The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not. HIV establishes a state of latency in vivo and this latent reservoir, although small, is difficult to eradicate. To be able to better understand this state of latency, and to develop strategies to eliminate it, many groups have developed in vitro models of HIV latency. However, notable differences exist among cell model systems because compounds that reactivate latent HIV in a particular system often fail to do so uniformly across different models. To begin to understand the biological characteristics that are inherent to each HIV model of latency, we compared the response properties of five primary T cell, four J-Lat cell models and those obtained with patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models.

The use of antiretroviral therapy in HIV type 1 (HIV-1)–infected patients does not lead to virus eradication. This is due, to a significant degree, to the fact that HIV-1 can establish a highly stable reservoir of latently infected cells. In this work, we describe an ex vivo experimental system that generates high levels of HIV-1 latently infected memory cells using primary CD4+ T cells. Using this model, we were able to dissect the T cell–signaling pathways and to characterize the long terminal repeat (LTR) cis-acting elements involved in reactivation of HIV-1 in memory CD4+ T cells. We conclude that Lck and nuclear factor of activated T cells (NFAT), but not NF-κB, are required for optimal latent virus reactivation in memory T cells. We also found that the cis-acting elements which are critical toward HIV-1 reactivation are the Sp1 and κB/NFAT transcription factor binding sites.

Background: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. Results: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT₅₀) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT₅₀ < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT₅₀ < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R² = 0.9344). Conclusions: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.

Homeostatic proliferation ensures the longevity of central memory T-cells by inducing cell proliferation in the absence of cellular differentiation or activation. This process is governed mainly by IL-7. Central memory T-cells can also be stimulated via engagement of the T-cell receptor, leading to cell proliferation but also activation and differentiation. Using an in vitro model of HIV-1 latency, we have examined in detail the effects of homeostatic proliferation on latently infected central memory T cells. We have also used antigenic stimulation via anti-CD3/anti-CD28 antibodies and established a comparison with a homeostatic proliferation stimulus, to evaluate potential differences in how either treatment affects the dynamics of latent virus populations. First, we show that homeostatic proliferation, as induced by a combination of IL-2 plus IL-7, leads to partial reactivation of latent HIV-1 but is unable to reduce the size of the reservoir in vitro. Second, latently infected cells are able to homeostatically proliferate in the absence of viral reactivation or cell differentiation. These results indicate that IL-2 plus IL-7 may induce a detrimental effect by favoring the maintenance of the latent HIV-1 reservoir. On the other hand, antigenic stimulation efficiently reactivated latent HIV-1 in cultured central memory cells and led to depletion of the latently infected cells via virus-induced cell death. HIV-1 latently infected cells are considered the last barrier towards viral eradication and cure. However, the low number of latently infected cells found in patients makes studies extremely difficult. Here, using a model of primary CD4 T-cells we study the behavior of latently infected central memory T cells when undergoing homeostatic proliferation. Homeostatic proliferation ensures the longevity of the central memory population, as it does not involve cellular differentiation. In the context of HIV infection, IL-7 has been reported to induce viral outgrowth from latently infected cells in different cellular models. However, those studies did not examine the relationship between cell proliferation and viral reactivation. We here report that the strong effect of IL-7 on the proliferation of memory cells counteracts this cytokine's modest ability to purge latent viruses. Thus, central memory cells are subject to homeostatic proliferation, a physiological effect that may contribute to the longevity of the latent reservoir in HIV-1 infected patients.

Antiretroviral therapy (ART) has rendered HIV-1 infection a treatable illness; however, ART is not curative owing to the persistence of replication-competent, latent proviruses in long-lived resting T cells. Strategies that target these latently infected cells and allow immune recognition and clearance of this reservoir will be necessary to eradicate HIV-1 in infected individuals. This review describes current pharmacologic approaches to reactivate the latent reservoir so that infected cells can be recognized and targeted, with the ultimate goal of achieving an HIV-1 cure.

HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G₂ and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G₂ checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G₂ arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G₂-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.