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FRET-based detection and quantification of HIV-1 Virion Maturation

Sarca, Anamaria Daniela 京都大学 DOI:10.14989/doctor.k23106

2021.03.23

概要

Several studies have shown that defective proviral HIV-1, which forms the majority of integrated HIV-1 DNA, can lead to the generation of viral proteins and virus-like particles (VLPs). These proteins serve as immunogens for chronic immune system stimulation, contributing to the higher burden of non-infectious chronic diseases seen even in virally suppressed people living with HIV. Furthermore, like HIV virion maturation, VLPs may be capable of Gag processing by the viral protease, increasing their immunogenicity. There is a need, therefore, for tools that can precisely assess maturation rates of both intact and defective HIV-1 particles.

The present thesis describes the development of a fluorescence microscopy tool that can assess virion maturation using Fluorescence Resonance Energy Transfer (FRET). HIV-1 Gag-iFRET (iFRET) is a NL4-3 derivate that contains a FRET fluorescent protein pair (CFP and YFP) inserted between the MA and CA domains of Gag, linked by protease cleavage sequences. In immature virions labeled with iFRET, CFP and YFP are linked and emit an intense FRET signal. In mature virions, they are cleaved apart and the FRET signal decreases. HIV-1 Gag-iFRET∆Pro (iFRET∆Pro), the protease deficient variant of iFRET, produces solely immature virions and was used as a FRET signal positive control.

Virions were produced using the iFRET or iFRET∆Pro constructs together with their NL4-3 or NL4-3∆Pro parent plasmids, respectively, at a 1:10 ratio. Western Blot analysis of both producer cells and virion lysates showed efficient FRET labeling and Gag-iFRET processing, but not Gag-iFRET∆Pro processing, as intended. Transmission Electron Microscopy (TEM) of iFRET labeled virions confirmed that they had similar mature and immature morphologies as parental NL4-3, at comparable ratios. iFRET∆Pro labeled virions displayed only immature morphologies.

Single virion images were then taken using both FRET (CFP) excitation and control YFP excitation, fluorescence intensity data was extracted from the images and semi-automatically processed to calculate the FRET ratio of every particle. Counts ranged between 17,000 to 77,000 virions. Using the iFRET∆Pro labeled virions as reference for FRET signal intensity range of immature virions, the proportion of immature virions in the iFRET samples was quantified to be 22.4% (±2.4%) [mean(±SD)]. TEM images in this study (18%) as well as other studies (10-20%) have shown comparable rates of immature virions.

To determine if the technique was sensitive to changes in the immature virion population, virus was produced under treatment with Darunavir, a protease inhibitor. The technique was capable of detecting dose-dependent shifts in immature virion population and allowed the calculation of a 50% effective concentration (EC50) against virion maturation of 7 nM Darunavir. The 50% inhibitory concentration of infectivity was 2.8 nM, showcasing that infectivity is not a robust surrogate for maturation inhibition.

The technique described in this thesis can perform faster and large-scale determinations of Gag maturation rates and will be useful for the study of VLPs and their role in immune system stimulation. Furthermore, HIV-1 Gag-iFRET labeling and imaging can be used to measure the direct effect on maturation of protease inhibitors and determine their EC50. These applications will deepen the knowledge in the HIV field regarding the long term, non-infectious consequences of living with HIV and regarding the complex pharmacodynamics of protease inhibitors.

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