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Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy.
J Virol. 2014 Jul; 88(14):7904-14.JV

Abstract

Human 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. Importance: HIV-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.

Authors+Show Affiliations

Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.Bioquant, University of Heidelberg, Heidelberg, Germany.Bioquant, IPMB, University of Heidelberg and DKFZ, Heidelberg, Germany.Bioquant, University of Heidelberg, Heidelberg, Germany.Bioquant, IPMB, University of Heidelberg and DKFZ, Heidelberg, Germany.Department of Chemistry and Biochemistry, Ludwig Maximilians University, Munich, Germany.Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany Barbara_Mueller@med.uni-heidelberg.de.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24789789

Citation

Rahman, Sheikh Abdul, et al. "Investigating the Role of F-actin in Human Immunodeficiency Virus Assembly By Live-cell Microscopy." Journal of Virology, vol. 88, no. 14, 2014, pp. 7904-14.
Rahman SA, Koch P, Weichsel J, et al. Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy. J Virol. 2014;88(14):7904-14.
Rahman, S. A., Koch, P., Weichsel, J., Godinez, W. J., Schwarz, U., Rohr, K., Lamb, D. C., Kräusslich, H. G., & Müller, B. (2014). Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy. Journal of Virology, 88(14), 7904-14. https://doi.org/10.1128/JVI.00431-14
Rahman SA, et al. Investigating the Role of F-actin in Human Immunodeficiency Virus Assembly By Live-cell Microscopy. J Virol. 2014;88(14):7904-14. PubMed PMID: 24789789.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Investigating the role of F-actin in human immunodeficiency virus assembly by live-cell microscopy. AU - Rahman,Sheikh Abdul, AU - Koch,Peter, AU - Weichsel,Julian, AU - Godinez,William J, AU - Schwarz,Ulrich, AU - Rohr,Karl, AU - Lamb,Don C, AU - Kräusslich,Hans-Georg, AU - Müller,Barbara, Y1 - 2014/04/30/ PY - 2014/5/3/entrez PY - 2014/5/3/pubmed PY - 2014/8/26/medline SP - 7904 EP - 14 JF - Journal of virology JO - J. Virol. VL - 88 IS - 14 N2 - Human 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. Importance: HIV-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. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/24789789/Investigating_the_role_of_F_actin_in_human_immunodeficiency_virus_assembly_by_live_cell_microscopy_ L2 - http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=24789789 DB - PRIME DP - Unbound Medicine ER -