A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

Biochemistry Insights ◽

10.4137/bci.s30379 ◽

2015 ◽

Vol 8s2 ◽

pp. BCI.S30379

Author(s):

Darin Abbadessa ◽

Cameron A. Smurthwaite ◽

Connor W. Reed ◽

Roland Wolkowicz

Keyword(s):

Drug Discovery ◽

Biomedical Research ◽

Envelope Protein ◽

Viral Infectivity ◽

Host Proteins ◽

Subcellular Compartment ◽

A Cell ◽

The Individual ◽

Cellular Compartments ◽

Hiv 1

Infectious diseases affect human health despite advances in biomedical research and drug discovery. Among these, viruses are especially difficult to tackle due to the sudden transfer from animals to humans, high mutational rates, resistance to current treatments, and the intricacies of their molecular interactions with the host. As an example of these interactions, we describe a cell-based approach to monitor specific proteolytic events executed by either the viral-encoded protease or by host proteins on the virus. We then emphasize the significance of examining proteolysis within the subcellular compartment where cleavage occurs naturally. We show the power of stable expression, highlighting the usefulness of the cell-based multiplexed approach, which we have adapted to two independent assays previously developed to monitor (a) the activity of the HIV-1-encoded protease or (b) the cleavage of the HIV-1-encoded envelope protein by the host. Multiplexing was achieved by mixing cells each carrying a different assay or, alternatively, by engineering cells expressing two assays. Multiplexing relies on the robustness of the individual assays and their clear discrimination, further enhancing screening capabilities in an attempt to block proteolytic events required for viral infectivity and spread.

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Recruitment of myosin VIII towards plastid surfaces is root-cap specific and provides the evidence for actomyosin involvement in root osmosensing

Functional Plant Biology ◽

10.1071/fp05004 ◽

2005 ◽

Vol 32 (8) ◽

pp. 721 ◽

Cited By ~ 28

Author(s):

Przemysław Wojtaszek ◽

Anna Anielska-Mazur ◽

Halina Gabryś ◽

František Baluška ◽

Dieter Volkmann

Keyword(s):

Cell Wall ◽

Root Cap ◽

Volume Control ◽

Animal Cells ◽

Active Involvement ◽

A Cell ◽

Osmotic Stimulus ◽

Tight Association ◽

The Individual ◽

Cellular Compartments

The existence of a cell wall–plasma membrane–cytoskeleton (WMC) continuum in plants has long been postulated. However, the individual molecules building such a continuum are still largely unknown. We test here the hypothesis that the integrin-based multiprotein complexes of animal cells have been replaced in plants with more dynamic entities. Using an experimental approach based on protoplast digestion mixtures, and utilising specific antibodies against Arabidopsis ATM1 myosin, we reveal possible roles played by plant-specific unconventional myosin VIII in the functioning of WMC continuum. We demonstrate rapid relocation (less than 5 min) of myosin VIII to statolith surfaces in maize root-cap cells, which is accompanied by the reorganisation of actin cytoskeleton. Upon prolonged stimulation, myosin VIII is also recruited to plasmodesmata and pit-fields of plasmolysing root cap statocytes. The osmotic stimulus is the major factor inducing relocation, but the cell wall–cytoskeleton interactions also play an important role. In addition, we demonstrate the tight association of myosin VIII with the surfaces of chloroplasts, and provide an indication for the differences in the mechanisms of plastid movement in roots and leaves of plants. Overall, our data provide evidence for the active involvement of actomyosin complexes, rooted in the WMC continuum, in the cellular volume control and maintenance of spatial relationships between cellular compartments.

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Cellular Compartments of Human Immunodeficiency Virus Type 1 Replication In Vivo: Determination by Presence of Virion-Associated Host Proteins and Impact of Opportunistic Infection

Journal of Virology ◽

10.1128/jvi.74.1.139-145.2000 ◽

2000 ◽

Vol 74 (1) ◽

pp. 139-145 ◽

Cited By ~ 49

Author(s):

Stephen D. Lawn ◽

Beverly D. Roberts ◽

George E. Griffin ◽

Thomas M. Folks ◽

Salvatore T. Butera

Keyword(s):

Human Immunodeficiency Virus ◽

Opportunistic Infection ◽

Human Immunodeficiency Virus Type ◽

Host Proteins ◽

Immunodeficiency Virus ◽

Cellular Compartments ◽

Hiv 1

ABSTRACT Antigens derived from host cells are detectable in the envelope of human immunodeficiency virus type 1 (HIV-1) and result in a distinctive viral phenotype reflecting that of the host cell. An immunomagnetic capture assay targeting discriminatory host proteins was developed to differentiate between HIV-1 derived from macrophages and lymphocytes. HIV-1 propagated in macrophages or lymphocytes in vitro was selectively captured by monoclonal antibodies directed against the virally incorporated cell-type-specific host markers CD36 (macrophages) and CD26 (lymphocytes). Furthermore, by targeting these markers, virus of defined cellular origin was selectively captured from a mixed pool of in vitro-propagated viruses. This technique was further refined in order to determine the impact of opportunistic infection on HIV-1 expression from these cellular compartments in vivo. Analysis of cell-free virus purified from plasma of patients with HIV-1 infection suggested that in those with an opportunistic infection, viral replication occurred in activated lymphocytes. Interestingly, there was also significant replication in activated macrophages in those patients with untreated pulmonary tuberculosis. Thus, in addition to lymphocytes, the macrophage cellular pool may serve as an important source of cell-free HIV-1 in patients with opportunistic infections that lead to marked macrophage activation. This novel viral capture technique may allow researchers to address a wide range of important questions regarding virus-host dynamics.

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Molecular Screening for Antagonists of HIV‐1 Viral Infectivity Factor Enables Drug Discovery

The FASEB Journal ◽

10.1096/fasebj.29.1_supplement.718.14 ◽

2015 ◽

Vol 29 (S1) ◽

Author(s):

Jason Salter ◽

Ryan Bennett ◽

Guillermo Morales ◽

Harold Smith

Keyword(s):

Drug Discovery ◽

Viral Infectivity ◽

Molecular Screening ◽

Viral Infectivity Factor ◽

Hiv 1

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Nonspecific Chronic Back Pain: Sixth Edition Approaches

Guides Newsletter ◽

10.1001/amaguidesnewsletters.2019.sepoct03 ◽

2019 ◽

Vol 24 (5) ◽

pp. 14-15

Author(s):

Jay Blaisdell ◽

James B. Talmage

Keyword(s):

Back Pain ◽

Chronic Back Pain ◽

Intervertebral Disk ◽

Key Factors ◽

Sixth Edition ◽

Whole Person ◽

Class 1 ◽

A Cell ◽

Intervertebral Disk Herniation ◽

The Individual

Abstract Ratings for “non-specific chronic, or chronic reoccurring, back pain” are based on the diagnosis-based impairment method whereby an impairment class, usually representing a range of impairment values within a cell of a grid, is selected by diagnosis and “specific criteria” (key factors). Within the impairment class, the default impairment value then can be modified using non-key factors or “grade modifiers” such as functional history, physical examination, and clinical studies using the net adjustment formula. The diagnosis of “nonspecific chronic, or chronic reoccurring, back pain” can be rated in class 0 and 1; the former has a default value of 0%, and the latter has a default value of 2% before any modifications. The key concept here is that the physician believes that the patient is experiencing pain, yet there are no related objective findings, most notably radiculopathy as distinguished from “nonverifiable radicular complaints.” If the individual is found not to have radiculopathy and the medical record shows that the patient has never had clinically verifiable radiculopathy, then the diagnosis of “intervertebral disk herniation and/or AOMSI [alteration of motion segment integrity] cannot be used.” If the patient is asymptomatic at maximum medical improvement, then impairment Class 0 should be chosen, not Class 1; a final whole person impairment rating of 1% indicates incorrect use of the methodology.

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A Clinical Trial to Evaluate the Safety and Immunogenicity of Recombinant HIV-1 Envelope Protein BG505 SOSIP.GT1.1 gp140 Vaccine, Adjuvanted in Healthy, HIV-uninfected Adults

Case Medical Research ◽

10.31525/ct1-nct04224701 ◽

2020 ◽

Author(s):

Keyword(s):

Clinical Trial ◽

Envelope Protein ◽

Hiv 1

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Proteomics: Opportunities and Challenges

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2010.3.4.1 ◽

2011 ◽

Vol 3 (4) ◽

pp. 1165-1172 ◽

Cited By ~ 1

Author(s):

Parag A Pathade ◽

Vinod A Bairagi ◽

Yogesh S. Ahire ◽

Neela M Bhatia

Keyword(s):

Biomedical Research ◽

Therapy Monitoring ◽

Dynamic State ◽

Data Sets ◽

Protein Markers ◽

Drug Target Discovery ◽

Proteomic Data ◽

Cell Tissue ◽

A Cell ◽

Analytical Tools

‘‘Proteomics’’, is the emerging technology leading to high-throughput identification and understanding of proteins. Proteomics is the protein equivalent of genomics and has captured the imagination of biomolecular scientists, worldwide. Because proteome reveals more accurately the dynamic state of a cell, tissue, or organism, much is expected from proteomics to indicate better disease markers for diagnosis and therapy monitoring. Proteomics is expected to play a major role in biomedical research, and it will have a significant impact on the development of diagnostics and therapeutics for cancer, heart ailments and infectious diseases, in future. Proteomics research leads to the identification of new protein markers for diagnostic purposes and novel molecular targets for drug discovery. Though the potential is great, many challenges and issues remain to be solved, such as gene expression, peptides, generation of low abundant proteins, analytical tools, drug target discovery and cost. A systematic and efficient analysis of vast genomic and proteomic data sets is a major challenge for researchers, today. Nevertheless, proteomics is the groundwork for constructing and extracting useful comprehension to biomedical research. This review article covers some opportunities and challenges offered by proteomics.

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Elucidating the Disparate Inhibitory Mechanisms of Novel 1-Heteroaryl1,3-Propanediamine Derivatives and Maraviroc towards C-C Chemokine Receptor 5: Insights for Structural Modifications in HIV-1 Drug Discovery

Medicinal Chemistry ◽

10.2174/1573406417666201208122110 ◽

2020 ◽

Vol 17 ◽

Author(s):

Patrick Appiah-Kubi ◽

Fisayo Andrew Olotu ◽

Mahmoud E. S. Soliman

Keyword(s):

Drug Discovery ◽

Chemokine Receptor ◽

Competitive Inhibition ◽

Dynamic Effect ◽

Triazole Ring ◽

Internal Dynamic ◽

V3 Loop ◽

Structural Modifications ◽

Hiv 1 ◽

Chemokine Receptor 5

Introduction: Blocking Human Immunodeficiency Virus type 1 (HIV-1) entry via C-C chemokine receptor 5 (CCR5) inhibition has remained an essential strategy in HIV drug discovery. This underlies the development of CCR5 blockers, such as Maraviroc, which, however, elicits undesirable side effects despite its potency. Background: Recent lead optimization efforts led to the discovery of novel 1-heteroaryl-1,3-propanediamine derivatives; Compd-21 and -34, which were ~3 times more potent than Maraviroc, with improved pharmacokinetics. However, atomistic molecular interaction mechanism of how slight structural variance between these inhibitors significantly affects their binding profiles have not been elucidated. Method: This study employed explicit lipid bilayer molecular dynamics (MD) simulations, and advance analyses to explore these inhibitory discrepancies. Results: Findings revealed that the thiophene moiety substitution common to Compd-21 and -34 enhanced their CCR5- inhibitory activities due to complementary high-affinity interactions with Trp862.60, Tyr1083.32, Tyr2516.51, Glu2837.39. These cumulatively accounted for their ΔGbind which were higher than Maraviroc. Binding dynamics further revealed that the compounds mediated direct competitive inhibition at CCR5 by blocking the gp120 V3 loop. Furthermore, constituent tropane and triazole moieties in the compounds commonly engaged in interactions with Glu2837.39 and Trp862.60, respectively. Structural analyses also revealed that both Compd-21 and -34 elicited distinct internal dynamic effect on CCR5 relative to Maraviroc. Conclusion: Structural modifications at the thiophene substituent and the addition of new functional groups to the triazole ring may enhance inhibitor competition with gp120 V3-loop. Findings herein highlighted would contribute to future structure-based design of inhibitors of HIV-1 CCR5 with improved potencies.

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A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies

Scientific Reports ◽

10.1038/s41598-021-84116-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jean-François Bruxelle ◽

Tess Kirilenko ◽

Nino Trattnig ◽

Yiqiu Yang ◽

Matteo Cattin ◽

...

Keyword(s):

Transgenic Mice ◽

Envelope Protein ◽

Protein Sequence ◽

Neutralizing Antibodies ◽

Human Antibody ◽

Broadly Neutralizing Antibodies ◽

Specific Antibodies ◽

Strong Binding ◽

Hiv Envelope ◽

Hiv 1

AbstractThe occurrence of oligomannose-specific broadly neutralizing antibodies (bnAbs) has spurred efforts to develop immunogens that can elicit similar antibodies. Here, we report on the antigenicity and immunogenicity of a CRM197-conjugate of a previously reported oligomannose mimetic. Oligomannose-specific bnAbs that are less dependent on interactions with the HIV envelope protein sequence showed strong binding to the glycoconjugates, with affinities approximating those reported for their cognate epitope. The glycoconjugate is also recognized by inferred germline precursors of oligomannose-specific bnAbs, albeit with the expected low avidity, supporting its potential as an immunogen. Immunization of human-antibody transgenic mice revealed that only a TLR4-stimulating adjuvant formulation resulted in antibodies able to bind a panel of recombinant HIV trimers. These antibodies bound at relatively modest levels, possibly explaining their inability to neutralize HIV infectivity. Nevertheless, these findings contribute further to understanding conditions for eliciting HIV-cross-reactive oligomannose-specific antibodies and inform on next steps for improving on the elicited response.

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Human TRIM5α: Autophagy Connects Cell-Intrinsic HIV-1 Restriction and Innate Immune Sensor Functioning

Viruses ◽

10.3390/v13020320 ◽

2021 ◽

Vol 13 (2) ◽

pp. 320 ◽

Cited By ~ 1

Author(s):

Alexandra P. M. Cloherty ◽

Anusca G. Rader ◽

Brandon Compeer ◽

Carla M. S. Ribeiro

Keyword(s):

Therapeutic Potential ◽

Innate Immune ◽

Health Concern ◽

Restriction Factor ◽

Viral Reservoirs ◽

Minimal Restriction ◽

Immunodeficiency Virus ◽

A Cell ◽

Species Specific ◽

Hiv 1

Human immunodeficiency virus-1 (HIV-1) persists as a global health concern, with an incidence rate of approximately 2 million, and estimated global prevalence of over 35 million. Combination antiretroviral treatment is highly effective, but HIV-1 patients that have been treated still suffer from chronic inflammation and residual viral replication. It is therefore paramount to identify therapeutically efficacious strategies to eradicate viral reservoirs and ultimately develop a cure for HIV-1. It has been long accepted that the restriction factor tripartite motif protein 5 isoform alpha (TRIM5α) restricts HIV-1 infection in a species-specific manner, with rhesus macaque TRIM5α strongly restricting HIV-1, and human TRIM5α having a minimal restriction capacity. However, several recent studies underscore human TRIM5α as a cell-dependent HIV-1 restriction factor. Here, we present an overview of the latest research on human TRIM5α and propose a novel conceptualization of TRIM5α as a restriction factor with a varied portfolio of antiviral functions, including mediating HIV-1 degradation through autophagy- and proteasome-mediated mechanisms, and acting as a viral sensor and effector of antiviral signaling. We have also expanded on the protective antiviral roles of autophagy and outline the therapeutic potential of autophagy modulation to intervene in chronic HIV-1 infection.

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