Abstract
Primary cells are often used to study viral replication and host-virus interactions as their antiviral pathways have not been altered or inactivated; however, their use is restricted by their short lifespan. Conventional methods to extend the life of primary cultures typically utilize viral oncogenes. Many of these oncogenes, however, perturb or inactivate cellular antiviral pathways, including the interferon (IFN) response. It has been previously shown that expression of the telomerase reverse transcriptase (TERT) gene extends the life of certain cell types. The effect that TERT expression has on the innate antiviral response to RNA- and DNA-containing viruses has not been examined. In the current study, we introduced the human TERT (hTERT) gene into a primary human embryonic lung (HEL-299) cell strain, which is known to respond to the type I IFN, IFN-β. We show that the resulting HEL-TERT cell line is capable of replicating beyond 100 population doublings without exhibiting signs of senescence. Treatment with IFN-β resulted in the upregulation of four model IFN stimulated genes (ISGs) in HEL-299 and HEL-TERT cells. Both cell lines supported the replication of herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV) and impaired the replication of both viruses upon IFN-β pretreatment. Introduction of the viral oncoprotein, simian virus 40 (SV40) large T-antigen, which is frequently used to immortalize cells, largely negated this effect. Taken together, our data indicate that expression of hTERT does not alter type 1 IFN signaling and/or the growth of two viruses, making this cell line a useful reagent for studying viral replication and virus-cell interactions.
Highlights
In performing studies that examine cellular immune responses to viral infections, it is often necessary to work with primary cells, as the efficacy of intrinsic and innate immune pathways are frequently diminished in immortalized cells [1,2,3,4]
We wanted to determine whether ectopic expression of human TERT (hTERT) in human embryonic lung (HEL)-299 cells would allow for a longer period of culturing
Transduction of HEL-299 cells with a retroviral vector that either expresses the green fluorescent protein or contains a deletion in hTERT failed to extend the life span of the HEL-299s. These results demonstrate that expression of hTERT significantly extends the life span of HEL-299 cells
Summary
In performing studies that examine cellular immune responses to viral infections, it is often necessary to work with primary cells, as the efficacy of intrinsic and innate immune pathways are frequently diminished in immortalized cells [1,2,3,4]. Telomeres are maintained by a complex known as telomerase, whose essential core consists of the catalytic subunit telomerase reverse transcriptase (TERT) and the telomerase RNA template component (TERC) [7,8]. Along with a number of other factors, TERT is loaded onto the 39 overhang of existing telomeric DNA and utilizes TERC as a template to add repeats of a guanine-rich sequence, 59TTAGGG39, in all vertebrates; concordantly, DNA primase and DNA polymerase are recruited to the new telomeric repeats, subsequently synthesizing the complementary 59 strand [6]. Once telomeres are reduced from their normal 15 kb length to ,4 kb, DNA damage sensors trigger p53- and pRb-dependent mechanisms that result in cellular senescence, inducing a G1 cell cycle arrest [11]
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