FRET-Based Detection and Quantification of HIV-1 Virion Maturation

Abstract

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

Evaluating HIV-1 Infectivity and Virion Maturation Across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay

“…The proportion of the total Gaussian distribution or kernel density estimation area overlapping with the HIV-1 Gag-iFRETDPro area was used to determine the proportion of immature virions. Image data analysis and subsequent calculations of mature and immature virion proportions were performed using an updated in-house MATLAB program [13].…”

“…Our previous research developed a unique tool for detecting virus maturation utilizing a fluorescence resonance energy transfer (FRET)-based technique [13]. This approach enables the semi-automatic evaluation of virion maturation through fluorescent microscopy, effectively minimizing human bias.…”

Insights Into Persistent HIV-1 Infection and Functional Cure: Novel Capabilities and Strategies

“…VLPs have previously been employed for immunization purposes and demonstrated that immature morphology enhanced immunogenicity of VLPs and strongly induced IFN-γ secretion and antibody production ( Álvarez-Fernández et al, 2012 ; Gonelli et al, 2019 ). Approximately 20% of the virions harboring intact protease still remain immature morphology after budding ( Sarca et al, 2021 ), which implies that VLPs generated from defective proviruses might contribute to chronic inflammation in PLWH. In addition, proviruses with defective major splice donors or hypermutations induced by APOBEC3 cytidine deaminase enzymes ( Izumi et al, 2008 ; Fukuda et al, 2019 ) can produce antigens against CTL and constitutively induce their activation ( Shankar et al, 2000 ; Monajemi et al, 2014 ; Figure 4 ).…”

Understanding viruses and viral infections by biophotonic methods

“…For deeper insight into cell-virion interaction more advanced fluorescence imaging methods, such as FRET, fluorescence recovery after photobleaching (FRAP) and fluorescence lifetime imaging (FLIM) are being used to visualize the virus-cell protein interactions, detection and quantification of the virus within the cell, surface receptor- binding, and so on [142,143]. For example, Gordon et al investigated SARS and MERS viral-human protein interaction using 3-Å cryoelectron microscopy, immunofluorescence, functional genetic screening and proteomics combined with cell biology [144].…”