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1
المؤلفون: Ayna, Alfadhli, CeAnn, Romanaggi, Robin Lid, Barklis, Eric, Barklis
المصدر: Virology. 579:54-66
مصطلحات موضوعية: Virology
الوصف: Trimers of the HIV-1 envelope (Env) protein perform receptor binding and virus-cell fusion functions during the virus life cycle. The cytoplasmic tail (CT) of Env forms an unusual baseplate structure, and is palmitoylated, rich in arginines, carries trafficking motifs, binds cholesterol, and interacts with host proteins. To dissect CT activities, we examined a panel of Env variants, including CT truncations, mutations, and an extension. We found that whereas all variants could replicate in permissive cells, viruses with CT truncations or baseplate mutations were defective in restrictive cells. We also identified a determinant in HIV-1 amphotericin sensitivity, and characterized variants that escape amphotericin inhibition via viral protease-mediated CT cleavage. Results additionally showed that full-length, his tagged Env can oligomerize and be co-assembled with CT truncations that delete portions of the baseplate, host protein binding sites, and trafficking signals. Our observations illuminate novel aspects of HIV-1 CT structure, interactions, and functions.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::996fc51a98201cfaff5a437565d66815Test
https://doi.org/10.1016/j.virol.2022.12.017Test -
2
المؤلفون: Ce Ann Romanaggi, Ayna Alfadhli, Robin Lid Barklis, Timothy A. Bates, Fikadu G. Tafesse, Ilaria Merutka, Eric Barklis
المصدر: Virology
مصطلحات موضوعية: viruses, Human immunodeficiency virus (HIV), Biology, Virus Replication, medicine.disease_cause, gag Gene Products, Human Immunodeficiency Virus, Article, Virus, Cell Line, Capsid, Protein Domains, Virology, medicine, Humans, Virus Release, Infectivity, Virus Assembly, Virion, Single-Domain Antibodies, Antigen recognition, Cell biology, HIV-1, biology.protein, Capsid Proteins, Antibody
الوصف: The capsid (CA) domain of the HIV-1 precursor Gag (PrGag) protein plays multiple roles in HIV-1 replication, and is central to the assembly of immature virions, and mature virus cores. CA proteins themselves are composed of N-terminal domains (NTDs) and C-terminal domains (CTDs). We have investigated the interactions of CA with anti-CA nanobodies, which derive from the antigen recognition regions of camelid heavy chain-only antibodies. The one CA NTD-specific and two CTD-specific nanobodies we analyzed proved sensitive and specific HIV-1 CA detection reagents in immunoassays. When co-expressed with HIV-1 Gag proteins in cells, the NTD-specific nanobody was efficiently assembled into virions and did not perturb virus assembly. In contrast, the two CTD-specific nanobodies reduced PrGag processing, virus release and HIV-1 infectivity. Our results demonstrate the feasibility of Gag-targeted nanobody inhibition of HIV-1.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2b7bfc335e63962840b0836106beb5b5Test
https://doi.org/10.1016/j.virol.2021.07.001Test -
3
المؤلفون: Robin Lid Barklis, Ayna Alfadhli, August O. Staubus, Eric Barklis
المصدر: Virology
مصطلحات موضوعية: viruses, Cell, HIV Infections, Matrix (biology), Biology, Virus Replication, gag Gene Products, Human Immunodeficiency Virus, Article, Virus, 03 medical and health sciences, Protein Domains, Virology, medicine, Humans, Permissive, 030304 developmental biology, 0303 health sciences, Virus Assembly, Cell Membrane, 030302 biochemistry & molecular biology, Virion, env Gene Products, Human Immunodeficiency Virus, Wild type, Cell biology, medicine.anatomical_structure, Cytoplasm, Cell culture, HIV-1, Function (biology), HeLa Cells
الوصف: Wild type (WT) HIV-1 envelope (Env) protein cytoplasmic tails (CTs) appear to be composed of membrane-proximal, N-terminal unstructured regions, and three C-terminal amphipathic helices. Previous studies have shown that WT and CT-deleted (ΔCT) Env proteins are incorporated into virus particles via different mechanisms. WT Env proteins traffic to cell plasma membranes (PMs), are rapidly internalized, recycle to PMs, and are incorporated into virions in permissive and restrictive cells in a Gag matrix (MA) protein-dependent fashion. In contrast, previously described ΔCT proteins do not appear to be internalized after their arrival to PMs, and do not require MA, but are only incorporated into virions in permissive cell lines. We have analyzed a new set of HIV-1 CT variants with respect to their replication in permissive and restrictive cells. Our results provide novel details as to how CT elements regulate HIV-1 Env protein function.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::aee3b22a8e6b1776b17902da10b1edabTest
https://doi.org/10.1016/j.virol.2019.09.008Test -
4
المصدر: J Mol Biol
مصطلحات موضوعية: Models, Molecular, Neuroblastoma RAS viral oncogene homolog, Gene isoform, Cellular membrane, Methylation, Article, Proto-Oncogene Proteins p21(ras), 03 medical and health sciences, Imaging, Three-Dimensional, 0302 clinical medicine, Prenylation, Structural Biology, Humans, Amino Acid Sequence, HRAS, Molecular Biology, 030304 developmental biology, 0303 health sciences, Effector, Chemistry, Cell Membrane, Hypervariable region, Cell biology, Membrane, 030217 neurology & neurosurgery
الوصف: Mutations of the Ras proteins HRAS, KRAS4A, KRAS4B, and NRAS are associated with a high percentage of all human cancers. The proteins are composed of highly homologous N-terminal catalytic or globular domains, plus C-terminal hypervariable regions (HVRs). Post-translational modifications of all RAS HVRs helps target RAS proteins to cellular membrane locations where they perform their signaling functions. For the predominant KRAS4 isoform, KRAS4B, post-translational farnesylation and carboxymethylation, along with a patch of HVR basic residues help foster membrane binding. Recent investigations implicate membrane-bound RAS dimers, oligomers, and nanoclusters as landing pads for effector proteins that relay RAS signals. The details of these RAS signaling platforms have not been elucidated completely, in part due to the difficulties in preparing modified proteins. We have employed properly farnesylated and carboxymethylated KRAS4B in lipid monolayer incubations to examine how the proteins assemble on membranes. Our results reveal novel insights into to how KRAS4B may organize on membranes.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::8c93cc7183a070ba53a3fd96587a0192Test
https://doi.org/10.1016/j.jmb.2019.07.025Test -
5
المؤلفون: Thomas O. Metz, Iris D. Zelnik, Lisa M. Bramer, Eric Barklis, R. Max Petty, Kent J. Bloodsworth, Jennifer E. Kyle, Ayna Alfadhli, Anthony H. Futerman, Robin Lid Barklis, Fikadu G. Tafesse, Hans C. Leier
المصدر: The Journal of Biological Chemistry
مصطلحات موضوعية: 0301 basic medicine, viruses, β-gal, β-galactosidase, HIV Infections, Biochemistry, chemistry.chemical_compound, NIH, National Institutes of Health, PIP2, phosphatidyl-4,5-bisphosphate, BME, β-mercaptoethanol, Sphingosine N-Acyltransferase, Ceramide synthase, CA, capsid, Infectivity, biology, Chemistry, Ceramide synthase 2, virus diseases, PS, phosphatidylserine, Cell biology, Vesicular stomatitis virus, PM, plasma membrane, PrGag, precursor Gag, SL, sphingolipid, Sphingomyelin, Research Article, Ceramide, CT, cytoplasmic tail, VSV, vesicular stomatitis virus, HexCer, hexosylceramide, Ceramides, 03 medical and health sciences, G, glycoprotein, fusion protein, Humans, ceramide, Molecular Biology, 030102 biochemistry & molecular biology, Tumor Suppressor Proteins, Membrane Proteins, HIV, Cell Biology, Virus Internalization, HEK293T, human embryonic kidney 293T, biology.organism_classification, Fusion protein, Sphingolipid, ceramide synthase, 030104 developmental biology, HEK293 Cells, CerS, ceramide synthase, Env, envelope, HIV-1, sphingolipid, Gene Deletion
الوصف: The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell–cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::d95d3b26a57ef253f47dc2293f746c62Test
https://pubmed.ncbi.nlm.nih.gov/33515546Test -
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المؤلفون: August O. Staubus, Ayna Alfadhli, Eric Barklis, Eric O. Freed, Philip R. Tedbury, Mariia Novikova
المصدر: Journal of Virology. 93
مصطلحات موضوعية: Models, Molecular, Protein Conformation, Immunology, Mutant, Virus Replication, gag Gene Products, Human Immunodeficiency Virus, Microbiology, Viral Matrix Proteins, 03 medical and health sciences, Cytosol, Protein Domains, Viral Envelope Proteins, Virology, Humans, Avidity, Binding site, 030304 developmental biology, 0303 health sciences, Binding Sites, biology, Structure and Assembly, Cell Membrane, 030302 biochemistry & molecular biology, Virion, RNA, Cell culture, Cytoplasm, Insect Science, Chaperone (protein), biology.protein, Biophysics, Protein Multimerization, Intracellular, Protein Binding
الوصف: The matrix (MA) domains of HIV-1 precursor Gag (PrGag) proteins direct PrGag proteins to plasma membrane (PM) assembly sites where envelope (Env) protein trimers are incorporated into virus particles. MA targeting to PM sites is facilitated by its binding to phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], and MA binding to cellular RNAs appears to serve a chaperone function that prevents MA from associating with intracellular membranes prior to arrival at the PI(4,5)P2-rich PM. Investigations have shown genetic evidence of an interaction between MA and the cytoplasmic tails (CTs) of Env trimers that contributes to Env incorporation into virions, but demonstrations of direct MA-CT interactions have proven more difficult. In direct binding assays, we show here that MA binds to Env CTs. Using MA mutants, matrix-capsid (MACA) proteins, and MA proteins incubated in the presence of inositol polyphosphate, we show a correlation between MA trimerization and CT binding. RNA ligands with high affinities for MA reduced MA-CT binding levels, suggesting that MA-RNA binding interferes with trimerization and/or directly or indirectly blocks MA-CT binding. Rough-mapping studies indicate that C-terminal CT helices are involved in MA binding and are in agreement with cell culture studies with replication-competent viruses. Our results support a model in which full-length HIV-1 Env trimers are captured in assembling PrGag lattices by virtue of their binding to MA trimers. IMPORTANCE The mechanism by which HIV-1 envelope (Env) protein trimers assemble into virus particles is poorly understood but involves an interaction between Env cytoplasmic tails (CTs) and the matrix (MA) domain of the structural precursor Gag (PrGag) proteins. We show here that direct binding of MA to Env CTs correlates with MA trimerization, suggesting models where MA lattices regulate CT interactions and/or MA-CT trimer-trimer associations increase the avidity of MA-CT binding. We also show that MA binding to RNA ligands impairs MA-CT binding, potentially by interfering with MA trimerization and/or directly or allosterically blocking MA-CT binding sites. Rough mapping implicated CT C-terminal helices in MA binding, in agreement with cell culture studies on MA-CT interactions. Our results indicate that targeting HIV-1 MA-CT interactions may be a promising avenue for antiviral therapy.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ad284924a947418d870702c00e004663Test
https://doi.org/10.1128/jvi.01079-19Test -
7
المؤلفون: Philip R. Tedbury, Vineet N. KewalRamani, Eric O. Freed, Ioannis Kagiampakis, Mariia Novikova, Eric Barklis, Ayna Alfadhli, Yuta Hikichi
المصدر: J Virol
مصطلحات موضوعية: Models, Molecular, Protein Conformation, alpha-Helical, viruses, T-Lymphocytes, Immunology, Mutant, Amino Acid Motifs, Gene Expression, Trimer, Virus Replication, Microbiology, gag Gene Products, Human Immunodeficiency Virus, Cell Line, Viral Matrix Proteins, Mice, Retrovirus, Viral envelope, Virology, Murine leukemia virus, Animals, Humans, Protein Interaction Domains and Motifs, biology, Virus Assembly, Structure and Assembly, Virion, env Gene Products, Human Immunodeficiency Virus, biology.organism_classification, Transmembrane protein, Cell biology, Leukemia Virus, Murine, Viral replication, Capsid, Amino Acid Substitution, Insect Science, Mutation, HIV-1, Protein Multimerization, HeLa Cells
الوصف: The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. The latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within and distant from the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations and by performing crystallographic studies of in vitro-assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly and establish the functional importance of trimeric MA for Env incorporation. IMPORTANCE The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication and the potential of the trimer interface as a therapeutic target.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::367c15c557022f36791cfb00aefca0f1Test
https://pubmed.ncbi.nlm.nih.gov/31619553Test -
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المؤلفون: Robin Lid Barklis, Eric Barklis, August O. Staubus, Andrew Mack, Ayna Alfadhli, Logan Harper
المصدر: Virology. 518:264-271
مصطلحات موضوعية: Phosphatidylinositol 4,5-Diphosphate, 0301 basic medicine, viruses, Mutant, Gene Products, gag, Biosensing Techniques, Matrix (biology), Biology, Article, 03 medical and health sciences, Virology, Pi, Sequence Deletion, chemistry.chemical_classification, Virus Assembly, Wild type, Colocalization, Group-specific antigen, 030104 developmental biology, Biochemistry, chemistry, HIV-1, Mutant Proteins, Glycoprotein, Biosensor, Protein Binding
الوصف: The matrix (MA) domain of the HIV-1 precursor Gag protein (PrGag) has been shown interact with the HIV-1 envelope (Env) protein, and to direct PrGag proteins to plasma membrane (PM) assembly sites by virtue of its affinity to phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). Unexpectedly, HIV-1 viruses with large MA deletions (ΔMA) have been shown to be conditionally infectious as long as they are matched with Env truncation mutant proteins or alternative viral glycoproteins. To characterize the interactions of wild type (WT) and ΔMA Gag proteins with PI(4,5)P2 and other acidic phospholipids, we have employed a set of lipid biosensors as probes. Our investigations showed marked differences in WT and ΔMA Gag colocalization with biosensors, effects on biosensor release, and association of biosensors with virus-like particles. These results demonstrate an alternative approach to the analysis of viral protein-lipid associations, and provide new data as to the lipid compositions of HIV-1 assembly sites.
الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::591c18f441a3cda6ba308ec4ae83d054Test
https://doi.org/10.1016/j.virol.2018.03.004Test -
9دورية أكاديمية
المؤلفون: Eric Barklis, Ayna Alfadhli, Doug Huseby, Eliot Kapit, Dalbinder Colman
المساهمون: The Pennsylvania State University CiteSeerX Archives
الوصف: This article cites 62 articles, 37 of which can be accessed free
وصف الملف: application/pdf
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10دورية أكاديمية
المؤلفون: Eric Barklis, Ayna Alfadhli, Tenzin Choesang Dhenub, Amelia Still
المساهمون: The Pennsylvania State University CiteSeerX Archives
الوصف: This article cites 41 articles, 29 of which can be accessed free
وصف الملف: application/pdf
