The Role of Lipid Rafts in Virus Assembly. Identification and Characterization of Microdomain Partitioning Factors of the HIV-1 Glycoprotein gp41 using Flim-FRET and Fluorescence Anisotropy Microscopy

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells.IMPORTANCEHIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

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Insight into the essential role of the Helicobacter pylori HP1043 orphan response regulator: genome-wide identification and characterization of the DNA-binding sites

Scientific Reports ◽

10.1038/srep41063 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 14

Author(s):

Simone Pelliciari ◽

Eva Pinatel ◽

Andrea Vannini ◽

Clelia Peano ◽

Simone Puccio ◽

...

Keyword(s):

Helicobacter Pylori ◽

Binding Sites ◽

Essential Role ◽

Response Regulator ◽

Dna Binding Sites ◽

Genome Wide ◽

Identification And Characterization ◽

Insight Into

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New Insights on the Versatile Role of the Cholesterol Binding Motif of the HIV-1 Glycoprotein Gp41

Biophysical Journal ◽

10.1016/j.bpj.2013.11.421 ◽

2014 ◽

Vol 106 (2) ◽

pp. 62a

Author(s):

Roland Schwarzer ◽

Andreas Herrmann ◽

Ilya Levental ◽

Andrea Gramatica

Keyword(s):

Binding Motif ◽

Glycoprotein Gp41 ◽

Hiv 1 ◽

Cholesterol Binding

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A small mitochondrial protein present in myzozoans is essential for malaria transmission

Open Biology ◽

10.1098/rsob.160034 ◽

2016 ◽

Vol 6 (4) ◽

pp. 160034 ◽

Cited By ~ 9

Author(s):

Dennis Klug ◽

Gunnar R. Mair ◽

Friedrich Frischknecht ◽

Ross G. Douglas

Keyword(s):

Drug Targets ◽

Mitochondrial Protein ◽

Sister Group ◽

Developmental Defect ◽

Mitochondrial Mass ◽

First Time ◽

Potential Drug Targets ◽

Identification And Characterization

Myzozoans (which include dinoflagellates, chromerids and apicomplexans) display notable divergence from their ciliate sister group, including a reduced mitochondrial genome and divergent metabolic processes. The factors contributing to these divergent processes are still poorly understood and could serve as potential drug targets in disease-causing protists. Here, we report the identification and characterization of a small mitochondrial protein from the rodent-infecting apicomplexan parasite Plasmodium berghei that is essential for development in its mosquito host. Parasites lacking the gene mitochondrial protein ookinete developmental defect ( mpodd ) showed malformed parasites that were unable to transmit to mosquitoes. Knockout parasites displayed reduced mitochondrial mass without affecting organelle integrity, indicating no role of the protein in mitochondrial biogenesis or morphology maintenance but a likely role in mitochondrial import or metabolism. Using genetic complementation experiments, we identified a previously unrecognized Plasmodium falciparum homologue that can rescue the mpodd(−) phenotype, thereby showing that the gene is functionally conserved. As far as can be detected, mpodd is found in myzozoans, has homologues in the phylum Apicomplexa and appears to have arisen in free-living dinoflagellates. This suggests that the MPODD protein has a conserved mitochondrial role that is important for myzozoans. While previous studies identified a number of essential proteins which are generally highly conserved evolutionarily, our study identifies, for the first time, a non-canonical protein fulfilling a crucial function in the mitochondrion during parasite transmission.

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Identification and Characterization of the Penicillin-Binding Protein 2a of Streptococcus pneumoniae and Its Possible Role in Resistance to β-Lactam Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.6.1745-1748.2000 ◽

2000 ◽

Vol 44 (6) ◽

pp. 1745-1748 ◽

Cited By ~ 20

Author(s):

Genshi Zhao ◽

Timothy I. Meier ◽

Joann Hoskins ◽

Kelly A. McAllister

Keyword(s):

Streptococcus Pneumoniae ◽

Binding Protein ◽

Penicillin Resistance ◽

Penicillin Binding Protein ◽

Insertional Mutant ◽

Identification And Characterization

ABSTRACT To further understand the role of penicillin-binding protein 2a (PBP 2a) of Streptococcus pneumoniae in penicillin resistance, we confirmed the identity of the protein as PBP 2a. The PBP 2a protein migrated electrophoretically to a position corresponding to that of PBP 2x, PBP 2a, and PBP 2b of S. pneumoniae and was absent in a pbp2ainsertional mutant of S. pneumoniae. We found that the affinities of PBP 2a for penicillins were lower than for cephalosporins and a carbapenem. When compared with other S. pneumoniae PBPs, PBP 2a exhibited lower affinities for β-lactam antibiotics, especially penicillins. Therefore, PBP 2a is a low-affinity PBP for β-lactam antibiotics in S. pneumoniae.

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Identification and Characterization of a New Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody

Journal of Virology ◽

10.1128/jvi.78.17.9233-9242.2004 ◽

2004 ◽

Vol 78 (17) ◽

pp. 9233-9242 ◽

Cited By ~ 63

Author(s):

Mei-Yun Zhang ◽

Xiaodong Xiao ◽

Igor A. Sidorov ◽

Vidita Choudhry ◽

Fatim Cham ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Human Antibody ◽

Immunodeficiency Virus ◽

Clade C ◽

Identification And Characterization ◽

Hiv 1 ◽

Soluble Cd4

ABSTRACT The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.

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