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Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging.

ABSTRACT: BACKGROUND:HIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell-cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined. RESULTS:To study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-?V1V2. The Env-isfGFP-?V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell-cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS. CONCLUSIONS:Env and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS.

SUBMITTER: Wang L

PROVIDER: S-EPMC6334456 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging.

Wang Lili L Izadmehr Sudeh S Kamau Edwin E Kong Xiang-Peng XP Chen Benjamin K BK

Retrovirology 20190115 1

<h4>Background</h4>HIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell-cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined.<h4>Results</h4>To study the trafficking of HIV-1 Env through the VS, we constructed a molecular ...[more]

PMID: 30646921

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Project description:UnlabelledRetroviruses spread more efficiently when infected and uninfected cells form tight, physical interfaces known as virological synapses (VSs). VS formation is initiated by adhesive interactions between viral Envelope (Env) glycoproteins on the infected cell and CD4 receptor molecules on the uninfected cell. How high-avidity Env-CD4 linkages are resolved over time is unknown. We describe here a tractable two-color, long-term (>24 h) live cell imaging strategy to study VS turnover in the context of a large cell population, quantitatively. We show that Env's conserved cytoplasmic tail (CT) can potently signal the recruitment of Gag capsid proteins to the VS, a process also dependent on residues within Gag's N-terminal matrix (MA) domain. Additionally, we demonstrate that Env's CT and Gag's MA domain both regulate the duration of interactions between viral donor and target cells, as well as the stability of this interaction over time (i.e., its capacity to resolve or form a syncytium). Finally, we report the unexpected finding that modulating extracellular fluid viscosity markedly impacts target T cell trafficking and thus affects the duration, stability, and turnover of virus-induced cell-cell contacts. Combined, these results suggest a stepwise model for viral cell-to-cell transmission wherein (i) Env-receptor interactions anchor target cells to infected cells, (ii) Env signals Gag's recruitment to the cell-cell contact dependent on an intact Env CT and Gag MA, and (iii) Env CT and Gag MA, in conjunction with extracellular forces, combine to regulate VS stability and infectious outcomes.ImportanceHIV-1 spreads efficiently at physical, cell-cell interfaces known as virological synapses (VSs). The VS provides for spatiotemporal coupling of virus assembly and entry into new host cells and may transmit signals relevant to pathogenesis. Disrupting this mode of transmission may be critical to the goal of abolishing viral persistence in infected individuals. We describe here a long-term live cell imaging strategy for studying virus-induced effects on cell behavior in the context of a large cell population. We demonstrate cooperative roles for viral Gag capsid proteins and Envelope glycoproteins in regulating VS formation and turnover. We also show that modulating fluid viscosity markedly affects T cell trafficking and VS stability. Thus, extracellular factors also play an important role in modulating the nature of infectious cell-cell interactions. In sum, our study provides new tools and insights relevant to exposing vulnerabilities in how HIV-1 and other viruses spread infection among cells, tissues, and people.

Project description:HIV-1 nucleotide substitution rates are central for understanding the evolution of HIV-1. Their accurate estimation is critical for analysis of viral dynamics, identification of divergence time of HIV variants, inference of HIV transmission clusters, and modeling of viral evolution.Intra-patient nucleotide substitution rates in HIV-1C gag and env gp120 V1C5 were analyzed in a longitudinal cohort of 32 individuals infected with a single viral variant. Viral quasispecies were derived by single genome amplification/sequencing from serially sampled blood specimens collected at median (IQR) of 5 (4-6) times per subject from enrollment (during Fiebig stages II to V) over a median (IQR) of 417 (351-471) days post-seroconversion (p/s). HIV-1C evolutionary rates were estimated by BEAST v.1.7 using a relaxed lognormal molecular clock model. The effect of antiretroviral therapy (ART) on substitution rates in gag and env was assessed in a subset of six individuals who started ARV therapy during the follow-up period.During primary HIV-1C infection, the intra-patient substitution rates were estimated at a median (IQR) of 5.22E-03 (3.28E-03-7.55E-03) substitutions per site per year of infection within gag, and 1.58E-02 (9.99E-03-2.04E-02) substitutions per site per year within env gp120 V1C5. The substitution rates in env gp120 V1C5 were higher than in gag (p<0.001, Wilcoxon signed rank test). The median (IQR) relative rates of evolution at codon positions 1, 2, and 3 were 0.73 (0.48-0.84), 0.67 (0.52-0.86), and 1.54 (1.21-1.71) in gag, and 1.01 (0.86-1.15), 1.05 (0.99-1.21), and 0.86 (0.67-0.94) in env gp120 V1C5, respectively. A first to the third position codon rate ratio >1.0 within env was found in 25 (78.1%) cases, but only in 4 (12.5%) cases in gag, while a second to the third position codon rate ratio >1.0 in env was observed in 26 (81.3%) cases, but in gag only in 2 (6.3%) cases (p<0.001 for both comparisons, Fisher's exact test). No ART effect on substitution rates in gag and env was found, at least within the first 3-4 months after ART initiation. Individuals with early viral set point ?4.0 log10 copies/ml had higher substitution rates in env gp120 V1C5 (median (IQR) 1.88E-02 (1.54E-02-2.46E-02) vs. 1.04E (7.24E-03-1.55E-02) substitutions per site per year; p=0.017, Mann-Whitney sum rank test), while individuals with early viral set point ?3.0 log10 copies/ml had higher substitution rates in gag (median (IQR) 5.66E-03 (3.45E-03-7.94E-03) vs. 1.78E-03 (4.57E-04-5.15E-03); p=0.028; Mann-Whitney sum rank test).The results suggest that in primary HIV-1C infection, (1) intra-host evolutionary rates in env gp120 V1C5 are about 3-fold higher than in gag; (2) selection pressure in env is more frequent than in gag; (3) initiation of ART does not change substitution rates in HIV-1C env or gag, at least within the first 3-4 months after starting ART; and (4) intra-host evolutionary rates in gag and env gp120 V1C5 are higher in individuals with elevated levels of early viral set point.

Dataset Information

Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging.

Publications

Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging.

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