Abstract
In studies of HIV-1, virus production is normally monitored by either a reverse transcriptase assay or a p24 antigen capture ELISA. However, these assays are costly and time-consuming for routine handling of a large number of HIV-1 samples. For example, sample dilution is always required in the ELISA procedure to determine p24 protein levels because of the very narrow range of detectable concentrations in this assay. Here, we establish a novel HIV-1 production assay system to solve the aforementioned problems by using a recently developed small peptide tag called HiBiT. This peptide is a fragment of NanoLuc luciferase and generates a strong luminescent signal when complemented with the remaining subunit. To employ this technology, we constructed a novel full-length proviral HIV-1 DNA clone and a lentiviral packaging vector in which the HiBiT tag was added to the C terminus of the integrase. Tagging the integrase with the HiBiT sequence did not impede the resultant virus production, infectivity, or susceptibility to an integrase inhibitor. EM revealed normal morphology of the virus particles. Most importantly, by comparing between ELISA and the HiBiT luciferase assay, we successfully obtained an excellent linear correlation between p24 concentrations and HiBiT-based luciferase activity. Overall, we conclude that HiBiT-tagged viruses can replace the parental HIV-1 and lentiviral vectors, which enables us to perform a super-rapid, inexpensive, convenient, simple, and highly accurate quantitative assay for HIV-1/lentivirus production. This system can be widely applied to a variety of virological studies, along with screening for candidates of future antiviral drugs.
Highlights
The HIV-1 proviral DNA clone pNL4-3 [1] has been highly frequently used for in vitroHIV-1 studies worldwide for the last 34 years
Quantitation of viral supernatants from cells transfected with proviral DNA clones, including pNL4-3, is commonly and routinely performed by conducting a standard reverse transcriptase (RT) assay [2] or p24 antigen capture ELISA [3], both of which are time-consuming, taking up to 4-5 hours or more
We transfected 293T cells with the resultant construct pNL-Luc2-IN/HiBiT-E(-) or the parental construct pNL-Luc2-E(-), subjected the cell lysates to Western blotting analyses, and found that the HiBiT tag sequence did not affect the expression of the HIV-1 Gag and Vif proteins (Fig. 1C)
Summary
The HIV-1 proviral DNA clone pNL4-3 [1] has been highly frequently used for in vitroHIV-1 studies worldwide for the last 34 years. We constructed a novel pNL4-3-based full-length proviral HIV-1 DNA clone and a lentiviral vector with a small luminescent peptide tag called HiBiT When this 11-amino-acid peptide tag binds to the larger counterpart protein called LgBiT, the full-length structure of the smallest luciferase protein called NanoLuc is formed [4] (Fig. 1A, upper panel). After expression of a HiBiT-tagged protein, the assay is performed by adding a lytic detection reagent containing LgBiT with the substrate, and the luminescence level of the HiBiT-tagged protein is quickly determined by a luminometer (Fig. 1A, lower panel) Using this system, we established a much faster (~15 minutes), accurate and cheap method, comparable to the recently established SG-PERT assay [5, 6] (as described in Discussion), demonstrating that this molecular tool would largely provide a cost/time-effective routine quantification of HIV-1 and lentiviral vectors
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