The Anti-HIV Pentameric Pseudopeptide HB-19 Binds the C-terminal End of Nucleolin and Prevents Anchorage of Virus Particles in the Plasma Membrane of Target Cells

AbstractHuman Cytomegalovirus (HCMV) is a highly prevalent herpesvirus that establishes lifelong latent infection in humans. It is the leading cause of congenital disabilities and a significant cause of disease in immunocompromised patients (1). HCMV significantly remodels host cell processes and membranes for the effective production of virus progeny (2). Their final morphogenesis process occurs in the cytoplasm, where capsids acquire the proteinaceous tegument layer and enveloping membrane. This involves budding into host membranes and subsequent exocytosis of assembled virions by fusion with the plasma membrane (3). Envelopment and exocytosis are thought to be mediated by small vesicles, leading to the continuous release of individually wrapped virions. However, groups of enveloped virus particles are also found inside multivesicular bodies (MVBs) (4, 5). Whether enveloped particles in MVBs reflect a productive envelopment and egress pathway or are targeted for lysosomal degradation has remained unknown.Using a novel correlative light and electron microscopy (CLEM) workflow that enables imaging of virus morphogenesis in whole cells, we present evidence that HCMV envelopment does occur at MVBs, generating large intracellular accumulations of enveloped virions in HFF cells. Virus-filled MVBs carried exosomal markers and were found to traverse the cytoplasm to the plasma membrane by volumetric, live-cell lattice light-sheet microscopy. Using a pH-sensitive biosensor, we show that virus particles were released in bulk by fusion of MVBs with the plasma membrane leading to ‘patches’ of particles on the plasma membrane.This hitherto undescribed envelopment and exocytosis pathway of HCMV, leading to the recurrent bulk release of virus particles, possibly involves the late-endosome/exosome pathway.Significance StatementHCMV is able to cause disease affecting various organs. Understanding how HCMV can infect a wide variety of cells is essential for developing antiviral strategies. Recent work suggests that HCMV particles with varying glycoprotein repertoires facilitate entry into different target cells. How this glycoprotein diversity at the single-particle-level is generated is unclear. Different envelopment and egress pathways might play a role.Here, using HFF cells, we present direct functional evidence that HCMV uses multivesicular bodies for the bulk release of virus particles into membrane-associated accumulations as a novel, alternative HCMV egress pathway. Future work will aim to illuminate how different egress pathways might lead to varying virion compositions.

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COVID-19: Update on Pathogenesis and Treatment Strategies

Current Immunology Reviews ◽

10.2174/1573395516999201001154837 ◽

2020 ◽

Vol 16 (1) ◽

pp. 1-5

Author(s):

Rakesh K. Chauhan ◽

Pramod K. Sharma ◽

Shikha Srivastava

Keyword(s):

Monoclonal Antibody ◽

Plasma Membrane ◽

Human Monoclonal Antibody ◽

Cardiac Injury ◽

Treatment Strategies ◽

Severe Pneumonia ◽

Ground Glass Opacity ◽

Golgi Membrane ◽

Virus Particles ◽

Global Pandemic

COVID-19 (Coronavirus disease) is the most contagious virus, which has been characterized as a global pandemic by WHO. The pathological cycle of COVID-19 virus can be specified as RNAaemia, severe pneumonia, along with the Ground-glass opacity (GGO), and acute cardiac injury. The S protein of Coronavirus has been reported to be involved in the entry of the virus into the host cell, which can be accomplished by direct membrane fusion between the virus and plasma membrane. In the endoplasmic reticulum or Golgi membrane, the newly formed enveloped glycoproteins are introduced. The spread of disease occurs due to contact and droplets unleashed by the vesicles holding the virus particles combined with the plasma membrane to the virus released by the host. The present manuscript describes the pathogenesis of COVID-19 and various treatment strategies that include drugs such as chloroquine and hydroxychloroquine, an anti-malarial drug, antibodies: SARS-CoV-specific human monoclonal antibody CR3022 and plasma treatment facilitate the therapeutic effect.

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Single-molecule imaging of HIV-1 envelope glycoprotein dynamics and Gag lattice association exposes determinants responsible for virus incorporation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910008116 ◽

2019 ◽

Vol 116 (50) ◽

pp. 25269-25277 ◽

Cited By ~ 10

Author(s):

Nairi Pezeshkian ◽

Nicholas S. Groves ◽

Schuyler B. van Engelenburg

Keyword(s):

Plasma Membrane ◽

Single Molecule ◽

Envelope Glycoprotein ◽

Virus Assembly ◽

Single Molecule Tracking ◽

Virus Particles ◽

Cell Plasma ◽

The Matrix ◽

Resolution Single ◽

Hiv 1

The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.

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Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus.

The Journal of Cell Biology ◽

10.1083/jcb.83.2.338 ◽

1979 ◽

Vol 83 (2) ◽

pp. 338-347 ◽

Cited By ~ 29

Author(s):

M Büechi ◽

T Bächi

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Plasma Membranes ◽

Sendai Virus ◽

Light And Electron Microscopy ◽

Distal Portion ◽

Double Labeling ◽

Virus Particles ◽

Viral Antigens ◽

Cytoplasmic Surface

A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double-labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.

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Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction

Journal of Virology ◽

10.1128/jvi.00137-17 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 23

Author(s):

Xianfeng Zhang ◽

Tao Zhou ◽

Jie Yang ◽

Yumei Lin ◽

Jing Shi ◽

...

Keyword(s):

Plasma Membrane ◽

Transmembrane Domain ◽

The Other ◽

Stable Expression ◽

Membrane Localization ◽

Plasma Membrane Localization ◽

Immunodeficiency Virus ◽

Alternatively Spliced ◽

Anti Hiv ◽

Hiv 1

ABSTRACT Among the five serine incorporator (SERINC) family members, SERINC5 (Ser5) was reported to strongly inhibit HIV-1 replication, which is counteracted by Nef. Ser5 produces 5 alternatively spliced isoforms: Ser5-001 has 10 putative transmembrane domains, whereas Ser5-004, -005, -008a, and -008b do not have the last one. Here, we confirmed the strong Ser5 anti-HIV-1 activity and investigated its isoforms' expression and antiviral activities. It was found that Ser5-001 transcripts were detected at least 10-fold more than the other isoforms by real-time quantitative PCR. When Ser5-001 and its two isoforms Ser5-005 and Ser5-008a were expressed from the same mammalian expression vector, only Ser5-001 was stably expressed, whereas the others were poorly expressed due to rapid degradation. In addition, unlike the other isoforms, which are located mainly in the cytoplasm, Ser5-001 is localized primarily to the plasma membrane. To map the critical determinant, Ser5 mutants bearing C-terminal deletions were created. It was found that the 10th transmembrane domain is required for Ser5 stable expression and plasma membrane localization. As expected, only Ser5-001 strongly inhibits HIV-1 infectivity, whereas the other Ser5 isoforms and mutants that do not have the 10th transmembrane domain show very poor activity. It was also observed that the Nef counteractive activity could be easily saturated by Ser5 overexpression. Thus, we conclude that Ser5-001 is the predominant antiviral isoform that restricts HIV-1, and the 10th transmembrane domain plays a critical role in this process by regulating its protein stability and plasma membrane targeting. IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express a small protein, Nef, to enhance viral pathogenesis in vivo. Nef has an important in vitro function, which is to make virus particles more infectious, but the mechanism has been unclear. Recently, Nef was reported to counteract a novel anti-HIV host protein, SERINC5 (Ser5). Ser5 has five alternatively spliced isoforms, Ser5-001, -004, -005, -008a, and -008b, and only Ser5-001 has an extra C-terminal transmembrane domain. We now show that the Ser5-001 transcripts are produced at least 10-fold more than the others, and only Ser5-001 produces stable proteins that are targeted to the plasma membrane. Importantly, only Ser5-001 shows strong anti-HIV-1 activity. We further demonstrate that the extra transmembrane domain is required for Ser5 stable expression and plasma membrane localization. These results suggest that plasma membrane localization is required for Ser5 antiviral activity, and Ser5-001 is the predominant isoform that contributes to the activity.

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Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽

10.1182/blood.v60.3.583.583 ◽

1982 ◽

Vol 60 (3) ◽

pp. 583-594 ◽

Cited By ~ 26

Author(s):

N Dainiak ◽

CM Cohen

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Mononuclear Cells ◽

Surface Membrane ◽

Freeze Fracture ◽

Membrane Vesicles ◽

Cell Types ◽

Target Cells ◽

Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

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The Interplay between HIV-1 Gag Binding to the Plasma Membrane and Env Incorporation

Viruses ◽

10.3390/v12050548 ◽

2020 ◽

Vol 12 (5) ◽

pp. 548 ◽

Cited By ~ 3

Author(s):

R. Elliot Murphy ◽

Jamil S. Saad

Keyword(s):

Plasma Membrane ◽

Envelope Glycoprotein ◽

Virus Production ◽

Mortality Rates ◽

Therapeutic Agents ◽

Structural Level ◽

Particle Assembly ◽

Virus Particles ◽

Molecular Determinants ◽

Hiv 1

Advancement in drug therapies and patient care have drastically improved the mortality rates of HIV-1 infected individuals. Many of these therapies were developed or improved upon by using structure-based techniques, which underscore the importance of understanding essential mechanisms in the replication cycle of HIV-1 at the structural level. One such process which remains poorly understood is the incorporation of the envelope glycoprotein (Env) into budding virus particles. Assembly of HIV particles is initiated by targeting of the Gag polyproteins to the inner leaflet of the plasma membrane (PM), a process mediated by the N-terminally myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). There is strong evidence that formation of the Gag lattice on the PM is a prerequisite for the incorporation of Env into budding particles. It is also suggested that Env incorporation is mediated by an interaction between its cytoplasmic tail (gp41CT) and the MA domain of Gag. In this review, we highlight the latest developments and current efforts to understand the interplay between gp41CT, MA, and the membrane during assembly. Elucidation of the molecular determinants of Gag–Env–membrane interactions may help in the development of new antiviral therapeutic agents that inhibit particle assembly, Env incorporation and ultimately virus production.

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Slaying the Trojan Horse: Natural Killer Cells Exhibit Robust Anti-HIV-1 Antibody-Dependent Activation and Cytolysis against Allogeneic T Cells

Journal of Virology ◽

10.1128/jvi.02461-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 97-109 ◽

Cited By ~ 31

Author(s):

Shayarana L. Gooneratne ◽

Jonathan Richard ◽

Wen Shi Lee ◽

Andrés Finzi ◽

Stephen J. Kent ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Target Cells ◽

Dependent Manner ◽

Inhibitory Receptors ◽

Cell Responses ◽

Anti Hiv ◽

Hiv 1

ABSTRACTMany attempts to design prophylactic human immunodeficiency virus type 1 (HIV-1) vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus bothin vitroandin vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of natural killer (NK) cells to respond in an Ab-dependent manner to allogeneic T cells bearing HIV-1 antigens. This study presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1+NK cell subset from HLA-Bw4+individuals exhibits an activation advantage over the KIR3DL1−subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses and are potentially valuable for designing Ab-dependent therapies and/or vaccines.IMPORTANCENK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression; however, their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated with HIV-1 gp120 were capable of activating NK cells and/or trigging cytolysis of the allogeneic target cells. This suggests ADCC may be able to assist in preventing infection with cell-associated HIV-1. In order to fully utilize NK cell-mediated Ab-dependent effector functions, it might also be important that educated NK cells, which hold the highest activation potential, can become activated against targets bearing HIV-1 antigens and expressing the ligands for self-inhibitory receptors. Here, we show that with Ab-dependent stimulation, NK cells expressing inhibitory receptors can mediate robust activation against targets expressing the ligands for those receptors.

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PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1916054117 ◽

2020 ◽

Vol 117 (17) ◽

pp. 9537-9545 ◽

Cited By ~ 10

Author(s):

Yajing Fu ◽

Sijia He ◽

Abdul A. Waheed ◽

Deemah Dabbagh ◽

Zheng Zhou ◽

...

Keyword(s):

Virus Particle ◽

Influenza A ◽

Leukemia Virus ◽

Surface Expression ◽

Cell Surface Expression ◽

Target Cells ◽

Viral Glycoprotein ◽

Selectin Binding ◽

Anti Hiv ◽

Hiv 1

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1–mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti–HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1–related monomeric E-selectin–binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1–mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.

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Neutrophil extracellular traps from healthy donors and HIV-1-infected individuals restrict HIV-1 production in macrophages

Scientific Reports ◽

10.1038/s41598-020-75357-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Andrés Mojoli ◽

Barbara Simonson Gonçalves ◽

Jairo R. Temerozo ◽

Bruno Cister-Alves ◽

Victor Geddes ◽

...

Keyword(s):

Virus Production ◽

Significant Inhibition ◽

Target Cells ◽

Extracellular Traps ◽

Healthy Donors ◽

Activated Neutrophils ◽

Immunity Mechanism ◽

Anti Hiv ◽

Hiv 1

Abstract Neutrophils release extracellular traps (NETs) after interaction with microorganisms and physiological or synthetic products. NETs consist of decondensed chromatin complexed with proteins, some of them with microbicidal properties. Because NETs can modulate the functioning of HIV-1 target cells, we aimed to verify whether they modify HIV-1 replication in macrophages. We found that exposure of HIV-1-infected macrophages to NETs resulted in significant inhibition of viral replication. The NET anti-HIV-1 action was independent of other soluble factors released by the activated neutrophils, but otherwise dependent on the molecular integrity of NETs, since NET-treatment with protease or DNase abolished this effect. NETs induced macrophage production of the anti-HIV-1 β-chemokines Rantes and MIP-1β, and reduced the levels of integrated HIV-1 DNA in the macrophage genome, which may explain the decreased virus production by infected macrophages. Moreover, the residual virions released by NET-treated HIV-1-infected macrophages lost infectivity. In addition, elevated levels of DNA-elastase complexes were detected in the plasma from HIV-1-infected individuals, and neutrophils from these patients released NETs, which also inhibited HIV-1 replication in in vitro infected macrophages. Our results reveal that NETs may function as an innate immunity mechanism able to restrain HIV-1 production in macrophages.

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