Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release

James Kirui,Eric O Freed

doi:10.1186/s12977-020-00521-5

James Kirui, Eric O Freed

Open Access

https://doi.org/10.1186/s12977-020-00521-5

Copy DOI

Abstract

BackgroundThe continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production.ResultsThe reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester.ConclusionThese results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small molecules that promote or inhibit HIV-1 particle production.

Highlights

The continued persistence of Human immunodeficiency virus type 1 (HIV-1) as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus
HIV-1 particle release is commonly measured by reverse transcriptase (RT) activity, p24 ELISA or p24 western blotting assays
The HIV-1 GagiNanoLuc vector allows for a single-step assay to measure virus particle production and is significantly more sensitive and cheaper compared to other assays

Summary

Introduction

The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Reporter viruses are an example of innovative tools that have been used to study HIV-1 replication. Several other HIV-1 reporter viruses have been developed using this approach [2, 3] With this strategy, the reporter virus infects target cells and expresses the reporter protein, allowing for detection and quantification of virus infection. Because the reporter protein is not packaged into progeny virions, the reporter cannot be used to detect virus released from the cell Another strategy that has been used to generate HIV-1 reporter virus involves inserting the reporter gene into the gene encoding the structural protein Gag, often between the matrix (MA) and capsid (CA) domains but in other regions of Gag as well [4,5,6,7].

Methods

Results

Discussion

Conclusion