scholarly journals Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells

2021 ◽  
Vol 17 (1) ◽  
pp. e1009190
Author(s):  
Gregory A. Sowd ◽  
Christopher Aiken
Keyword(s):  
T Cells ◽  
Particle Production ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Virus Production ◽  
Particle Assembly ◽  
Gag Protein ◽  
Viral Spread ◽  
Hiv 1

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication.

scholarly journals Kinesin KIF4 Regulates Intracellular Trafficking and Stability of the Human Immunodeficiency Virus Type 1 Gag Polyprotein

2008 ◽  
Vol 82 (20) ◽  
pp. 9937-9950 ◽  
Author(s):  
Nathaniel W. Martinez ◽  
Xiaoxiao Xue ◽  
Reem G. Berro ◽  
Geri Kreitzer ◽  
Marilyn D. Resh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Particle Assembly ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.

scholarly journals Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kaushik Inamdar ◽  
Feng-Ching Tsai ◽  
Rayane Dibsy ◽  
Aurore de Poret ◽  
John Manzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Particle Assembly ◽  
Gag Protein ◽  
Cell Plasma ◽  
Curved Membranes ◽  
Hiv 1

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. SiRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

scholarly journals Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release

2020 ◽  
Author(s):  
James Kirui ◽  
Eric Freed
Keyword(s):  
Virus Particle ◽  
High Throughput Screening ◽  
Particle Production ◽  
Health Concern ◽  
Reporter Virus ◽  
Particle Assembly ◽  
Gag Protein ◽  
Minimal Impact ◽  
Particle Release ◽  
Hiv 1

Abstract Background The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. Results The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. Conclusion These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small-molecules that promote or inhibit HIV-1 particle production.

Structural determinants of virion assembly and release in the C-terminus of the M-PMV capsid protein

2021 ◽  
Author(s):  
Marlene V. Buckmaster ◽  
Kaneil K. Zadrozny ◽  
Barbie K. Ganser-Pornillos ◽  
Owen Pornillos ◽  
Stephen P. Goff
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structural Determinants ◽  
Particle Assembly ◽  
Gag Protein ◽  
Virion Assembly ◽  
C Terminus ◽  
Hiv 1

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located downstream of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the ‘clasp motif’, important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and Mason-Pfizer Monkey Virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide comparable function. The importance of the sequences spanning the CA-NC cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant protein in vitro , and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant virus in vivo . The mutants revealed major defects in virion assembly and release in 293T and HeLa cells, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. Importance The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short ‘clasp’ motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.

scholarly journals The Conserved Tyr176/Leu177 Motif in the α-Helix 9 of the Feline Immunodeficiency Virus Capsid Protein Is Critical for Gag Particle Assembly

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 816
Author(s):  
César A. Ovejero ◽  
Silvia A. González ◽  
José L. Affranchino
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Particle Production ◽  
Equine Infectious Anemia Virus ◽  
Particle Assembly ◽  
Immunodeficiency Virus ◽  
Immature Particle ◽  
Gag Assembly ◽  
Hiv 1

The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag–Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices. Structural studies performed in HIV-1 indicate that the CA-CTD helix 9 establishes homodimeric interactions that contribute to the formation of the hexameric Gag lattice in immature virions. Interestingly, the mature CA core also shows inter-hexameric associations involving helix 9 residues W184 and M185. The CA proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) exhibit, at equivalent positions in helix 9, the motifs Y176/L177 and L169/F170, respectively. In this paper, we investigated the relevance of the Y176/L177 motif for FIV assembly by introducing a series of amino acid substitutions into this sequence and studying their effect on in vivo and in vitro Gag assembly, CA oligomerization, mature virion production, and viral infectivity. Our results demonstrate that the Y176/L177 motif in FIV CA helix 9 is essential for Gag assembly and CA oligomerization. Notably, mutations converting the FIV CA Y176/L177 motif into the HIV-1 WM and EIAV FL sequences allow substantial particle production and viral replication in feline cells.

scholarly journals Kinetic Analysis of Human Immunodeficiency Virus Type 1 Assembly Reveals the Presence of Sequential Intermediates

2000 ◽  
Vol 74 (13) ◽  
pp. 5845-5855 ◽  
Author(s):  
Marc Tritel ◽  
Marilyn D. Resh
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Particle Production ◽  
Particle Assembly ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Membrane Bound ◽  
Hiv 1

ABSTRACT The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag protein precursor, but the molecular details of these processes remain poorly defined. In this study, we have combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in the lentivirus assembly process. We show that newly synthesized HIV-1 Gag rapidly forms cytoplasmic protein complexes that are resistant to detergent treatment, sensitive to protease digestion, and degraded intracellularly. A subpopulation of newly synthesized Gag binds membranes within 5 to 10 min and over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on Optiprep density gradients. These complexes likely represent assembly intermediates because they are not observed with assembly-defective Gag mutants and can be chased into extracellular viruslike particles. At steady state, nearly all of the Gag is present as membrane-bound complexes in various stages of assembly. The identification of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via an ordered process. Assembly intermediates should serve as attractive targets for the design of antiviral agents that interfere with the process of particle production.

scholarly journals HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory T cells

2021 ◽  
Author(s):  
Ann-Kathrin Reuschl ◽  
Maitreyi Shivkumar ◽  
Dejan Mesner ◽  
Laura J. Pallett ◽  
José Afonso Guerra-Assunção ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Biology ◽  
Memory T Cells ◽  
Virus Production ◽  
Cell Loss ◽  
Productive Infection ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) replicates in CD4+ T cells leading to profound T cell loss, immunological dysfunction and AIDS. Determining how HIV-1 shapes the immunological niche in which it resides to create a permissive environment is central to informing efforts to limit pathogenesis, disturb viral reservoirs and achieve a cure. A key roadblock in understanding HIV-T cell interactions is the requirement to activate CD4+ T cells in vitro in order to make them permissive to infection. This dramatically alters T cell biology, obscuring native virus-host interactions. Here we show that HIV-1 cell-to-cell spread permits efficient and productive infection of resting CD4+ T cells without the need for prior activation. Infection is preferential for resting memory T cells, is observed with both CXCR4-tropic virus and CCR5-tropic transmitter-founder viruses and results in virus production and onward spreading infection. Strikingly, we find that HIV-1 infection of resting memory CD4+ T cells primes for induction of a tissue-resident memory (TRM)-like phenotype evidenced by upregulation of TRM markers CD69/CXCR6 alongside co-expression of CD49a, PD-1, CD101 as well as transcription factor Blimp-1. Furthermore, we reveal that HIV-1 initiates a transcriptional program that overlaps with the core TRM transcriptional signature. This reprograming depends on the HIV-1 accessory protein Vpr. We propose that HIV-1 infection drives a CD4+ TRM-phenotype potentially sequestering infected cells within tissues to support viral replication and persistence.

scholarly journals Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53

2021 ◽  
Author(s):  
Kaushik Inamdar ◽  
Feng-Ching Tsai ◽  
Aurore de Poret ◽  
Rayane Dibsy ◽  
John Manzi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Particle Production ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Particle Assembly ◽  
Gag Protein ◽  
Cell Plasma ◽  
Curved Membranes ◽  
Hiv 1

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. Partial gene editing of IRSp53 induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein localizes preferentially to IRSp53 I-BAR domain induced curved membranes on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

scholarly journals Modulation of Susceptibility to HIV-1 Infection by the Cytotoxic T Lymphocyte Antigen 4 Costimulatory Molecule

1999 ◽  
Vol 191 (11) ◽  
pp. 1987-1998 ◽  
Author(s):  
James L. Riley ◽  
Katia Schlienger ◽  
Patrick J. Blair ◽  
Beatriz Carreno ◽  
Nancy Craighead ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocyte ◽  
Cd4 T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Costimulatory Molecule ◽  
Lymphocyte Antigen ◽  
Viral Spread ◽  
Hiv 1

CD4 T cells activated in vitro by anti-CD3/28–coated beads are resistant to infection by CC chemokine receptor 5 (CCR5)-dependent HIV-1 isolates. In vivo, antigen-presenting cells (APCs) activate CD4 T cells in part by signaling through the T cell receptor and CD28, yet cells stimulated in this manner are susceptible to HIV-1 infection. We show that cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement counteracts the CD28 antiviral effects, and that the ratio of CTLA-4 to CD28 engagement determines the susceptibility of HIV-1 infection. Furthermore, unopposed CTLA-4 signaling provided by CD28 blockade promotes vigorous HIV-1 replication, despite minimal T cell proliferation. Finally, CTLA-4 antibodies decrease the susceptibility of antigen-activated CD4 T cells to HIV, suggesting a potential approach to prevent or limit viral spread in HIV-1–infected individuals.

scholarly journals Generation and validation of a highly sensitive bioluminescent HIV-1 reporter vector that simplifies measurement of virus release

2020 ◽  
Author(s):  
James Kirui ◽  
Eric Freed
Keyword(s):  
Virus Particle ◽  
High Throughput Screening ◽  
Particle Production ◽  
Health Concern ◽  
Reporter Virus ◽  
Particle Assembly ◽  
Gag Protein ◽  
Minimal Impact ◽  
Particle Release ◽  
Hiv 1

Abstract Background The continued persistence of HIV-1 as a public health concern due to the lack of a cure calls for the development of new tools for studying replication of the virus. Here, we used NanoLuc, a small and extremely bright luciferase protein, to develop an HIV-1 bioluminescent reporter virus that simplifies functional measurement of virus particle production. Results The reporter virus encodes a Gag protein containing NanoLuc inserted between the matrix (MA) and capsid (CA) domains of Gag, thereby generating virus particles that package high levels of the NanoLuc reporter. We observe that inserting the NanoLuc protein within HIV-1 Gag has minimal impact on Gag expression and virus particle release. We show that the reporter virus recapitulates inhibition of HIV-1 particle release by Gag mutations, the restriction factor tetherin, and the small-molecule inhibitor amphotericin-B methyl ester. Conclusion These results demonstrate that this vector will provide a simple and rapid tool for functional studies of virus particle assembly and release and high-throughput screening for cellular factors and small-molecules that promote or inhibit HIV-1 particle production.

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