Adaptation of the Fluorogenic 5′-Nuclease Chemistry to a PCR-Based Reverse Transcriptase Assay

In the quest for an effective vaccine against human immunodeficiency virus (HIV), live attenuated virus vaccines have proven to be very effective in the experimental model system of simian immunodeficiency virus (SIV) in macaques. However, live attenuated HIV vaccines are considered unsafe for use in humans because the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. As an alternative approach, we earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (DOX). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient DOX administration. Since the effectiveness and safety of such a conditionally live AIDS vaccine should be tested in macaques, we constructed a similar DOX-dependent SIVmac239 variant in which the Tat-TAR (trans-acting responsive) transcription control mechanism was functionally replaced by the DOX-inducible Tet-On regulatory mechanism. Moreover, this virus can be used as a tool in SIV biology studies and vaccine research because both the level and duration of replication can be controlled by DOX administration. Unexpectedly, the new SIV variant required a wild-type Tat protein for replication, although gene expression was fully controlled by the incorporated Tet-On system. This result suggests that Tat has a second function in SIV replication in addition to its role in the activation of transcription.

Section: Methodsmentioning

confidence: 99%

Construction of a Doxycycline-Dependent Simian Immunodeficiency Virus Reveals a Nontranscriptional Function of Tat in Viral Replication

Das

Klaver

Harwig

et al. 2007

“…In addition, when the contaminants have the potential to infect human cells, such as MLV-X and GALV, they could introduce a further biohazard, either by representing a direct risk of infection to the operator or by altering the virulence and tropism of infectious agents following phenotypic mixing and/or recombination. Because of their high sensitivity and their ability to detect divergent retroviruses, PCR-based RT assays are the ideal tools for monitoring retrovirus contaminations (2,6,20,22,33,39). Given the simplicity and low cost of these assays, screenings should be performed routinely to identify accidental retrovirus laboratory contaminations or to detect replication-competent recombinants in retrovirus vector-transduced cells.…”

mentioning

confidence: 99%

Identification of Gammaretroviruses Constitutively Released from Cell Lines Used for Human Immunodeficiency Virus Research

Takeuchi

McClure

Pizzato

2008

240

185

Three human cell lines used in human immunodeficiency virus research were found to be contaminated with previously undetected retroviruses. On the bases of partial nucleotide sequence, capsid protein antigenicity, vector mobilization, and receptor usage studies, these contaminants were shown to be replication competent and to belong to the Gammaretrovirus genus. While the TZM-bl cells harbor ecotropic murine leukemia virus (MLV), Jurkat J6 cells were found to release xenotropic MLV and the A3.01/F7 cells to produce gibbon ape leukemia virus. These findings highlight the importance of routine testing of cell lines for retrovirus contamination to prevent potential experimental artifacts and allow correct biohazard assessment.

“…Virus-containing culture supernatants were collected and ultracentrifuged over 20% sucrose as previously described (20), and purified virus was incubated with the S. aureus cells. Both total and bound PERV were quantified by a product-enhanced reverse transcriptase (RT) assay that utilizes an internal TaqMan probe (TM-PERT) (8). As shown in Fig.…”

mentioning

confidence: 99%

Human CD59 Incorporation into Porcine Endogenous Retrovirus Particles: Implications for the Use of Transgenic Pigs for Xenotransplantation

Takefman

Spear

Saifuddin

et al. 2002

Transgenic pigs have been engineered to express human CD59 (hCD59) in order to suppress hyperacute rejection of xenotransplants in human recipients. In this study, porcine endogenous retrovirus (PERV) was produced in a porcine cell line expressing hCD59 in order to examine the effect of this complement control protein on PERV neutralization by human sera. hCD59 was found to be incorporated into PERV particles produced from engineered ST-IOWA cells. PERV incorporation of hCD59 resulted in a dramatic inhibition of complement-mediated virolysis by human serum. However, incorporation of hCD59 had no effect on neutralization of PERV by human serum, as measured in infectivity assays. Our results suggest that the use of organs from hCD59 transgenic pigs will inhibit complement-mediated virolysis, but will not compromise the protective effects of human sera on the neutralization of PERV particles.