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1.
Viruses ; 16(6)2024 May 26.
Article in English | MEDLINE | ID: mdl-38932142

ABSTRACT

HIV-1 protease inhibitors are an essential component of antiretroviral therapy. However, drug resistance is a pervasive issue motivating a persistent search for novel therapies. Recent reports found that when protease activates within the host cell's cytosol, it facilitates the pyroptotic killing of infected cells. This has led to speculation that promoting protease activation, rather than inhibiting it, could help to eradicate infected cells and potentially cure HIV-1 infection. Here, we used a nanoscale flow cytometry-based assay to characterize protease resistance mutations and polymorphisms. We quantified protease activity, viral concentration, and premature protease activation and confirmed previous findings that major resistance mutations generally destabilize the protease structure. Intriguingly, we found evidence that common polymorphisms in the hinge domain of protease can influence its susceptibility to premature activation. This suggests that viral heterogeneity could pose a considerable challenge for therapeutic strategies aimed at inducing premature protease activation in the future.


Subject(s)
Drug Resistance, Viral , HIV Infections , HIV Protease , HIV-1 , Polymorphism, Genetic , HIV Protease/genetics , HIV Protease/metabolism , HIV-1/genetics , HIV-1/drug effects , HIV-1/enzymology , Humans , Drug Resistance, Viral/genetics , HIV Infections/virology , HIV Infections/drug therapy , HIV Infections/genetics , HIV Protease Inhibitors/pharmacology , Mutation
2.
Viruses ; 16(5)2024 05 18.
Article in English | MEDLINE | ID: mdl-38793683

ABSTRACT

This review focuses on the emerging field of flow virometry, the study and characterization of individual viral particles using flow cytometry instruments and protocols optimized for the detection of nanoscale events. Flow virometry faces considerable technical challenges including minimal light scattering by small viruses that complicates detection, coincidental detection of multiple small particles due to their high concentrations, and challenges with sample preparation including the inability to easily "wash" samples to remove unbound fluorescent antibodies. We will discuss how the field has overcome these challenges to reveal novel insights into viral biology.


Subject(s)
Flow Cytometry , Virion , Flow Cytometry/methods , Humans , Viruses , Animals
3.
J Virol ; 96(9): e0219821, 2022 05 11.
Article in English | MEDLINE | ID: mdl-35438536

ABSTRACT

HIV-1 encodes a viral protease that is essential for the maturation of infectious viral particles. While protease inhibitors are effective antiretroviral agents, recent studies have shown that prematurely activating, rather than inhibiting, protease function leads to the pyroptotic death of infected cells, with exciting implications for efforts to eradicate viral reservoirs. Despite 40 years of research into the kinetics of protease activation, it remains unclear exactly when protease becomes activated. Recent reports have estimated that protease activation occurs minutes to hours after viral release, suggesting that premature protease activation is challenging to induce efficiently. Here, monitoring viral protease activity with sensitive techniques, including nanoscale flow cytometry and instant structured illumination microscopy, we demonstrate that the viral protease is activated within cells prior to the release of free virions. Using genetic mutants that lock protease into a precursor conformation, we further show that both the precursor and mature protease have rapid activation kinetics and that the activity of the precursor protease is sufficient for viral fusion with target cells. Our finding that HIV-1 protease is activated within producer cells prior to release of free virions helps resolve a long-standing question of when protease is activated and suggests that only a modest acceleration of protease activation kinetics is required to induce potent and specific elimination of HIV-infected cells. IMPORTANCE HIV-1 protease inhibitors have been a mainstay of antiretroviral therapy for more than 2 decades. Although antiretroviral therapy is effective at controlling HIV-1 replication, persistent reservoirs of latently infected cells quickly reestablish replication if therapy is halted. A promising new strategy to eradicate the latent reservoir involves prematurely activating the viral protease, which leads to the pyroptotic killing of infected cells. Here, we use highly sensitive techniques to examine the kinetics of protease activation during and shortly after particle formation. We found that protease is fully activated before virus is released from the cell membrane, which is hours earlier than recent estimates. Our findings help resolve a long-standing debate as to when the viral protease is initially activated during viral assembly and confirm that prematurely activating HIV-1 protease is a viable strategy to eradicate infected cells following latency reversal.


Subject(s)
HIV Protease , HIV-1 , Enzyme Activation/physiology , HIV Infections/virology , HIV Protease/metabolism , HIV-1/drug effects , HIV-1/enzymology , Humans , Protease Inhibitors/pharmacology
4.
J Extracell Vesicles ; 10(8): e12112, 2021 06.
Article in English | MEDLINE | ID: mdl-34188786

ABSTRACT

In late 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. SARS-CoV-2 and the disease it causes, coronavirus disease 2019 (COVID-19), spread rapidly and became a global pandemic in early 2020. SARS-CoV-2 spike protein is responsible for viral entry and binds to angiotensin converting enzyme 2 (ACE2) on host cells, making it a major target of the immune system - particularly neutralizing antibodies (nAbs) that are induced by infection or vaccines. Extracellular vesicles (EVs) are small membraned particles constitutively released by cells, including virally-infected cells. EVs and viruses enclosed within lipid membranes share some characteristics: they are small, sub-micron particles and they overlap in cellular biogenesis and egress routes. Given their shared characteristics, we hypothesized that EVs released from spike-expressing cells could carry spike and serve as decoys for anti-spike nAbs, promoting viral infection. Here, using mass spectrometry and nanoscale flow cytometry (NFC) approaches, we demonstrate that SARS-CoV-2 spike protein can be incorporated into EVs. Furthermore, we show that spike-carrying EVs act as decoy targets for convalescent patient serum-derived nAbs, reducing their effectiveness in blocking viral entry. These findings have important implications for the pathogenesis of SARS-CoV-2 infection in vivo and highlight the complex interplay between viruses, extracellular vesicles, and the immune system that occurs during viral infections.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/therapy , Extracellular Vesicles/chemistry , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , COVID-19/immunology , COVID-19/virology , Flow Cytometry , HEK293 Cells , Humans , Immunization, Passive , Protein Binding , Spike Glycoprotein, Coronavirus/analysis , COVID-19 Serotherapy
5.
Sci Rep ; 10(1): 18101, 2020 10 22.
Article in English | MEDLINE | ID: mdl-33093566

ABSTRACT

HIV encodes an aspartyl protease that is activated during, or shortly after, budding of viral particles from the surface of infected cells. Protease-mediated cleavage of viral polyproteins is essential to generating infectious viruses, a process known as 'maturation' that is the target of FDA-approved antiretroviral drugs. Most assays to monitor protease activity rely on bulk analysis of millions of viruses and obscure potential heterogeneity of protease activation within individual particles. In this study we used nanoscale flow cytometry in conjunction with an engineered FRET reporter called VIral ProteasE Reporter (VIPER) to investigate heterogeneity of protease activation in individual, patient-derived viruses. We demonstrate previously unappreciated interpatient variation in HIV protease processing efficiency that impacts viral infectivity. Additionally, monitoring of protease activity in individual virions distinguishes between drug sensitivity or resistance to protease inhibitors in patient-derived samples. These findings demonstrate the feasibility of monitoring enzymatic processes using nanoscale flow cytometry and highlight the potential of this technology for translational clinical discovery, not only for viruses but also other submicron particles including exosomes, microvesicles, and bacteria.


Subject(s)
Drug Resistance, Viral , Flow Cytometry/methods , HIV Infections/virology , HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , HIV-1/enzymology , Virion/enzymology , HIV Infections/drug therapy , HIV Infections/enzymology , HIV-1/drug effects , HIV-1/isolation & purification , Humans , Jurkat Cells , Virion/drug effects , Virion/isolation & purification
6.
Retrovirology ; 15(1): 44, 2018 07 03.
Article in English | MEDLINE | ID: mdl-29970186

ABSTRACT

BACKGROUND: Viral reprogramming of host cells enhances replication and is initiated by viral interaction with the cell surface. Upon human immunodeficiency virus (HIV) binding to CD4+ T cells, a signal transduction cascade is initiated that reorganizes the actin cytoskeleton, activates transcription factors, and alters mRNA splicing pathways. METHODS: We used a quantitative mass spectrometry-based phosphoproteomic approach to investigate signal transduction cascades initiated by CCR5-tropic HIV, which accounts for virtually all transmitted viruses and the vast majority of viruses worldwide. RESULTS: CCR5-HIV signaling induced significant reprogramming of the actin cytoskeleton and mRNA splicing pathways, as previously described. In addition, CCR5-HIV signaling induced profound changes to the mRNA transcription, processing, translation, and post-translational modifications pathways, indicating that virtually every stage of protein production is affected. Furthermore, we identified two kinases regulated by CCR5-HIV signaling-p70-S6K1 (RPS6KB1) and MK2 (MAPKAPK2)-that were also required for optimal HIV infection of CD4+ T cells. These kinases regulate protein translation and cytoskeletal architecture, respectively, reinforcing the importance of these pathways in viral replication. Additionally, we found that blockade of CCR5 signaling by maraviroc had relatively modest effects on CCR5-HIV signaling, in agreement with reports that signaling by CCR5 is dispensable for HIV infection but in contrast to the critical effects of CXCR4 on cortical actin reorganization. CONCLUSIONS: These results demonstrate that CCR5-tropic HIV induces significant reprogramming of host CD4+ T cell protein production pathways and identifies two novel kinases induced upon viral binding to the cell surface that are critical for HIV replication in host cells.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , HIV Infections/metabolism , HIV Infections/virology , HIV-1/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, CCR5/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , CD4-Positive T-Lymphocytes/immunology , Cytoskeleton/metabolism , HIV Infections/immunology , Host-Pathogen Interactions , Humans , Immunologic Memory , Phosphoproteins/metabolism , Proteomics/methods , Receptors, CXCR4/metabolism , Viral Tropism , Virus Replication
7.
Retrovirology ; 14(1): 4, 2017 01 24.
Article in English | MEDLINE | ID: mdl-28114951

ABSTRACT

BACKGROUND: HIV-1 hijacks host cell machinery to ensure successful replication, including cytoskeletal components for intracellular trafficking, nucleoproteins for pre-integration complex import, and the ESCRT pathway for assembly and budding. It is widely appreciated that cellular post-translational modifications (PTMs) regulate protein activity within cells; however, little is known about how PTMs influence HIV replication. Previously, we reported that blocking deacetylation of tubulin using histone deacetylase inhibitors promoted the kinetics and efficiency of early post-entry viral events. To uncover additional PTMs that modulate entry and early post-entry stages in HIV infection, we employed a flow cytometric approach to assess a panel of small molecule inhibitors on viral fusion and LTR promoter-driven gene expression. RESULTS: While viral fusion was not significantly affected, early post-entry viral events were modulated by drugs targeting multiple processes including histone deacetylation, methylation, and bromodomain inhibition. Most notably, we observed that inhibitors of the Rho GTPase family of cytoskeletal regulators-including RhoA, Cdc42, and Rho-associated kinase signaling pathways-significantly reduced viral infection. Using phosphoproteomics and a biochemical GTPase activation assay, we found that virion-induced signaling via CD4 and CCR5 activated Rho family GTPases including Rac1 and Cdc42 and led to widespread modification of GTPase signaling-associated factors. CONCLUSIONS: Together, these data demonstrate that HIV signaling activates members of the Rho GTPase family of cytoskeletal regulators that are required for optimal HIV infection of primary CD4+ T cells.


Subject(s)
CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/virology , HIV/physiology , Receptors, CCR5/metabolism , Signal Transduction , rho GTP-Binding Proteins/metabolism , Cells, Cultured , Host-Pathogen Interactions , Humans , Virus Integration , Virus Internalization
8.
J Virol ; 88(18): 10803-12, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25008921

ABSTRACT

UNLABELLED: Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat will be given in the context of antiretroviral therapy (ART), infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4(+) T cells to infection by HIV in a dose- and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells but rather enhances the kinetics and efficiency of postentry viral events, including reverse transcription, nuclear import, and integration, and enhances viral production in a spreading-infection assay. Selective inhibition of the cytoplasmic class IIb HDAC6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4(+) T cells that is distinct from their well-characterized effects on nuclear histone acetylation and long-terminal-repeat (LTR) transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV. IMPORTANCE: HDAC inhibitors, particularly vorinostat, are currently being investigated clinically as part of a "shock-and-kill" strategy to purge latent reservoirs of HIV. We demonstrate here that vorinostat increases the susceptibility of uninfected CD4(+) T cells to infection with HIV, raising clinical concerns that vorinostat may reseed the viral reservoirs it is meant to purge, particularly under conditions of suboptimal drug exposure. We demonstrate that vorinostat acts following viral fusion and enhances the kinetics and efficiency of reverse transcription, nuclear import, and integration. The effect of vorinostat was recapitulated using the cytoplasmic histone deacetylase 6 (HDAC6) inhibitor tubacin, revealing a novel and previously unknown cytoplasmic mechanism of HDAC inhibitors on HIV replication that is distinct from their well-characterized effects of long-terminal-repeat (LTR)-driven gene expression. Moreover, our results suggest that treatment of patients with class I-specific HDAC inhibitors could induce latent viruses without increasing the susceptibility of uninfected cells to HIV.


Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , HIV-1/drug effects , HIV-1/physiology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , CD4-Positive T-Lymphocytes/chemistry , CD4-Positive T-Lymphocytes/drug effects , Gene Expression Regulation, Viral/drug effects , HIV Long Terminal Repeat , HIV-1/genetics , Humans , Kinetics , Reverse Transcription/drug effects , Virus Integration/drug effects , Virus Latency/drug effects , Virus Replication/drug effects , Vorinostat
9.
J Virol ; 88(9): 4976-86, 2014 May.
Article in English | MEDLINE | ID: mdl-24554663

ABSTRACT

UNLABELLED: CD4(+) and CD8(+) memory T cells with stem cell-like properties (T(SCM) cells) have been identified in mice, humans, and nonhuman primates and are being investigated for antitumor and antiviral vaccines and immunotherapies. Whether CD4(+) T(SCM) cells are infected by human immunodeficiency virus (HIV) was investigated by using a combination HIV reporter virus system in vitro and by direct staining for HIV p24 antigen ex vivo. A proportion of T(SCM) cells were found to express the HIV coreceptors CCR5 and CXCR4 and were infected by HIV both in vitro and in vivo. Analysis of viral outcome following fusion using the combination reporter virus system revealed that T(SCM) cells can become productively or latently infected, although the vast majority of T(SCM) cells are abortively infected. Knockdown of the HIV restriction factor SAMHD1 using Vpx-containing simian immunodeficiency virus (SIV) virion-like particles enhanced the productive infection of T(SCM) cells, indicating that SAMHD1 contributes to abortive infection in these cells. These results demonstrate that CD4(+) T(SCM) cells are targets for HIV infection, that they become productively or latently infected at low levels, and that SAMHD1 expression promotes abortive infection of this important memory cell subset. IMPORTANCE: Here we demonstrate the susceptibility of CD4(+) memory stem cells (T(SCM) cells) to infection by HIV in vitro and in vivo, provide an in-depth analysis of coreceptor expression, demonstrate the infection of naïve and memory CD4(+) T cell subsets with both CCR5- and CXCR4-tropic HIV, and also perform outcome analysis to calculate the percentage of cells that are productively, latently, or abortively infected. Through these outcome studies, we determined that the vast majority of T(SCM) cells are abortively infected by HIV, and we demonstrate that knockdown of SAMHD1 significantly increases the frequency of infection of this CD4(+) T cell subset, indicating that SAMHD1 is an active restriction factor in T(SCM) cells.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , HIV-1/growth & development , Monomeric GTP-Binding Proteins/immunology , Monomeric GTP-Binding Proteins/metabolism , Stem Cells/virology , Gene Expression , Healthy Volunteers , Humans , Receptors, CCR5/biosynthesis , Receptors, CXCR4/biosynthesis , Receptors, HIV/biosynthesis , SAM Domain and HD Domain-Containing Protein 1
10.
J Virol Methods ; 195: 164-9, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24025341

ABSTRACT

Fusion between the viral membrane of human immunodeficiency virus (HIV) and the host cell marks the end of the HIV entry process and the beginning of a series of post-entry events including uncoating, reverse transcription, integration, and viral gene expression. The efficiency of post-entry events can be modulated by cellular factors including viral restriction factors and can lead to several distinct outcomes: productive, latent, or abortive infection. Understanding host and viral proteins impacting post-entry event efficiency and viral outcome is critical for strategies to reduce HIV infectivity and to optimize transduction of HIV-based gene therapy vectors. Here, we report a combination reporter virus system measuring both membrane fusion and viral promoter-driven gene expression. This system enables precise determination of unstimulated primary CD4+ T cell subsets targeted by HIV, the efficiency of post-entry viral events, and viral outcome and is compatible with high-throughput screening and cell-sorting methods.


Subject(s)
CD4-Positive T-Lymphocytes/virology , Gene Expression , HIV/physiology , Reverse Transcription , Virology/methods , Virus Integration , Virus Uncoating , Cells, Cultured , Genes, Reporter , Humans , Staining and Labeling/methods
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