De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro

Abstract

While exploring previous findings that ex vivo treatment of lymphoid cells from Hepatitis C virus (HCV)-infected individuals with T cell-stimulating mitogens augments detection of the residing virus, an in vitro HCV replication system was established, in which mitogen-induced T cell-enriched cultures served as HCV targets and the derived T cells multiplied virus during repeated serial passage. HCV replication was ascertained by detecting HCV RNA positive and negative strands, HCV NS5a and E2 proteins, release of HCV virions and nucleocapsids (confirmed by immunoelectron microscopy) and de novo infection of mitogen-induced T cells prepared from healthy donors. Further, affinity-purified normal human T lymphocytes were also susceptible to HCV infection in vitro and HCV replication was detected in pure T cells isolated from a patient with chronic hepatitis C. These results document that T cells can support propagation of HCV both in vivo and in vitro. The infection system established offers a valuable tool for in vitro studies on the entire cycle of HCV replication, virus cytopathogenicity and evaluation of antiviral agents against wild-type HCV in the natural host-cell milieu.

Hepatitis C Virus Infection of Human T Lymphocytes Is Mediated by CD5

“…To identify factors facilitating T cell susceptibility to infection with wild-type HCV, we explored a previously established T lymphocyte-based HCV infection system in which plasma from patients with CHC serves as infectious inocula (22,23). In a search for the most suitable and readily accessible human T cell targets other than mitogen-induced primary T lymphocytes (22,23), the susceptibilities of Molt4, Jurkat, CEM, and PM1 human lymphoblastic T cell lines to infection with genotype 1 HCV were investigated.…”

“…Then, the cells were extensively washed, suspended in fresh culture medium, and cultured for 4 to 5 days postinfection (p.i.). In the case of PBMC and primary T cells exposed to HCV, the culture medium was alternatively supplemented with PHA (5 g/ml; Sigma-Aldrich) or PHA and rIL-2 (20 IU/ml; Roche), and the cells were maintained in culture for 14 days, as reported by in our previous studies (22,23).…”

“…Primary T lymphocytes were affinity purified from monocyte-depleted PBMC by negative selection using MACS magnetic microbeads (Miltenyi Biotec, Auburn, CA), as reported previously (22,32). The T cells were 97 to 98% pure by flow cytometry.…”

“…were dissolved in ice-cold RIPA buffer (1% NP- 40 CD5 5=-TCAAGCGTCAAAAGTCTGCC-3= 5=-AGCCACACTGGAGGTTGTTG-3= CD81 5=-ACAAGGACCAGATCGCCAAG-3= 5=-AGTCAAGCGTCTCGTGGAAG-3= IFN-␣ 5=-CAGCCTGAGTAACAGGAGGA-3= 5=-GCAGATGAGTCCTTTGTGGT-3= IFN-␥ 5=-TCAGCTCTGGATCGTTTTGG-3= 5=-TGTTTTAGCTGCTGGGGACA-3= IRF3 5=-TACGTGAGGCATGTGCTGA-3= 5=-AGTGGGTGGCTGTTGGAAAT-3= IRF7 5=-GCCCTTGCCTCCCCTGTTAT-3= 5=-CCACTGCAGCCCCTCATAG-3= 50 mmol/liter Tris, pH 8.0; Sigma-Aldrich), and the proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) at 20 g protein/lane. After blotting onto a nitrocellulose membrane by wet transfer using the Bio-Rad SD cell system (Bio-Rad Laboratories, Mississauga, Ontario, Canada) (22,27), the blots were exposed to 5% skim milk in Tris-buffered saline (pH 7.4) for 1 h at room temperature and then exposed overnight at 4°C to anti-E2 mouse MAb diluted 1:500 (provided by Arvind Patel, University of Glasgow, Glasgow, United …”

“…Analysis of HCV compartmentalization in infected patients demonstrated virus replication in both T and B lymphocyte subsets (7,16,28,31,33). The susceptibility of normal human T lymphocytes to infection in vitro with patient-derived HCV and their ability to support the entire cycle of HCV replication have been shown (22,23). The propensity of HCV to infect the immune system is consistent with a significantly greater prevalence of lymphoproliferative disorders, such as non-Hodgkin's lymphoma and mixed cryoglobulinemia, and perhaps mucosa-associated lymphoid tissue lymphoma, in patients infected with HCV (4,10,13,40,42).…”