Natural Killer (NK) cells play an important role in host antiviral response against HIV-1, notably by mediating the elimination of infected cells by Antibody Dependant Cellular Cytotoxicity (ADCC). This relies on the capacity of their Fcγ receptor to recognize antibody-coated infected cells and to stimulate NK cells effector functions. These functions are also tightly regulated by an array of activating, co-activating and inhibitory receptors recognizing molecules present at the surface of target cells. To escape NK cell responses, HIV-1 accessory proteins Nef and Vpu downregulate the ligands of several NK cell activating/co-activating receptors, including NKG2D, DNAM-1 and NTB-A. NTB-A belongs to the Signaling Lymphocyte Activating Molecule (SLAM) family of receptors, which was associated with improved NK cell effector functions. Vpu-mediated downregulation of NTB-A was shown to prevent NK cells degranulation and ADCC responses. Considering the role of SLAMs in stimulating NK cell functions, we evaluated whether HIV-1 modulated CD48, the ligand of another SLAM receptor, 2B4. Using a panel of infectious molecular clones defective or not for Nef and/or Vpu expression, we found that Vpu downregulates CD48 from the surface of infected primary CD4+ T cells. This modulation depends of Vpu’s transmembrane domain and dual phosphoserine motif. Using a NK cell redirection assay, we observed a functional redundancy in the ability of NTB-A and 2B4 receptors to promote NK cells degranulation. Lastly, we confirmed the role of Vpu-mediated CD48 downmodulation in the capacity of infected primary CD4+ T cells to evade ADCC responses. Our results support the redundancy of NTB-A and 2B4 in stimulating ADCC against HIV-1-infected cells. Taken together, these results improve our understanding of how HIV-1 succeeds in evading ADCC responses by showing that Vpu must downregulate multiple SLAM ligands to protect infected cells from this response.

Background: The Th17-polarized CD4+ T-cells are enriched in viral reservoirs (VR) in people living with HIV-1 (PLWH) receiving viral-suppressive antiretroviral therapy (ART). We previously demonstrated that the transcriptional signature associated with HIV permissiveness in Th17 cells includes the circadian clock components/regulators BMAL1 and RORC2, the last identified as a positive regulator of HIV replication/outgrowth. REV-ERB, another circadian clock component, acts as a transcriptional repressor of BMAL1 (a transcriptional activator binding to E-boxes in the HIV LTR) and RORC2 (the master regulator of Th17 polarization). Thus, we hypothesized that REV-ERB restricts HIV-1 transcription/replication and effector functions in Th17 cells via mechanisms involving BMAL1/RORC2 repression.
Methods: Memory CD4+ T-cells of HIV-uninfected individuals were exposed to replication-competent and single-round HIV-1 constructs upon αCD3/αCD28 triggering in vitro. Early and late reverse transcripts, and integrated HIV-DNA were quantified by nested real-time PCR. HIV-1 replication was quantified by FACS and ELISA. A quantitative viral outgrowth assay (VOA) was performed using memory CD4+ T-cells of ART-treated PLWH. The REV-ERB agonist SR9011 and the antagonist SR8278 were used to modulate REV-ERB activity. Gene expression was quantified by real-time RT-PCR.
Results: HIV-1 replication boosted by TCR activation in memory CD4+ T-cells coincided with a significant downregulation of REV-ERB and an increase of RORC2 mRNA expression. SR9011 decreased RORC2 and IL-17A but not BMAL1 expression and reduced HIV-1 replication in vitro and viral outgrowth in cells of ART-treated PLWH. Despite the reported REV-ERB antagonism-mediated increase in HIV transcription in cell line, SR8278 reduced HIV replication in vitro and viral outgrowth in part via decreasing the expression of the HIV-1 co-receptor CCR5.
Conclusions: These results provide evidence that REV-ERB modulates HIV replication/outgrowth in CD4+ T-cells by mechanisms involving at least in part the repression of RORC2 and/or CCR5 expression, thus identifying REV-ERB as a novel therapeutic target in HIV cure/remission strategies.

Introduction: Gene therapy using a combination of antiviral RNAs could induce long-term remission from HIV-1 infection. Short hairpin (sh)RNAs designed against conserved regions of the HIV-1 genome exploit the RNA interference pathway to precisely target HIV-1 RNA for degradation. Their sequence and pairing with an RNA polymerase III promoter (7SK, U6, or H1) must be tested to ensure that the resulting promoter-shRNA cassette is safe and effective. Here, we optimized top-performing shRNAs and investigated a possible mechanism for promoter-dependent shRNA efficacy.

Methods: We assessed short-term efficacy of cassettes by cotransfecting shRNAs with the HIV-1 molecular clone pNL4-3 in HEK 293T cells, then measuring viral production. Long-term efficacy was assessed by expressing cassettes from lentiviral vectors in SUP-T1 cells before infection with HIV-1 NL4-3 to compare replication kinetics. We probed cassette cytotoxicity with cell metabolism and competitive cell growth assays. Guide strand expression was quantified with Northern blots.

Results: 7SK, U6, and H1 promoter efficacies were tested with one shRNA in clinical trials and one targeting a conserved sequence in the HIV-1 Gag coding region. shRNAs expressed from 7SK and U6 were best able to inhibit viral production. We screened other top-performing shRNAs and identified three that significantly delayed NL4-3 replication when expressed from H1, and even more so when expressed from 7SK and U6 promoters. Cell growth was negatively impacted by 7SK and U6-containing cassettes, but the extent varied depending on the shRNA identity. One shRNA impacted cell growth less than others, suggesting that these effects are partly sequence specific. 7SK and U6 promoters produced the most guide strands, indicating that cassette efficacy is partly dictated by transcriptional efficiency.

Conclusion: 7SK and U6 generate the most effective shRNAs, and the shRNA that impacted cell growth the least is the best molecule from our panel to pursue for future gene therapies.

Human immunodeficiency virus 1 (HIV-1) is transmitted by contact with infected fluids, including genital secretions or blood. As a result, HIV-risk groups include heterosexual individuals (HET), men-who-have-sex-with-men (MSM), people who inject drugs (PWID) and people who received contaminated blood transfusions (CBT). When HIV-1 is transmitted, typically only a single clone, or in rare cases, a small number of HIV-1 clones establish the new infection, creating a transmission bottleneck for the virus. The viral clone that establishes infection is called the transmitted/founder (T/F) virus. Specific traits that permit successful transmission have not been well characterized.

This project aims to determine transmission fitness between T/F viruses from different transmission routes through in vitro competitions. Subsequently, phenotypic assays were used to analyze the contribution of select factors to transmission fitness. Competitions on human cervical tissues suggested that tissues favored T/F viruses over chronic viruses and glycosylation might play a role in the process. We also found that T/F viruses from HET and MSM groups often outcompeted T/F viruses from PWID group in T helper type 1 (Th1) cells, while viruses from HET and PWID groups dominated infection in Th17 cells. T/F viruses showed different phenotypic characteristics between different transmission routes and from chronic viruses. T/F viruses from HET group required more stringent cellular co-receptor conformations to enter susceptible cells compared to others. Furthermore, T/F viruses exhibited more rapid cell entry than chronic viruses. However, there is no significant difference in envelope expression level across T/F virus groups and chronic viruses, which indicates that differences between T/F and chronic viruses in this study are more likely due to envelope structure or glycosylation patterns/levels. This project will establish the key viral phenotypes contributing to successful virus transmission to inform the design of a robust anti-HIV vaccine and will help the improvement of antiretroviral therapy strategy.

The surface of HIV displays just one viral protein (glycoprotein 120) but is also decorated with numerous host-derived proteins. Virion-incorporated host proteins have gained much interest in the field because of their preserved functionality and ability to bind to their cognate receptors, thus, altering HIV infectivity and homing. Different laboratory techniques with varying sensitivities are available to detect and quantify these HIV-incorporated host proteins. In this study, we are providing a comparison of sensitivities and detection thresholds for three commonly used techniques, to provide useful information to researchers studying virion-incorporated proteins.
Herein we compared results from conventional virus capture assays and Western blotting (on whole virus lysates), alongside novel techniques in flow virometry (FVM). More specifically, we employed a unique FVM protocol developed by our lab to detect and quantify HIV virion-incorporated proteins on native virions that were stained directly from culture supernatants. We compared these techniques for their ability to detect four host proteins (CD14, CD38, CD59 and CD162) known to be incorporated at high levels into HIV virions. For this study, we generated four pseudovirus stocks, each expressing one host protein by co-transfecting viral expression plasmids and host protein expression plasmids in HEK293T cells. To provide a controlled comparison, all techniques were employed in parallel on identical viral stocks with normalized virus inputs.
Our data show that each technique has advantages and caveats for detecting virion-incorporated proteins, with different thresholds for detection. Additionally, there is a large variance in total virus input required for reliability of each technique, which may be an important consideration when determining which technique to use in future biological studies. Flow virometry detection provided distinct advantages because it enabled highly reproducible quantification of virion-incorporated proteins (number of proteins per virus particle), with the lowest sample requirements and reagent costs, and minimal hands-on experimental time.

Downregulation of CD4 by HIV-1 protects infected cells from antibody-dependent cellular cytotoxicity (ADCC). However, productively-infected cells shed gp120 which can then bind CD4 at the surface of uninfected bystander CD4+ T cells, thus sensitizing them to ADCC mediated by HIV+ plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. CD4 engages gp120 within a cavity located at the interface between the inner and outer domains of gp120 known as the Phe43 cavity. The FDA-approved small molecule temsavir acts as an HIV-1 attachment inhibitor. Temsavir binds under the Env β20-β21 loop and within the Phe43 cavity, therefore preventing Env-CD4 interaction. We recently demonstrated that temsavir alters the overall antigenicity of Env by affecting its processing and glycosylation. Here, we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents gp120 shed from productively-infected cells to interact with uninfected bystander CD4+ cells. This protects uninfected bystander CD4+ T cells from ADCC responses mediated by HIV+ plasma and prevents a gp120-induced cytokine burst. Mechanistically, this depends on the capacity of temsavir to reduce soluble gp120-CD4 interaction at the surface of bystander cells, but also to control the levels and the antigenicity of shed gp120. Consistent with its ability to decrease Env proteolytic cleavage, we show that temsavir significantly reduces gp120 shedding. In agreement with temsavir’s impact on Env antigenicity, gp120 released from temsavir-treated cells exhibits a reduced capacity to interact with CD4 and to coat primary CD4+ T cells. Altogether, our study reveals how temsavir block the immunomodulatory activities of shed gp120 that are dependent on CD4 interaction. Our results suggest that the clinical benefits provided by temsavir could extend beyond blocking viral entry.