Glycosyl-Phosphatidylinositol-Anchored Anti-HIV Env Single-Chain Variable Fragments Interfere with HIV-1 Env Processing and Viral Infectivity

Misra, Anisha; Gleeson, Emile I.; Wang, Weiming; Ye, Chaobaihui; Zhou, Paul; Kimata, Jason T.

doi:10.1128/jvi.02080-17

Cited by 10 publications

(7 citation statements)

References 80 publications

(97 reference statements)

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“…It has been reported that processed HIV-1 gp120/gp41, but not gp160, is preferentially incorporated into virions via MA-dependent mechanisms (37, 38). Our current results are consistent with this model, showing that impaired gp160 cleavage in the absence of Vpr leads to reduced levels of gp120/gp41 and thus lower levels of gp120/gp41 in the virion (24, 39–42). As we have previously reported, reduced gp120/gp41 in the virions led to reduced infectivity of virions (defined as infectious activity per virion particle) (43, 44).…”

Section: Discussionsupporting

confidence: 92%

Vpr Enhances HIV-1 Env Processing and Virion Infectivity in Macrophages by Modulating TET2-Dependent IFITM3 Expression

Wang

2019

mBio

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HIV-1 Vpr enhances viral replication in human macrophages via multiple mechanisms that are not clearly defined. It does not affect HIV-1 virion production during the first round of infection. We have recently discovered that Vpr targets the DNA demethylase TET2 for degradation, which leads to sustained interleukin-6 (IL-6) expression and elevated HIV-1 replication. We report here that Vpr enhanced Env processing in infected macrophages, associated with increased Env incorporation into virions with higher infectivity. Interestingly, IFITM3 was constitutively expressed in macrophages in a TET2-dependent fashion. We showed that Vpr-enhanced Env processing depended genetically on TET2 and IFITM3. We further showed that Vpr reduced IFITM3 expression by reducing demethylation of the IFITM3 promoter in macrophages, associated with degradation of TET2 and reduced TET2 binding to the IFITIM3 promoter. Our findings indicate that the Vpr-TET2 axis enhances HIV-1 replication in macrophages via two independent mechanisms: reduced IFTIM3 expression to enhance Env processing and virion infectivity and sustained IL-6 expression to increase HIV-1 replication. The Vpr-TET2 axis may provide a novel target to develop therapeutics to inhibit HIV-1 infection and pathogenesis. IMPORTANCE How Vpr enhances HIV-1 replication in macrophages is still unclear. We report here that Vpr enhanced HIV-1 Env processing during the first round of HIV-1 replication, resulting in virions with higher Env incorporation and viral infectivity. These higher-quality viral particles contributed to elevated infection during the second round and spreading infection in macrophages and other HIV-1 target cells. We have recently discovered that TET2 is a novel host factor degraded by Vpr, which leads to sustained IL-6 expression in macrophages. Interestingly, Vpr-enhanced HIV-1 Env processing depended on both the IFITIM3 and TET2 genes. The constitutive expression of IFITIM3 expression in macrophages was maintained by TET2, which demethylated the IFITIM3 promoter. We conclude that the Vpr degrades TET2 to enhance HIV-1 replication in macrophages by reducing IFITIM3 expression to increase viral Env processing, virion incorporation, and infectivity and by sustaining IL-6 expression to increase HIV-1 gene expression. The Vpr-TET2 axis may serve as a novel target to develop anti-HIV drugs to inhibit HIV-1 infection and pathogenesis.

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Section: Discussionsupporting

confidence: 92%

Vpr Enhances HIV-1 Env Processing and Virion Infectivity in Macrophages by Modulating TET2-Dependent IFITM3 Expression

Wang

2019

mBio

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“…If positive, supernatants were passed through 0.45-μm syringe filters, aliquoted, and frozen at −80°C. The infectious titers of the stocks were determined by limiting dilution infection analysis using TZM-bl reporter cells and luciferase assay as described ( Misra et al, 2018 ). Infectious virus recovered from CD4 + T cells at 196 and 200wpi was named C/196 and C/200, respectively.…”

Section: Methodsmentioning

confidence: 99%

In vivo Serial Passaging of Human–Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication

et al. 2021

Self Cite

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We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

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“…Previous studies suggested that GPI-anchored anti-gp120 scFv X5 (GPI-X5) and nanobody (GPI-m36.4) can interfere with the expression and processing of viral Env glycoproteins and the infectivity of progeny HIV-1virions, underlying a multifaceted mode of antiviral action [ 26 , 36 ]. Herein, we also utilized similar experimental protocols to characterize the antiviral mechanism of GPI-10E8.…”

Section: Resultsmentioning

confidence: 99%

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

Chen

Jin

Tang

et al. 2021

Emerging Microbes & Infections

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Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell–cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4 + T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

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Glycosyl-Phosphatidylinositol-Anchored Anti-HIV Env Single-Chain Variable Fragments Interfere with HIV-1 Env Processing and Viral Infectivity

Cited by 10 publications

References 80 publications

Vpr Enhances HIV-1 Env Processing and Virion Infectivity in Macrophages by Modulating TET2-Dependent IFITM3 Expression

Vpr Enhances HIV-1 Env Processing and Virion Infectivity in Macrophages by Modulating TET2-Dependent IFITM3 Expression

In vivo Serial Passaging of Human–Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

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