MODE-K cells were as able as the permissive HeLa cells in supporting MV polymerase activity. The restriction phenotype of MODE-K cells could be alleviated by providing, in trans, either N-P-L or N-P functional protein complexes but not by P-L complexes or individual N, P, and L proteins. Several human x mouse (HeLa x MODE-K) somatic hybrid clones expressing human CD46 were isolated and found to be either nonpermissive or permissive according to their human chromosomal contents. The MV-restricted phenotype exhibited by the MODE-K cell line suggests that a cellular factor(s) can control MV transcription, possibly by stabilizing the incoming virus polymerase templates.

Restricted species tropism of maedi-visna virus strain EV-1 is not due to limited receptor distribution.

The distribution of receptors for maedi-visna virus (MVV) was studied using co-cultivation assays for virus fusion and PCR-based assays to detect the formation of virus-specific reverse transcription products after virus entry. Receptors were present on cell lines from human, monkey, mouse, chicken, quail, hamster and ovine sources. Thus, the distribution of the receptor for MVV is more similar to that of the amphotropic type C retroviruses than to that of other lentiviruses. The receptor was sensitive to proteolysis by papain, but was resistant to trypsin. Chinese hamster ovary (CHO) and lung cells (V79 TOR) did not express functional receptors for MVV. The receptor was mapped to either chromosome 2 or 4 of the mouse using somatic cell hybrids. This allowed several candidates (e.g. MHC-II, CXCR4) that have been proposed for the MVV receptor to be excluded.

Efficient infection of brain microvascular endothelial cells by an in vivo-selected neuroinvasive SIVmac variant.

A neuroinvasive/neuropathogenic SIV variant termed SIVmac182 was previously isolated and characterized (Watry et al, 1994). This neuroinvasive strain was derived from the uncloned strain SIVmac251 through serial animal passage of infected microglia, unlike previously reported neurovirulent strains. Importantly, the virus described here was isolated from a strain which already demonstrates limited neuroinvasiveness in vivo, through a route of inoculation which exerts selective pressure for variants in the periphery that can naturally cross the blood-brain barrier and gain access to the brain. Examination of animal tissues indicated that the neuroinvasive strain was capable of replicating in brain microvascular endothelial cells (BMEC). Therefore, we developed an in vitro model of BMEC infection in which to examine mechanisms of virus neuroinvasiveness and neuropathogenicity as well as to address mechanisms of HIV-induced dementia. Results obtained with this in vitro system indicate that growth in BMEC may predict neuroinvasiveness in vivo, and furthermore, that brain passage of virus results in the generation of neuroinvasive strains which demonstrate an increased efficiency of BMEC infection in vitro.

Pathogenesis of lymphocyte-tropic and macrophage-tropic SIVmac infection in the brain.

SIVmac239 replicates productivity in activated CD4+ T lymphocytes, but inefficiently in macrophages from rhesus macrophages. Inoculation of the virus into animals results in an acute, highly productive burst of virus replication in activated T lymphocytes in lymphoid tissues and infected cells invade the central nervous system (CNS). This phase lasts a few weeks and is eventually followed by development of immunosuppression of different degrees of severity, opportunistic infections, and tumors related to the loss of T lymphocytes. On rare occasions, infected immunosuppressed animals develop encephalitis and/or interstitial pneumonia, syndromes that are associated with selection of mutant viruses that replicate efficiently in macrophages of these tissues. Usually, however, brains of animals dying with AIDS caused by SIVmac239 appear histologically normal. Is the brain infected with virus? We report here on a macaque dying with AIDS, a neuroinvasive tumor and interstitial pneumonia associated with macrophage-tropic virus. Except for focal infiltration of tumor cells, the brain was normal histologically. We examined the virus and viral DNA from different tissues and found that lymphocytes but not macrophages from lymph nodes and spleen yielded virus, whereas macrophages but not lymphocytes from the lung produced virus. No virus was recovered from the brain but small amounts of viral p27 were present in the brain homogenate. Viral sequences were present in the brain as determined by PCR from tissue DNA. Comparison showed that the viral sequences in the brain closely resembled those from the spleen. Presumably, the virus caused a minimally productive infection detectable by production of small amounts of p27, but was not accompanied by any histopathological changes. It is unclear why the macrophage-tropic virus in the lung failed to 'take-off' in the brain of this animal. To determine whether this virus had encephalitic potential, we inoculated the lung homogenate containing cell-free, macrophage tropic virus into a young pigtail macaque, a species known to be sensitive to primate lentiviral infections. This animal developed severe encephalitis 10 weeks later. Virus from the brain was very similar to the inoculum virus, proving its encephalitic potential. Possible reasons for the differences in neurovirulence of this virus between the two animals remain speculative.