Chronically infected T-cell lines become handy for a novel assay measuring the reservoir of replication-competent HIV-1

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Discovery of novel, more accurate and cheaper assays to quantitatively define the reservoir of replication-competent HIV-1 (further referred to as the reservoir) is a top priority for research aiming at achieving a ‘Functional Cure’ of this infection [1]. In this regard, the ‘gold standard’ approach for estimating the size of the reservoir is the viral outgrowth assay (VOA) based on the quantification of infectious HIV particles (virions) released by patients’ resting CD4+ T lymphocytes under maximal stimulatory conditions. However, some prominent investigators who have significantly standardized the VOA have earlier reported an approximately 60-fold gap between the size of the potentially intact proviral sequences and the capacity of the VOA to estimate the amount of infectious virus [2]. In this regard, a recent article entitled ‘Novel assay reveals a large, inducible, replication competent HIV-1 reservoir in resting CD4+ T cells’ published by Sanyal et al.[3] sheds new light on this relevant subject.
A crucial feature of this novel assay is the clear-cut discrimination of infectious vs. noninfectious virus that is not considered relevant to achieve this goal [1]. Of interest, the specificity and sensitivity of this assay is based on chronically infected cell lines generated from the acute infection of the CEM T-lymphocytic cell line in the late 1980s at the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA as sources of infectious and noninfectious viruses. In particular, the ACH-2 cell line [4] was used as a source of infectious virus, whereas the 8E5 cell line [5] was correctly considered as harboring a reverse transcriptase-defective HIV-1 provirus. Counterintuitively, cocultivation of the highest tested number of 8E5 cells (64 000 cells/106 peripheral blood mononuclear cells) with HeLa-derived TZM-bl reporter cells generated a weakly positive signal unlike other cells infected with defective proviruses that were totally negative in the assay readout. We fully support the validation of the TZM-bl-based (TZA) assay, as a new assay that could be even more selective in discriminating infectious vs. noninfectious HIV-1 than originally described by the authors who do not comment these findings; furthermore, we would like to offer a likely explanation for this seemingly discrepant result.
We here propose that the 8E5 cell line could be considered a source of atypical, yet infectious virions further validating the capacity of the novel TZA assay to distinguish between infectious and defective viral particles. If indeed 8E5 cells contain a single copy of a reverse transcriptase-defective provirus, Quillent et al.[6] demonstrated in 1993 that consequently to a spontaneous correction of the proviral genome by a single nucleotide insertion, the 8E5 cell line produces a weakly infectious virus that could be transmitted to other CD4+ cells, a finding later confirmed by independent investigators [7,8].
In our opinion, the use of ACH-2 cells as positive control for infectious virus suggests some additional considerations. In fact, like 8E5, also ACH-2 cells contain a single copy of a provirus that cannot be activated transcriptionally by either endogenous or exogenous Tat (the major endogenous transcriptional activator of the provirus) because of a defective Tat-binding RNA sequence known as trans-activation region (TAR) region, as described by Emiliani et al.[9]. Nonetheless, ACH-2 release virions that are fully capable of infecting new CD4+ target cells thereby activating the heterologous provirus harbored in TZM-bl cells through a newly synthesized Tat protein binding to an intact TAR region as described in the study by Sanyal et al.[3].

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