Patent Grant
12194087
The contents of the computer readable text file with following details (filename: GMUN_003_01US_ST25.txt, date recorded: Oct. 7, 2022, file size 11.7 kilobytes) submitted electronically herewith are incorporated herein by reference in their entirety. The sequences enlisted in .txt file are identical to sequences present in
The Invention relates generally to method of blocking viral infectivity to target cells, more specifically to the method of inhibiting Human Immunodeficiency Viruses 1 (HIV-1) replication through expression of a P-selectin glycoprotein ligand-1 (PSGL-1).
Various references, including patents, patent applications, accession numbers, technical articles, and scholarly articles are cited throughout the specification. Each reference is incorporated by reference herein, in its entirety and for all purposes.
PSGL-1 (P-selectin glycoprotein ligand-1) also known as SELPLG or CD162, is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins (1-3). PSGL-1 is primarily expressed on the surface of lymphoid and myeloid cells (1, 4, 5), and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium to promote cell migration into inflamed tissues (6-9). PSGL-1 also serves as a surface receptor for enterovirus 71 (EV71) infection of leukocytes (10). In a mouse model of chronic viral infection, PSGL-1 has been reported to regulate T-cell checkpoints (11) and has been shown to be an IFN-γ regulated factor involved in Th1-mediated antiviral activity (8, 12). During T-cell differentiation, culturing T cells in the Th1 cytokine IFN-, and IL-12 promoted PSGL-1 expression preferentially in the IFN-γ producing T-cell population (8), suggesting that PSGL-1 could be an IFN-γ regulated factor involved in Th1-mediated antiviral activity.
PSGL-1 was reported to co-cluster with HIV-1 Gag at sites of assembly in the T-cell uropodin Gag-expressing cells (13). In addition, PSGL-1 was recently identified, through proteomic profiling of HIV-1-infected T cells, as an IFN-γ. induced restriction factor that blocks HIV-1 reverse transcription early post-entry and inactivates progeny virion infectivity during viral assembly (12). Emerging evidence has shown that the anti-HIV-1 activity of PSGL-1 was antagonized by the HIV-1 accessory protein Vpu (12). It is therefore not clear how HIV-1 overcomes PSGL-1 restriction at early time points during DNA synthesis. Furthermore, the mechanism of PSGL-1 inactivation of virion infectivity remains to be elucidated.
Furthermore, till date there has been no literature, elucidating the anti-viral effect of expressed PSGL-1 within a virus producing cells There is also dearth of broad-spectrum viral vaccine.
Embodiments relates generally to a method of expressing a PSGL-1 or a mutant thereof in a virus producing cell and blocking virus infection by inactivating infectivity of a released virion from the virus producing cell. PSGL-1 can inactivate viruses in cell culture conditions. It is found that during the production of murine leukemia virus (MLV) viruses, if PSGL-1 DNA was co-transfected with MLV DNA, PSGL-1, the virions released became non-infectious. These results suggest that PSGL-1 can be used to inactivate other viral particles.
In an embodiment, PSGL-1 is found to inactivate Human Immunodeficiency Viruses 1 (HIV-1).
In an embodiment, PSGL-1 is found to inactivate murine leukemia virus (MLV) viruses. It was found that during the production of murine leukemia virus (MLV) viruses, if PSGL-1 DNA was co-transfected with MLV DNA, PSGL-1, the virions released became non-infectious.
In an embodiment, PSGL-1 inactivate other viral particles.
An embodiment relates to the molecular details of PSGL-1 restriction of viral infection particularly HIV-1 infection.
In an embodiment, PSGL-1, a mucin-like glycoprotein expressed on blood CD4 T cells, restricts HIV-1 virion infectivity primarily by interfering with particle binding to target cells.
In an embodiment, PSGL-1 does not inhibit virion release.
In an embodiment, PSGL-1 is a potent inhibitor: it almost completely inactivates HIV-1 particle infectivity at a vector-to-proviral DNA ratio of 0.05:1.
An embodiment relates to a method for targeting a viral infection by expressing a selectin glycoprotein like PSGL-1 or a CD43 or a mutant thereof in a virus producing cell and blocking a virus infection by preventing binding of a released virion to a target cell and inactivating the infectivity of the released virion.
An embodiment relates to the method the PSGL or CD43 blocks the virus infection by structurally hindering binding of the released virion.
An embodiment relates to the molecular details of PSGL-1 restriction of viral infection particularly HIV-1 infection. Embodiments demonstrate that PSGL-1, a mucin-like glycoprotein expressed on blood CD4 T cells, restricts HIV-1 virion infectivity primarily by interfering with particle binding to target cells. PSGL-1 does not inhibit virion release. PSGL-1 is a remarkably potent inhibitor: it almost completely inactivates HIV-1 particle infectivity at a vector-to-proviral DNA ratio of 0.05:1.
An embodiment relates PSGL-1 is surface glycoproteins inhibits HIV spreading infection. PSGL-1 blocks the binding of virus particles to target cells. Another embodiment relates CD43 is surface glycoproteins inhibits HIV spreading infection. CD43 blocks the binding of virus particles to target cells.
An embodiment relates to a method for targeting a viral infection by expressing a selectin glycoprotein like PSGL-1 or CD43 or a mutant thereof in a virus producing cell and blocking a virus infection by preventing binding of a released virion to a target cell and inactivating the infectivity of the released virion. In the method the PSGL or CD43 blocks the virus infection by structurally hindering binding of the released virion.
An embodiment relates to a broad-spectrum anti-viral product comprising: a vector expressing a selectin glycoprotein like PSGL-1 or CD43 or a mutant thereof in a virus producing cell; and blocking a virus infection by inactivating infectivity of a released virion from the virus producing cell.
An embodiment relates to a vaccine comprising live attenuated, inactivated, or non-infectious virus particles, wherein the virus particles are produced in virus producing cells, and wherein a selectin glycoprotein like PSGL-1 or CD43 or a mutant thereof is expressed in the virus producing cells.
An embodiment relates to a method comprising expressing a vector comprising a PSGL-1 (P-selectin glycoprotein ligand-1) or a mutant thereof in a VPC (virus producing cell); and blocking a virus infection by inactivating an infectivity of a released virions from the VPC.
In an embodiment, the PSGL-1 comprises an extracellular N-terminal domain that is configured to block the virus infection.
In an embodiment, the extracellular N-terminal domain of PSGL-1 comprises a decameric repeat: wherein the decameric repeat is configured to block the virus infection.
In an embodiment, the PSGL-1 is configured to block the infectivity of the released virions by blocking a binding of the released virions to a target cell.
In an embodiment, the PSGL-1 is configured to block the infectivity through a structural hindrance of the released virions binding to a target cell.
In an embodiment, the PSGL-1 is expressed by an expression vector into the VPC; inactivating infectivity of the released virions are independent of an envelope glycoprotein of the VPC; the VPC comprises a host cell infected with a virus; the virus infection comprises a human immunodeficiency virus HIV, a murine leukemia virus, an influenza virus or similar thereof.
Another embodiment relates to a method comprising: expressing a glycoprotein or a mutant thereof in a VPC (virus producing cell); blocking a virus infection by preventing binding of a released virions to a target cells; inactivating infectivity of the released virions; and targeting a viral infection.
In other embodiments: the glycoprotein comprises a PSGL-1 (P-selectin glycoprotein ligand-1), a CD43, a mutant of the PSGL-1 or a mutant of the CD43; the glycoprotein is configured to block infectivity of the released virions by preventing the binding of the released virions to the target cells: the glycoprotein or the mutant therefor is expressed by an expression vector into the VPC; the glycoprotein is configured to block the virus infection by structurally hindering binding of the released virions; the VPC comprises a host cell infected with a virus; the virus comprises a human immunodeficiency virus (HIV), a murine leukemia virus, an influenza virus or similar thereof.
Another embodiment relates to a broad-spectrum anti-viral product comprising: a vector expressing a glycoprotein or a mutant thereof in a VPC (virus producing cell); and blocking a virus infection by inactivating infectivity of a released virion from the VPC.
In other embodiments the glycoprotein comprises a PSGL-1 (P-selectin glycoprotein ligand-1), a CD43, a mutant of the PSGL-1 or a mutant of the CD43; about 1 μg of the PSGL-1 produces about 100 fold decrease in anti-viral activity of the released virion from the VPC; about 3 μg of the PSGL-1 produces about 8000 fold decrease in anti-viral activity of the released virion from the VPC. A CD43 expression vector is configured to block the virus infection: about 0.5 to 400 ng of a CD43 expression vector is configured inactivate infectivity of a released virion from the VPC; the VPC comprises a host cell infected with a virus. In an embodiment the virus infection comprises a human immunodeficiency virus (HIV), a murine leukemia virus (MLV), an influenza virus or similar thereof.
Another embodiment relates to a vaccine comprising, a viral particle is configured to a live attenuated or an inactivated or a non-infectious, wherein the viral particle are produced in a VPC, and wherein the VPC comprises a vector expressing a glycoprotein or a mutant thereof. In other embodiments: the glycoprotein comprises a PSGL-1 (P-selectin glycoprotein ligand-1) or a CD43 or a mutant thereof; the VPC are a host cell infected with a virus; the viral particle comprises a human immunodeficiency virus (HIV), a murine leukemia virus (MLV), an influenza virus or similar thereof.
Another embodiment relates to a use of a glycoprotein or a mutant thereof in a therapeutic treatment of a virus infection, wherein, a vector expressing the glycoprotein or a mutant thereof in a VPC (virus producing cell); and blocking a virus infection by inactivating infectivity of a released virion from the VPC, wherein the glycoprotein comprises a PSGL-1 (P-selectin glycoprotein ligand-1), a CD43, a mutant of the PSGL-1 or a mutant of the CD43.
An embodiment relates a finding that PSGL-1 can inactivate both HIV and other viruses e.g. MLV (murine leukemia virus), suggesting that PSGL-1 can inactivate a large number of viruses. PSGL-1 could be used in the preparation of a non-infectious virion particles by the expressing or overexpressing PSGL-1, in virus producing cells to inactivate viral infectivity. Such virions particles could be used as a vaccine to treat or prevent disease related to HIV infection or other viruses infection. Also, the application also elucidated the mechanism of PSGL-1 inactivation of virion infectivity.
An embodiment relates to a selectin glycoprotein like PSGL-1 can inactivate both HIV and other viruses such as MLV (murine leukemia virus, influenza A virus), suggesting that PSGL-1 can inactivate a large number of viruses. The use of overexpression of PSGL-1 in viral producing cells to inactivate viral infectivity. The released non-infectious virion particles can be used as a viral vaccine.
FIG.3B: Virions produced in the presence of PSGL-1 or the empty vector were assayed for attachment to target HeLa JC.53 cells at 4° C., for 2 hours. Cells were washed and then analyzed by western blot for bound p24.
Definitions and General Techniques
For simplicity and clarity of illustration, the drawing figures illustrate the general manner of descriptions and techniques.
The terms “first.” “second.” “third.” “fourth.” and the like in the description and in the claims, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms “include.” and “have.” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, device, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such process, method, system, article, device, or apparatus.
The terms “left,” “right,” “front,” “back,” “top,” “bottom,” “over,” “under,” and the like in the description and in the claims, if any, are used for descriptive purposes and not necessarily for describing permanent relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments of the apparatus, methods, and/or articles of manufacture described herein are, for example, capable of operation in other orientations than those illustrated or otherwise described herein.
No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include items, and may be used interchangeably with “one or more.” Furthermore, as used herein, the term “set” is intended to include items (e.g., related items, unrelated items, a combination of related items, and unrelated items, etc.), and may be used interchangeably with “one or more.” Where only one item is intended, the term “one” or similar language is used. Also, as used herein, the terms “has.” “have.” “having.” or the like are intended to be open-ended terms. Further, the phrase “based on” is intended to mean “based, at least in part, on” unless explicitly stated otherwise.
The present invention may be embodied in other specific forms without departing from its spirit or characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
As defined herein, “approximately” can, in some embodiments, mean within plus or minus ten percent of the stated value. In other embodiments, “approximately” can mean within plus or minus five percent of the stated value. In further embodiments, “approximately” can mean within plus or minus three percent of the stated value. In yet other embodiments, “approximately” can mean within plus or minus one percent of the stated value.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, health monitoring described herein are those well-known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. The nomenclatures used in connection with, and the procedures and techniques of embodiments herein, and other related fields described herein are those well-known and commonly used in the art.
By “glycoprotein” is meant by a protein which contain oligosaccharide chains (glycans) covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or post-translational modification.
The term “PSGL-1” as used herein refers to P-selectin glycoprotein ligand-1, which is a membrane protein that mediates the surface tethering and rolling of Th1 T cells for tissue migration, and has been suggested to be up-regulated by Th1 cytokines such as interferon gamma. PSGL-1, also known as SELPLG or CD162, is primarily expressed on the surface of lymphoid and myeloid cells and binds to all three members of the selectin family of proteins, P-, E-, and L-selectin. PSGL-1 is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of the endothelium to promote leukocyte migration into inflamed tissues. In a mouse model of chronic viral infection, PSGL-1 has been reported to be an immune factor regulating T-cell checkpoints. In addition, PSGL-1 serves as a receptor for enterovirus 71 (EV71) infection of leukocytes. PSGL-1 has also been shown to be an INF-γ-regulated factor involved in Th 1-mediated antiviral activity. During T-cell differentiation, culturing T cells in the Th1 cytokine INF-γ and IL-12 promoted PSGL-1 expression preferentially in the INF-γ-producing T-cell population, suggesting that PSGL-1 could be an INF-γ-regulated factor involved in Th1-mediated antiviral activity. PSGL-1 was reported to co-cluster with HIV-1 Gag at sites of assembly in the T-cell uropod.
A “selectin” is a family of cell adhesion molecules (or CAMs). Selectins comprise a family of three members (E-, P-, and L-selectin) that are differentially expressed by leukocytes and endothelial cells, and are involved in the early steps of leukocyte extravasation.
An “Interferons or IFNs” are a group of signaling proteins made and released by host cells in response to the presence of several viruses. A virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.
An “Interleukin (IL)”, any of a group of naturally occurring proteins that mediate communication between cells. Interleukins regulate cell growth, differentiation, and motility. They are particularly important in stimulating immune responses, such as inflammation.
A term “T-cell” is a type of lymphocyte, which develops in the thymus gland and plays a central role in the immune response. T-cells can be distinguished from other lymphocytes by the presence of a T-cell receptor on the cell surface.
The term “ligand” is a substance that forms a complex with a biomolecule to serve a biological purpose.
The term “CD4 (cluster of differentiation 4)” is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and \ dendritic cells.
The term “target cell” or “host cell” means a cell that is to be transformed using the methods and compositions of the invention.
The term “virus or virion” as used herein refers to a submicroscopic infectious agent that is unable to grow or reproduce outside a host cell. It is non-cellular but consisting of a core of DNA or RNA surrounded by a protein coat. A virus is a small parasite that cannot reproduce by itself. Once it infects a susceptible cell, however, a virus can direct the cell machinery to produce more viruses. Virus and virion as used herein are synonymous.
The term “pseudotyping” as used herein refers to a process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle. With this method, the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the virus particles. Pseudotyped particles do not carry the genetic material to produce additional viral envelope proteins, so the phenotypic changes cannot be passed on to progeny viral particles. For example, pseudotyping allows one to specify the character of the envelope proteins. A frequently used protein is the glycoprotein G of the Vesicular stomatitis virus (VSV), short VSV-G. These envelope proteins transduce to all cell types.
The term “virus particle or virion particle” as used herein refers to a complete infectious agent that consists of an RNA or DNA core with a protein coat sometimes with external envelopes and that is the extracellular infective form of a virus.
The term “HIV” as used herein refers to human immunodeficiency virus. Infection of subjects with HIV can result in diseases, including acquired immune deficiency syndrome (AIDS).
The term “subject” as used herein refers to a human individual. This individual could be a patient requiring prophylaxis and/or medical treatment.
The term “vaccine” as used herein refers to a suspension of antigens derived from viruses or bacteria that, upon administration, will produce active immunity and provide protection against those viruses or bacteria or related viruses or bacteria
The term “virus producing cell” as used herein refers to cell that a virus has infected and whose cell machinery the virus can direct to produce more viruses.
The term “expressing or overexpressing PSGL-1” in virus producing cells as used herein refers to causing the protein PSGL-1 to be synthesized by the cell in larger quantities than would normally be expressed by the cell on its own.
The term “non-infectious virus particle” as used herein refers to a virus particle that has been rendered unable to infect a host cell and cause one or more effects, such as disease or death of the host cell.
The term “attenuated virus particle” as used herein refers to a virus particle that has been weakened in its ability to infect a host cell and cause one or more effects, such as disease or death of the host cell.
The term “inactivated virus particle” as used herein refers to a virus particle that has been rendered unable to infect a host cell and cause one or more effects, such as disease or death of the host cell.
The term “vector” as used herein refers to an agent (i.e., a DNA construct) that can be used to introduce into a cell or organism genetic material that directs the synthesis of a protein that is encoded by the genetic material.
The term “human” as used herein refers to one that is not chimeric (e.g., not “humanized”) and not from (either in whole or in part) a nonhuman species
The term “Jurkat cells” as used herein refers to an immortalized line of human T lymphocyte cells that are used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptors susceptible to viral entry, particularly HIV.
An embodiment relates to molecular details of PSGL-1 restriction of viral such as but not limited to HIV-1 infection of human CD4 T cells.
An embodiment relates to a method for expressing a PSGL-1 or a mutant thereof in a virus producing cell; and blocking virus infection by inactivating infectivity of a released virion from the virus producing cell.
An embodiment relates to a method for expressing a PSGL-1 or a mutant thereof in a virus producing cell; and blocking virus infection by inactivating infectivity of a released virion from the virus producing cell.
An embodiment relates to the molecular details of PSGL-1 restriction of HIV-1 infection of human CD4 T cells were investigated.
An embodiment relates in a single cycle of HIV-1 infection, ectopic expression of PSGL-1 in target cells did not inhibit HIV infection.
In an embodiment, the presence of PSGL-1 in virus-producing cells inhibits the processing and incorporation of the HIV-1 envelope (Env) glycoprotein, disrupting progeny virion attachment to target cells.
In an embodiment VSV-G (glycoprotein G of the Vesicular stomatitis virus) pseudotyping of HIV-1 virions, particle binding and infectivity were blocked by PSGL-1 without reducing VSV-G incorporation into virions.
In an embodiment, PSGL-1 in virions likely blocks viral infectivity through structural hindrance of particle binding to target cells. Functional mapping of PSGL-1 demonstrated that the extracellular, N-terminal domain of PSGL-1, but not PSGL-1 dimerization, is required for its anti-HIV-1 activity.
In an embodiment, a monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV infectivity.
In an embodiment, E-selectin-binding glycoprotein CD43 blocks HIV infectivity with a potency similar to that of PSGL-1, suggesting a shared property of selectin ligands in blocking viral infectivity.
In an embodiment, PSGL-1 in addition to disrupting the binding of HIV-1 virions to target cells, PSGL-1 expression in the virus-producer cell is also capable of interfering with the infectivity of another retrovirus-MLV (murine leukemia viruses)—and the influenza A virus.
In one embodiment, PSGL-1-mediated suppression of virus infectivity extends to another retrovirus-murine leukemia virus—and to influenza A virus.
In one embodiment PSGL-1 can inactivate viruses in cell culture conditions. It was found that during the production of MLV viruses, if PSGL-1 DNA was co-transfected with MLV DNA. PSGL-1, the virions released became non-infectious. These results suggest that PSGL-1 can be used to inactivate other viral particles. The methods can be used to help the development of new methods for making live attenuated vaccines for viruses such as EAV (Equine Amemia virus) or Influenza viruses.
An embodiment relates to, a viral vaccine comprising live attenuated, inactivated, or non-infectious virus particles or virions, wherein the virus particles or virions are produced in virus producing cells, and wherein a selectin glycoprotein like PSGL-1 or CD43 or mutants thereof is expressed or overexpressed in the virus producing cells.
In an embodiment, the viral vaccine could be for human immunodeficiency virus (HIV), a murine leukemia virus (MLV), an influenza virus, EAV (Equine Amemia virus) or Influenza viruses or similar thereof.
An embodiment features a system comprising a virus vaccine comprising live attenuated, inactivated, or non-infectious viral particles.
Another embodiment features a system comprising a virus vaccine comprising live attenuated, inactivated, or non-infectious viral particles, wherein the viral particles are produced in virus producing cells, and wherein PSGL-1 is expressed or overexpressed in the virus producing cells. Furthermore, the system does not require a chemical that destroy a structure of the viral producing cells. The virus infection is a human immunodeficiency virus (HIV), a murine leukemia virus (MLV), an influenza virus or similar thereof.
An embodiment features a system comprising a virus vaccine comprising live attenuated, inactivated, or non-infectious viral particles, wherein the viral particles are produced in virus producing cells, and wherein CD43 is expressed or overexpressed in the virus producing cells. Furthermore, the system does not require a chemical that destroy a structure of the viral producing cells. The virus infection is a human immunodeficiency virus (HIV), a murine leukemia virus (MLV), an influenza virus or similar thereof.
An embodiment relates to a system capable of performing a method comprising administering a vaccine comprising live attenuated, inactivated, or non-infectious HIV particles to a subject in need of the vaccine; and treating or preventing one or more disease states in the subject resulting from HIV infection.
An embodiment relates to a system capable of performing a method comprising administering a vaccine comprising live attenuated, inactivated, or non-infectious HIV particles, wherein the HIV particles are produced in virus producing cells, and wherein PSGL-1 is expressed or overexpressed in the virus producing cells, to a subject in need of the vaccine; and treating or preventing one or more disease states in the subject resulting from HIV infection.
An embodiment relates to a method, comprising expressing or overexpressing PSGL-1 in HIV producing cells; isolating HIV particles from the HIV producing cells; and preparing the isolated HIV particles as a HIV vaccine. The HIV particles may be non-infectious, attenuated, or inactivated. Furthermore, the method does not require a chemical that destroy a structure of the HIV producing cells.
An embodiment relates to a use of PSGL-1 or CD43 in virions to block the infectivity of HIV-1 particles or other virus particles by preventing the binding of particles to target cells.
An embodiment relates to a broad-spectrum anti-viral product comprising: a vector expressing a selectin glycoprotein or like PSGL-1 or CD43 a mutant thereof in a virus producing cell; and blocking a virus infection by inactivating infectivity of a released virion from the virus producing cell.
In one embodiment PSGL-1 restricts viral infection through a novel “kill and release” mechanism, overexpression of PSGL-1 promotes virion release, and the released virions lose infectivity. PSGL-1 restricts HIV spreading through direct virion incorporation, which inactivates progeny virion attachment and entry into CD4 T cells.
In an embodiment, PSGL-1 restricts HIV infection through promoting the release of non-infectious virion particles. Given that the released virion particles are not able to attach to target cells, these particles are likely endocytosed by antigen presenting cells, and processed as antigens for MHC class II antigen presentation to stimulate anti-viral humoral immunity.
Another embodiment PSGL-1 inhibits the infectivity of VSV-G pseudo-typed lentiviruses, demonstrating that PSLG-1 potentially has broad anti-viral activity against different viruses. PSGL-1 can be used to inactivate viral infectivity for producing live-attenuated viral vaccines.
An embodiment relates to PSGL-1 restricts HIV infection through promoting the release of non-infectious virion particles. Given that the released non-infectious virion particles are not able to attach to target CD4 T cells, these particles are likely endocytosed by antigen presenting cells, and processed as antigens for MHC class II antigen presentation to stimulate anti-HIV humoral immunity.
Yet another embodiment PSGL-1 promotes the release of non-infectious virions (“kill and release”). The inactivation by PSGL-1 of virion infectivity may occur through blocking the protease processing of virion proteins. Some of the most abundant cellular proteins in the virion particles are actin and cofilin (10-15% and 2-10% of gag). HIV reverse transcriptase, nuclear capsid, and Nef have been shown to bind to actin directly. It is possible that F-actin may serve as a scuffled protein to organize the proper positioning of various virion proteins and their precursors. PSGL-1 may interfere with the organization of virion proteins and affect their proper processing by protease.
An embodiment relates to a PSGL-1, a mucin-like glycoprotein expressed on blood resting CD4 T cells, restricts HIV-1 virion infectivity. In this respect, PSGL-1-mediated restriction resembles that imposed by the Apobec3G, SERINC5, MARCH, GBP5, and 90K proteins, which also target virion infectivity. In contrast to the Vpu-antagonized restriction factor BST2/tetherin that tethers virion particles to the cell surface, PSGL-1 does not inhibit virion release, PSGL-1-imprinted virions lose the ability to attach to, and infect, target cells. PSGL-1 is a remarkably potent inhibitor of HIV-1 infectivity; it almost completely inactivated WT HIV-1 (WT is HIV derivative like Vpu) particle infectivity at a vector-to-proviral DNA ratio of 0.05. Given this high potency, it appears that HIV-1 uses two of its accessory proteins, Vpu and Nef, to antagonize PSGL-1.
An embodiment provides novel insights into the ability of the host cell to interfere with HIV-1 infection, and the biological function of lentiviral accessory proteins. Further elucidation of the mechanism by which PSGL-1 restricts HIV-1 infection may offer new therapeutic strategies for targeting HIV-1 replication.
An embodiment relates to PSGL-1, a mucin-like glycoprotein expressed on blood CD4 T cells, restricts HIV-1 virion infectivity primarily by interfering with particle binding to target cells. Human cells possess an antiviral activity that inhibits the release of retrovirus particles, and other enveloped virus particles, and is antagonized by the HIV-1 accessory protein, Vpu. This antiviral activity can be constitutively expressed or induced by interferon-α, and it consists of protein-based tethers called ‘tetherins’, that cause retention of fully formed virions on infected cell surfaces. The HIV-1 accessory protein Vpu counteracts a host factor that restricts virion release from infected cells. The interferon-induced cellular protein BST-2/HM1.24/CD317 is such a factor. BST-2 is downregulated from the cell surface by Vpu, and BST-2 is specifically expressed in cells that support the Vpu phenotype. Exogenous expression of BST-2 inhibits HIV-1 virion release, while suppression of BST-2 relieves the requirement for Vpu.
Another embodiment states that in contrast to the Vpu-antagonized restriction factor BST2/tetherin, which tethers virus particles to the cell surface, PSGL-1 does not inhibit virion release. PSGL-1 is a remarkably potent inhibitor; it almost completely inactivates WT HIV-1 particle infectivity at a vector-to-proviral DNA ratio of 0.05:1 (illustrated in
An embodiment relates to PSGL-1 expression is strongly inhibitory in our HIV-1 spreading assay, in which much of the viral transmission likely occurs through cell-to-cell contact at virological synapses. Although the effects of PSGL-1 on HIV-1 cell-cell transmission have not been studied, given that PSGL-1 is recruited to sites of viral assembly by Gag, we speculate that it is likely present and inhibitory at the virological synapse. Interestingly, it has been shown that overexpression of PSGL-1 in cells also inhibits antibody binding to multiple cell surface receptors; the molecular mechanism has been suggested to be steric hindrance of antibody access to surface molecules, resulting from the extended structure and heavy O-glycosylation of the extracellular domains of PSGL-1.
An embodiment relates to the detrimental effect of PSGL-1 in hindering particle binding to target cells, one would expect viruses to evolve strategies to remove PSGL-1 from the virion surface. HIV-1 appears to use at least two viral accessary proteins, Vpu and Nef, to downregulate PSGL-1 from the surface (illustrated in
In another embodiment, in addition to disrupting the binding of HIV-1 virions to target cells, we observed that PSGL-1 expression in the virus-producer cell is also capable of interfering with the infectivity of another retrovirus—MLV—and the influenza A virus. We and others have provided evidence that HIV-1 antagonizes PSGL-1 through the activities of Vpu and Nef. Further work will be necessary to determine whether other retroviruses and non-retroviral enveloped viruses have also acquired defense mechanisms to counteract the road block imposed by PSGL-1.
An embodiment relates to a binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized. A critical function of the chemokine coreceptor signalling in facilitating HIV infection of resting CD4 T cells. Static cortical actin in resting T cells represents a restriction, and HIV utilizes the Gαi-dependent signalling from the chemokine coreceptor CXCR4 to activate a cellular actin depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a post entry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. Viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.
An embodiment relates to a most common method utilizes reporter cell lines based on the HIV LTR (Long Terminal Repeat) promoter. The early HIV gene product Tat amplifies expression from the LTR; however, there is a background transcriptional activity that is independent of Tat. Furthermore, LTR activity can be influenced by cellular activation states. Recently constructed a Rev-dependent expression vector, and as a test of this construct's functionality, integrated this vector into a continuous T cell line. This novel indicator cell has no measurable background signal, is not affected by elevated metabolic states, and yet responds robustly to the presence of HIV. The line is able to complete TCID50 assays in 3-5 days, and appears sensitive to both CCR5- and CXCR4-utilizing viruses.
An embodiment relates to an electron microscopy of rotary-shadowed PSGL-1 purified from human neutrophils indicated that it is a highly extended molecule with an extracellular domain that is-50 nm long. Both individual PSGL-1 molecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed. The extracellular domain of PSGL-1 has 318 residues, including a signal peptide from residues 1-18 and a propeptide from residues 19-41. PSGL-1 is a long, extended molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane. This location may facilitate interactions of PSGL-1 with P-selectin under shear stress. P-selectin project its lectin domain well above the membrane to mediate optimal attachment of neutrophils under shear forces. The length of P-selectin may: (a) facilitate interactions with PSGL-1 on flowing neutrophils, and (b) increase the intermembrane distance where specific bonds form, minimizing contacts between the glycocalyces that result in cell-cell repulsion. PSGL-1 amino acid sequence of the transmembrane and cytoplasmic domains are well conserved and that, despite a poor conservation of PSGL-1 N-terminus, L- and P-selectin binding sites are evolutionary conserved. Functional assays reveal a critical role for post-translational modifications in regulating mammalian PSGL-1 interactions with selectins.
An embodiment relates to a PSGL-1 extracellular domain contains decameric repeats, which extend L- and P-selectin binding sites far above the plasma membrane. Decamers may play a role in regulating PSGL-1 interactions with selectins. Deletion of decamers abrogated soluble L-selectin binding and cell rolling on L-selectin, whereas their substitution partially reversed these diminutions. P-selectin-dependent interactions with PSGL-1 were less affected by decamer deletion. Decamers are required to stabilize L-selectin-dependent rolling. Importantly, adhesion assays performed on recombinant decamers demonstrated that they directly bind to E-selectin and promote slow rolling. The role of decamers is to extend PSGL-1 N terminus far above the cell surface to support and stabilize leukocyte rolling on L- or P-selectin. In addition, they function as a cell adhesion receptor, which supports approximately 80% of E-selectin-dependent rolling.
An embodiment relates to a selectins are carbohydrate-binding molecules involved in constitutive lymphocyte homing and chronic and acute inflammation processes. Th1 lymphocytes participate in cell-mediated inflammatory reactions, where the selectins play a role and predominate in delayed-type hypersensitivity (DTH) reactions of the skin. The glycosylated form of CD43 expressed in Th1 cells is a functional E-selectin-specific ligand in vitro. The relevance of CD43 as an E-selectin ligand in vitro and in vivo. Both PSGL-1 and CD43 are major E-selectin ligands and are likely to be important during leukocyte recruitment in the development of inflammatory reactions.
An embodiment relates to a glycosylation affects many essential T cell processes and is intrinsically controlled throughout the lifetime of a T cell. CD43 and CD45 are the two most abundant glycoproteins on the T cell surface and are decorated with O- and N-glycans. Global T cell glycosylation and specific glycosylation of CD43 and CD45 are modulated during thymocyte development and T cell activation; T cells control the type and abundance of glycans decorating CD43 and CD45 by regulating expression of glycosyltransferases and glycosidases. Additionally, T cells regulate glycosylation of CD45 by expressing alternatively spliced isoforms of CD45 that have different glycan attachment sites. The glycophenotype of CD43 and CD45 on T cells influences how T cells interact with the extracellular environment, including how T cells interact with endogenous lectins. The CD43 expressed on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.
In an embodiment, HIV-1 infection, or expression of either Vpu or Nef, downregulates 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. PSGL-1 inhibits the infectivity of other viruses such as murine leukemia virus and influenza A virus. PSGL-1 is a broad-spectrum antiviral host factor with a novel mechanism of action.
Cells and Viruses
Peripheral blood buffy coats from HIV-1-negative adults were purchased from the New York Blood Center or received from the NIH Blood Bank. CD4+ T cells were isolated by negative selection using the Dynabeads Untouched magnetic separation kit (Invitrogen) or as previously described. CD4+ T cells were cultured in RPMI 1640 plus 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin (Invitrogen). Resting CD4 T cells were activated by culturing in PHA (2 μg/ml) plus IL-2 (2 ng/ml) (PepTech). HEK293T cells (ATCC) and HeLaJC.53 cells (kindly provided by Dr. David Kabat) were maintained in Dulbecco-modified Eagle's medium (DMEM) (Invitrogen) containing 10% FBS and 1× penicillin-streptomycin (Invitrogen). PSGL-1-HeLaJC53 and Empty-HeLaJC53 cells were cultured in DMEM supplemented with 10% FBS and 550 μg/ml hygromycin B (Invitrogen). TZM-bl cells (Dr. John C. Kappes, and Dr. Xiaoyun Wu were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. TZM-bl cells were cultured in DMEM containing 10% FBS and 1× penicillin-streptomycin (Invitrogen). Jurkat cells (NIH AIDS Reagent Program) were cultured in RPMI 1640 supplemented with 2 mM L-glutamine. 10% FBS and 1× penicillin-streptomycin (Invitrogen). HIV Rev-dependent GFP indicator cells Rev-A3R5-GFP (Virongy) were cultured in RPMI 1640 plus 10% FBS supplemented with 1 μg/ml G418 (Sigma-Aldrich) and 1 μg/ml puromycin (Sigma-Aldrich). A3R5.7 cells (NIH AIDS Reagent Program) were cultured in RPMI-1640 containing 10% FBS. 1% L-Glutamine, 1× penicillin-streptomycin, and 1 μg/mL G418 (Invitrogen). CEM-SS cells (NIH AIDS Reagent Program) were cultured in RPMI-1640 with 10% FBS. To construct PSGL-1-HeLaJC53 cells, HeLaJC.53 cells were seeded into a 6-well plate and cultured in DMEM with 10% FBS. Cells were transfected with 2 μg pCMV3-PSGL-1 or pCMV3-Empty DNA using Jetprime transfection reagent (Polyplus) as recommended by the manufacturer. Transfected cells were cultured and selected with DMEM containing 10% FBS and 550 μg/ml of hygromycin B (Invitrogen) to generate stably transfected cells.
Plasmids, Vectors, Transfection, and Virion Production and Purification
The infectious HIV-1 molecular clone pNL4-3, codon-optimized Vpu expression vector (pcDNA-Vphu). Nef expression vector (pNef-ER), and NL4-3 ΔVpu infectious molecular clone (pNL-U35) were obtained from the NIH AIDS Reagent Program, pCMV3-PSGL-1 and pCMV3-Empty vectors were obtained from Sinobiological, pRetroPSGL-1-NT, pRetroPSGL-1-CT, and pRetroPSGL-1 were synthesized and cloned into pMSCVneo vector by GeneScript, pPSGL-1-3A, pPSGL-1-6A, pPSGL-1-C310A, pPSGL-1-ACT, and pPSGL-1(Wt) were kindly provided by Dr. Akira Ono, pPSGL-1ΔDR was kindly provided by Drs. Caroline Spertini and Olivier Spertini. pCMV3-CD43 was obtained from Sinobiological, pSV-ψ-MLV-env- was from NIH AIDS Reagent Program, pNLΔΨEny (gp160) and pHCMV-G expressing the HIV-1 Env and the vesicular stomatitis virus G glycoprotein, respectively, were described previously. The GFP-expressing retroviral vector pRetroQ-AcGFP1-N1 was obtained from Clontech, pNL4-3ΔNef was described previously. The env-defective pNL4-3 derivative pNL4-3/KFS was described previously.
The procedure for transfection of HEK293T cells to produce HIV-1 particles was described previously. For GFP reporter MLV particle assembly, pRetroQ-AcGFP1-N1 (0.5 μg), pSV-ψ-MLV-env-(0.375 μg), and pHCMV-G (0.125 μg) were co-transfected with either pCMV3-PSGL-1 or pCMV3-Empty vector (at indicated dosages) in a 6-well plate. Virus supernatants were collected at 48 hours post transfection. For transient transfection of HeLaJC.53 cells. 0.5 million cells were transfected with 2 μg of either pCMV3-Empty or pCMV-PSGL-1 using the transfection reagent Jetprime (Polyplus) as recommended by the manufacturer. Following transfection, cells were cultured for the indicated times until analysis. For the p24 release assay in HEK293T cells, cells were cotransfected with 1 μg of HIV-1 NL4-3 and indicated doses of pCMV3-PSGL-1 or pCMV3-Empty DNA using Lipofectamine 2000 (Invitrogen). Supernatant was collected at 48 hours post-transfection. To purify virions by ultracentrifugation, supernatants harvested from transfected HEK293T cells first filtered through a 0.45 μm filter, then concentrated by Vivaspin20 concentrator. Concentrated viruses were purified by ultra-speed centrifugation through a gradient of 6-18% OptiPrep solution (Sigma-Aldrich) (40.000 rpm for 2 hours, SW41Ti rotor from Beckman), followed by a second round of ultracentrifugation to pellet the virus (20,000 rpm for 1.5 hours, SW41Ti).
FACS Analysis
For PSGL-1 surface staining, 0.5-1 million cells were stained with anti-PSGL1 antibody (KPL-1) (BD Pharmingen) followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen). For surface staining of infected blood resting CD4+ T cells, HIV-1 infection was done using 125 ng to 320 ng p24 gag equivalents of NLENG1-ES-IRES GPF reporter virus (pseudotyped with HIV-1 NL4-3 envelope) per million cells. Cells were washed and cultured in 10% FBS RPMI with IL-7 (2 ng/mL). On the indicated days, cells were harvested and stained at 4° C., for 30 min with AF687 anti-PSGL-1 antibody (KPL-1, BD Pharmingen) and analyzed by flow cytometry. For surface PSGL-1 staining of Jurkat, CEMSS, and A3R5.7 cells. 0.5 million cells were stained with FITC-conjugated anti-PSGL-1 antibody (Abcam) and analyzed by flow cytometry. For HIV-1-infected Jurkat T-cell surface staining. 0.5 million cells were infected with different volumes of HIV-1 NL4-3. At 3 days post infection, cells were stained with anti-PSGL-1 antibody [KPL-1] (BD Pharmingen), followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen) and flow cytometry analysis. For HEK293T cells. 0.5 million cells were co-transfected with different dosages (1 μg to 4 μg) of HIV NL4-3 Vpu or HIV NL4-3 Nef, and 100 ng of pCMV3-PSGL-1 using Lipofectamine 2000 (Invitrogen). Cells were stained at 48 hours post-transfection with anti-PSGL-1 antibody [KPL-1] (BD Pharmingen), followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen).
Western Blots
The following antibodies were from the NIH AIDS Reagent Program: anti-HIV-1 p24 monoclonal antibody (183-H12-5C), anti-HIV Env (16H3) antibody, anti-HIV-1 gp41 monoclonal antibody (2F5), anti-HIV-1 gp41 monoclonal antibody (10E8), and anti-HIV immune globulin (HIVIG). Cells or virus pellets were solubilized in lysis buffer containing 50 mM Tris-HCl (pH 7.4). 150 mM NaCl. 1 mM EDTA. 0.5% Triton X-100, and protease inhibitor cocktail (Roche Life Science. Basel, Switzerland) or LDS lysis buffer (Invitrogen).
Proteins were denatured by boiling in sample buffer and subjected to SDS-PAGE, transferred to PVDF or nitrocellulose membrane, and incubated overnight at 4° C., with one of the following primary antibodies: anti-PSGL-1 monoclonal antibody (clone KPL-1, BD Pharmingen) (1:1000 dilution): anti-PSGL-1 TC-2 monoclonal antibody (Abcam) (1:1000 dilution); anti-PSGL-1 polyclonal antibody (Abcam) (1:1000 dilution); anti-PSGL-1-C terminal polyclonal antibody (anti-PSGL-1 amino acid 350) to the C-terminus) (Abcam) (1 μg/ml): anti-GAPDH goat polyclonal antibody (Abcam) (1:1000 dilution); anti-CD45RA antibody (BD Biosciences); anti-HIV envelope antibodies (183-H12-5C, 16H3, 2F5), HIVIG, and anti-CD63 polyclonal antibody (System Biosciences) (1:1000 dilution). Membranes were then incubated with HRP-labeled goat anti-mouse IgG (KPL) (1:2500 dilution) or anti-rabbit IgG (Cell Signaling) (1:2000 dilution) for 60) min at room temperature. Chemiluminescence signal was detected by using West Pico or West Femto chemiluminescence reagent (Thermo Fisher Scientific). Images were captured with a CCD camera (FluorChem 9900 Imaging Systems) (Alpha Innotech). Protein bands were also quantified using Imagelab-Chemidoc (Bio-Rad Laboratories, France). On some occasions, western blot was also performed using infrared imaging (Odyssey infrared imager. LI-cor Biosciences) with IRDye goat anti-mouse or rabbit 680 or 800 cw labeled antibodies (Li-corBiosciences) (1:5000 diluted in blocking buffer) for 1 h at 4° C. The blots were washed three times for 15 minutes and scanned with Odyssey Infrared Imager (Li-cor Biosciences). The ratios of gp120/p24 and gp160/p24 were quantified in virions, and the ratio of gp120/gp160 and expression of PSGL-1 were quantified in cell and virus fractions. To quantify virus release efficiency. HEK293T cells were transfected with the indicated plasmids (WT pNL4-3, pNL43ΔVpu, or pNL43ΔNef) in the absence or presence of PSGL-1 expression vector using Lipofectamine 2000 (Invitrogen) or polyethylenimine (PEI) transfection reagent (Sigma-Aldrich). At 30 to 48 hours after the addition of DNA, virus-containing supernatant was harvested for p24 ELISA, or filtered and pelleted in an ultracentrifuge for analysis. The viral release efficiency (VRE) was calculated as the amount of virion-associated Gag as a fraction of total (cell- and virion-associated) Gag quantified from Western blot analysis.
p24 ELISA
HIV-1 p24 released into the cell culture supernatant was detected by an in-house p24 ELISA kit. Briefly, microtiter plates (Sigma-Aldrich) were coated with anti-HIV-1 p24 monoclonal antibody (183-H12-5C) (NIH AIDS Reagent Program). Samples were incubated for 2 hours at 37° C., followed by washing and incubating with biotinylated anti-HIV immune globulin (HIVIG) (NIH AIDS Reagent Program) for 1 hour at 37° C. Plates were then washed and incubated with avidinperoxidase conjugate (R&D Systems) for 1 hour at 37° C., followed by washing and incubating with tetramethylbenzidine (TMB) substrate. Plates were kinetically read using an ELx808 automatic microplate reader (Bio-Tek Instruments) at 630 nm.
Viral Entry Assay (BLAM Assay)
The viral entry assay was performed as previously described. Briefly, viruses were generated by co-transfection of HEK293T cells with three plasmids: pNL4-3, pAdvantage (Promega) and pCMV4-3BlaM-Vpr (kindly provided by Dr. Warner C. Greene) (at a ratio of 6:1:2). Supernatant was harvested at 48 hours post-transfection, concentrated, and then used for infection as suggested. Flow cytometry was performed using a Becton Dickinson LSR II (Becton Dickinson). B-lactamase and CCF2 measurements were performed using a 407-nm violet laser with emission filters of 525/50 nm (green fluorescence) and 440/40 nm (blue fluorescence), respectively. Green and blue emission spectra were separated using a 505LP dichroic mirror. The UV laser was turned off during the analysis. Data analysis was performed using FlowJo software (FlowJo).
Viral Attachment Assay
Virion particles produced in the presence of PSGL-1 or the empty vector were incubated with HelaJC53 cells (pre-chilled at 4° C., for 1 hour) at 4° C., for 2 hours. The cells were then washed extensively (5 times) with cold PBS buffer and then lysed with LDS lysis buffer (Invitrogen) for analysis by Western blot.
Infectivity Assays
For flow cytometry-based infectivity assay, virus particles were produced in HEK293T cells by co-transfection with pNL4-3, pNL4-3ΔVpu, or pNL4-3ΔNef with pCMV3-PSGL-1, pCMVCD43, or pCMV3-Empty, or by cotransfection with pNL4-3/KFS, pHCMV-G, and pCMV3-PSGL-1 or pCMV3-Empty vector (using the indicated plasmid inputs) in a 6-well plate with Lipofectamine 2000 (Invitrogen). Viral particles were also produced in CEM-SS cells by electroporation. Briefly, CEM-SS cells (2 millions) were electroporated with pNL4-3 (2 μg) plus 400 ng of pCMV3-PSGL-1 or pCMV3-Empty using a T cell electroporation kit (Lonza). Viral particles were harvested at 3 days post electroporation. Rev-A3R5-GFP cells were infected with each of the indicated viruses (0.2-0.5 million cells/infection). The cells were then washed and cultured in fresh media. Flow cytometry analysis of GFP expression was performed on the indicated days. The percentage of GFP+ cells was quantified.
For luciferase-based, single-cycle infectivity assays, RT-normalized virus stocks were used to infect the CD4+/CXCR4+/CCR5+HeLa derivative TZM-bl. This indicator cell line contains integrated copies of the B-galactosidase and luciferase genes under the control of the HIV-1 LTR. Infection efficiency was determined by measuring luciferase activity 2 days post-infection. For infectivity assays in HeLa JC53-PSGL-1 and HeLa-JC53-empty cell lines, the cells were seeded in 6-well plates at a density of 0.2×106/well 24 hours prior to infection. Cells were infected with the indicated p24 equivalents of either WT NL4-3. NL43ΔVpu, or NL43ΔNef. Viral replication was quantified by virion p24 released into the medium by p24 ELISA. For MLV (murine leukemia virus) virion infectivity. MLV-GFP reporter virus was assembled by co-transfecting HEK293T cells (in 6-well plate) with pSV-Ψ-MLV-env-(0.375 μg), pRetroQAcGFP1-N1 (0.5 μg), pHCMV-G (0.125 μg), and pCMV3-PSGL1 or an empty vector at the indicated dosages. An equal amount of DNA was used all transfections. Viral supernatants were harvested 48 h post transfection and used to infect HEK293T cells for 6 hours in the presence of Infectin (Virongy, Manassas, VA). Cells were washed to remove virus and Infectin, and cultured for 48 hours for flowcytometry analyses.
To determine the effect of PSGL-1 on influenza A virus replication, HEK293T and MDCK cells were co-cultured at approximately 70% confluence in 6-well plates and transfected with either empty vector or pCMV3-PSGL-1 (both vectors at 1.0 or 3.0 μg), together with an eight plasmid influenza A/WSN/33 reverse genetics system (RGS) system (1.0 μg of each plasmid). Transfection reaction was prepared with PEI (polyethylenimine). Culture supernatants were collected at 16 and 24 h post-transfection and titrated in MDCK cells to determine end-point titers (TCID50 per ml).
shRNA Knockdown of PSGL-1
Lentiviral vectors carrying shRNAs against PSGL-1 or non-target control (NTC) (Sigma MISSION shRNA. PSGL-1 TRCN0000436811 or shRNA NTC) were purchased from Sigma. Virion particles were assembled by cotransfecting HEK293T cells with 0.5 μg of pHCMV-G. 1.5 μg pCMV-AR8.2, and 2 μg of lentiviral vectors using Lipofectamine 3000 (Invitrogen). Supernatant was collected at 48 hours post co-transfection, and then filtered through 0.45 um filter. Virion particles were used to transduce Jurkat T cells for 6 hours. Cells were then washed twice and cultured in fresh media for 3 days, and then selected in puromycin (4 μg/ml) for one to two weeks. PSGL-1 knockdown was confirmed by surface staining with an anti-PSGL-1 antibody (KPL-1) (BD Pharmingen) followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen). PSGPL-1 knockdown or NTC control cells (2×106) were also transfected with 2 μg of HIV-1(NL4-3) DNA by electroporation using a T cell electroporation kit (Lonza). Viruses were harvested and used for the infection of Rev-A3R5-GFP cells (20 ng p24 per infection). Flow cytometry analysis of GFP expression was performed on the indicated days. Lentiviral vector-mediated ShRNA knockdown of PSGL-1 in primary CD4 T-cells was performed as described previously. Briefly, blood resting CD4 T cells were purified by negative depletion, transiently stimulated with anti-CD3/CD28 beads (1-2 beads per cell) for 12 hours, and then transduced with the lentiviral vectors carrying shRNAs against PSGL-1 or non-target control (NTC) (Sigma MISSION shRNA. PSGL-1 TRCN0000436811 or shRNA NTC). Following transduction, the beads were removed at 12 hours, and cells were cultured for 3 days, and then analyzed from surface PSGL-1 expression. Cells were also subsequently transfected with HIV-1(NL4-3) DNA by electroporation using a T-cell electroporation kit (Lonza). HIV-1 viral replication was monitored by harvesting cell culture supernatant, and HIV p24 was detected by an in-house p24 ELISA kit.
The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
PSGL-1 is expressed on primary CD4 T cells and elicits anti-HIV activity in HeLa JC.53 cells. To study the mechanisms of PSGL-1-mediated restriction of HIV-1 replication, we first quantified PSGL-1 expression on both primary blood CD4 T cells and transformed T-cell lines. We observed that human blood resting CD4 T cells express high levels of PSGL-1, and T-cell activation (IL-2 plus PHA) down-regulates PSGL-1 (illustrated in
It is possible that HIV infection of primary resting CD4 T cells may downregulate PSGL-1 as seen in transformed T-cell lines (illustrated in
Vpu has been identified as a viral factor that mediates intracellular PSGL-1 degradation. However, both Vpu and Nef are known broad-spectrum modulators of cell-surface receptors. Thus, we also examined the role Nef in HIV-mediated PSGL-1 downregulation, and observed a dose-dependent downmodulation of surface PSGL-1 levels by both Vpu and Nef, consistent with previous reports (illustrated in
To confirm the anti-HIV activity of PSGL-1, we established PSGL-1-stably transfected HeLa JC.53 cells (HeLaJC53-PSGL-1) (illustrated in
Given the observed differences between Vpu and Nef in downregulating PSGL-1 (illustrated in
PSGL-1 does not block viral release but inactivates virion infectivity. PSGL-1 has been suggested to block viral reverse transcription early post-infection, and inactivate virion infectivity late in the replication cycle. Thus, we examined the role of PSGL-1 in a single HIV-1 replication cycle. We infected PSGL-1-stably transfected HeLa JC.53 cells (HeLaJC53-PSGL-1) or the control cells (HeLaJC53-Vector) with an HIV-1 Env-pseudotyped, single-cycle virus. HIV (gp160) (illustrated in
We observed small effects of PSGL-1 expression on HIV-1 virion release, from slight enhancement at low doses (below: 100 ng) to no effect at high doses (200 and 400 ng). When normalized to the levels of intracellular Gag. PSGL-1 did not inhibit virion release at any dose tested (illustrated in
The potent inactivation of HIV-1 infectivity was confirmed by quantifying HIV infectivity in the HeLa cell-derived TZM-bl cell line (illustrated in
To further examine the role of endogenous PSGL-1 in virus replication, we performed lentiviral vector-mediated stable shRNA knockdown of PSGL-1 in Jurkat T cells, which express low levels of PSGL-1 (illustrated in
The levels of PSGL-1 on Jurkat are comparable with those on activated primary blood CD4 T cells (illustrated in
PSGL-1 blocks virion attachment to target cells. To examine whether PSGL-1 expression in the producer cell, and its incorporation into virus particles (illustrated in
The HIV-1 entry inhibitor AMD3100 provided a positive control for entry inhibition. The results indicated that PSGL-1-imprinted virus particles were severely impaired in their ability to enter target cells (illustrated in
We also performed a virion attachment assay, and observed that PSGL-1-imprinted virions bearing either VSV-G or HIV-1 Env were impaired in their ability to attach to target cells (illustrated in
The extracellular, N-terminal domain of PSGL-1 is required for blocking HIV-1 infectivity. Structurally, PSGL-1 has a relatively rigid and elongated extracellular domain that extends nearly 60 nm from the cell surface. To test the hypothesis that the presence of PSGL-1 on virus particles may sterically hinder the binding of particles to target cells (illustrated in
Cellular expression and virion incorporation of PSGL-1 mutants were confirmed by surface staining and western blots (illustrated in
We further tested a panel of previously studied PSGL-1 mutants (illustrated in
An additional mutant. PSGL-1-C310A (sequence no. 7), abolishes PSGL-1 dimerization. Given that the cytoplasmic tail mutations may decrease virion incorporation of PSGL-1, we used a high amount of the mutant PSGL-1 DNA (500 ng) to co-transfect with HIV-1 DNA (1 μg) to ensure that even with a lower level of virion incorporation of PSGL-1, it would still be sufficient to confer a restrictive phenotype. Using this high PSGL-1 dose, we were able to demonstrate that all the PSGL-1 C-terminal mutants, along with the dimerization mutant C310A (sequence no. 7), maintained their capacity to restrict HIV-1 (illustrated in
PSGL-1 is a broad-spectrum antiviral host factor. Given that the PSGL-1 intracellular domain deletion mutant, PSGL-1-ACT, effectively blocks HIV-1 infectivity in the absence of coclustering with HIV-1 Gag, it is possible that PSGL-1 does not rely on specific interactions with an HIV-1 protein to restrict particle infectivity. Indeed. PSGL-1 inhibition of viral attachment to cells is independent of the presence of viral envelope protein (illustrated in
PSGL-1-related selectin ligand CD43 also restricts HIV infectivity. Given that PSGL-1 restriction of HIV infectivity does not require its dimerization (illustrated in
The embodiments describe a new approach to producing vaccines or broad spectrum anti-viral products comprising live attenuated, inactivated, or non-infectious virion particles, such as HIV particles. Other embodiments are also within the scope of the following claims.
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/828,381, entitled “THE USE OF PSGL-1 (P-SELECTIN GLYCOPROTEIN LIGAND-1) TO INACTIVATE ALL ENVELOPED VIRUSES FOR PRODUCING LIVE-ATTENUATED VACCINES.” filed Apr. 2, 2019, the disclosure of which is hereby incorporated by reference herein in its entirety. This application is related to U.S. patent application Ser. No. 16/271,100, filed Feb. 8, 2019, entitled “METHOD AND SYSTEM FOR INACTIVATING VIRUS INFECTIVITY FOR PRODUCING LIVE-ATTENUATED VACCINES.” which is incorporated herein in its entirety by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 11116833 | Wu | Sep 2021 | B2 |
| 11382967 | Wu | Jul 2022 | B2 |
| Entry |
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| Fu, Y., et al., Apr. 2020, PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells, PNAS 117(17):9537-9545. |
| He, S., et al., 2021, PSGL-1 inhibits the incorporation of SARS-CoV and SARS-CoV-2 spike glycoproteins into pseudovirions and impairs pseudovirus attachment and infectivity, Viruses 13, 46, p. 1-8. |
| Number | Date | Country | |
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| 20200316190 A1 | Oct 2020 | US |
| Number | Date | Country | |
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| 62828381 | Apr 2019 | US |
