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.
Highlights
While the acquired immunodeficiency syndrome (AIDS) is a deadly disease caused by infection with human immunodeficiency virus type 1 (HIV-1), AIDS-related deaths have been reduced due to the tremendous efforts that have gone into researching the virus itself and ways to counteract it (Centers for Disease Control and Prevention (CDC), 2006)
To microscopically visualize the viral core generation, we designed a novel bifunctional HIV-1 labeling system that consists of a tandem of cyan- and yellowemitting fluorescent protein pair as fluorescence resonance energy transfer (FRET) donor and acceptor and named it HIV-1 Gag-iFRET (Figure 1A)
We bridged an optimized intramolecular FRET pair, ECFP C11 (CFP) and circularly permutated Venus with a new N-terminus starting at Asp-173 (Nagai et al, 2004) with an HIV-1 protease cleavage site, and inserted them between the MA and CA domains of HIV-1 Gag
Summary
While the acquired immunodeficiency syndrome (AIDS) is a deadly disease caused by infection with human immunodeficiency virus type 1 (HIV-1), AIDS-related deaths have been reduced due to the tremendous efforts that have gone into researching the virus itself and ways to counteract it (Centers for Disease Control and Prevention (CDC), 2006). The major obstacle to achieving a cure for HIV-1 is the existence of latently infected reservoir cells within memory CD4 T cells and macrophages that can persist even during cART (Chun et al, 1997; Finzi et al, 1997; Siliciano et al, 2003; Hassan et al, 2016; Wong et al, 2019). Latent HIV-1 persistent reservoirs are established early in the acute phase of infection (Finzi et al, 1997, 1999; Daar et al, 1998; Zhang et al, 2000; Whitney et al, 2014; Henrich et al, 2017; Colby et al, 2018). Since most defective proviruses preserve the 5 end of intact proviral sequences encoding gag and gag-pol (Ho et al, 2013; Imamichi et al, 2016; Hiener et al, 2017), they may be able to assemble and release virus-like particles into the extracellular space
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