Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

Hendrix, Jelle; Baumgärtel, Viola; Schrimpf, Waldemar; Ivanchenko, Sergey; Digman, Michelle A.; Gratton, Enrico; Kräusslich, Hans‐Georg; Müller, Bárbara; Lamb, Don C.

doi:10.1083/jcb.201504006

Cited by 87 publications

(125 citation statements)

References 83 publications

(131 reference statements)

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“…In support of this idea, crosslinking experiments performed in living cells revealed that the major Gag-interacting RNAs in cytosol are host ncRNAs, particularly tRNAs and 7SL RNA (Kutluay et al 2014). Although the tRNA crosslinks were largely dependent on Matrix, a Gag domain dispensable for forming oligomers, crosslinks to most other host RNAs were strongly reduced by mutations in nucleocapsid (NC), a domain important for oligomer formation (Kutluay et al 2014;Hendrix et al 2015).…”

Section: B Cmentioning

confidence: 79%

“…There is much evidence that RNA binding promotes Gag oligomerization (Campbell and Vogt 1995;Rein et al 2011;Hendrix et al 2015); however, the identities of the responsible RNAs remain unknown. As noted previously (Eckwahl et al 2015;Eckwahl et al 2016), newly synthesized RNAs that have not bound their usual protein partners may be more likely than components of mature RNPs to have the 15-16 nt of protein-free RNA that is needed for Gag multimerization in vitro (Campbell and Rein 1999;Ma and Vogt 2002).…”

Section: B Cmentioning

confidence: 99%

“…One role could be to promote Gag oligomerization, an early step in virus assembly that occurs in cytosol, before arrival of Gag at the plasma membrane (Kutluay and Bieniasz 2010;Hendrix et al 2015). Although oligomerization is enhanced by RNA (Campbell and Vogt 1995;Rein et al 2011;O'Carroll et al 2012;Sundquist and Krausslich 2012), virus-like particle (VLP) formation remains efficient when gRNA is absent or its packaging is prevented (Levin et al 1974;Mann et al 1983).…”

Section: Introductionmentioning

confidence: 99%

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Analysis of the human immunodeficiency virus-1 RNA packageome

Eckwahl

Arnion

Kharytonchyk

et al. 2016

RNA

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All retroviruses package cellular RNAs into virions. Studies of murine leukemia virus (MLV) revealed that the major host cell RNAs encapsidated by this simple retrovirus were LTR retrotransposons and noncoding RNAs (ncRNAs). Several classes of ncRNAs appeared to be packaged by MLV shortly after synthesis, as precursors to tRNAs, small nuclear RNAs, and small nucleolar RNAs were all enriched in virions. To determine the extent to which the human immunodeficiency virus (HIV-1) packages similar RNAs, we used high-throughput sequencing to characterize the RNAs within infectious HIV-1 virions produced in CEM-SS T lymphoblastoid cells. We report that the most abundant cellular RNAs in HIV-1 virions are 7SL RNA and transcripts from numerous divergent and truncated members of the long interspersed element (LINE) and short interspersed element (SINE) families of retrotransposons. We also detected precursors to several tRNAs and small nuclear RNAs as well as transcripts derived from the ribosomal DNA (rDNA) intergenic spacers. We show that packaging of a pre-tRNA requires the nuclear export receptor Exportin 5, indicating that HIV-1 recruits at least some newly made ncRNAs in the cytoplasm. Together, our work identifies the set of RNAs packaged by HIV-1 and reveals that early steps in HIV-1 assembly intersect with host cell ncRNA biogenesis pathways.

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Section: B Cmentioning

confidence: 79%

Section: B Cmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Analysis of the human immunodeficiency virus-1 RNA packageome

Eckwahl

Arnion

Kharytonchyk

et al. 2016

RNA

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“…Tight confinement further distorts the distribution, because this boundary condition excludes the largest step sizes. The artifacts introduced by confinement and in-frame motion were not encountered in previous applications of image correlation spectroscopy, because those experiments focused on slow motion (26,(31)(32)(33)(34), or else minimized pixel dwell time, as in raster image correlation spectroscopy (29). On the other hand, here in the regime of fast, confined motion, the two independent effects of blur and confinement manifest themselves as two independent diffusion-coefficient measurement biases.…”

Section: Introductionmentioning

confidence: 96%

“…All of these methods compute the correlation function of an entire fluorescence imaging movie instead of relying on localization and tracking, and like fluorescence correlation spectroscopy, these powerful image correlation approaches can be extended to measure 2D maps of heterogeneous diffusion (18,26) and very fast diffusion (27). Particle image correlation spectroscopy measures diffusion dynamics by computing a similar correlation function from data that have already been processed with the Gaussian localization used in SPT and other methods (28).…”

Section: Introductionmentioning

confidence: 99%

Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy

Rowland

Tuson

Biteen

2016

Biophysical Journal

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By following single fluorescent molecules in a microscope, single-particle tracking (SPT) can measure diffusion and binding on the nanometer and millisecond scales. Still, although SPT can at its limits characterize the fastest biomolecules as they interact with subcellular environments, this measurement may require advanced illumination techniques such as stroboscopic illumination. Here, we address the challenge of measuring fast subcellular motion by instead analyzing single-molecule data with spatiotemporal image correlation spectroscopy (STICS) with a focus on measurements of confined motion. Our SPT and STICS analysis of simulations of the fast diffusion of confined molecules shows that image blur affects both STICS and SPT, and we find biased diffusion rate measurements for STICS analysis in the limits of fast diffusion and tight confinement due to fitting STICS correlation functions to a Gaussian approximation. However, we determine that with STICS, it is possible to correctly interpret the motion that blurs single-molecule images without advanced illumination techniques or fast cameras. In particular, we present a method to overcome the bias due to image blur by properly estimating the width of the correlation function by directly calculating the correlation function variance instead of using the typical Gaussian fitting procedure. Our simulation results are validated by applying the STICS method to experimental measurements of fast, confined motion: we measure the diffusion of cytosolic mMaple3 in living Escherichia coli cells at 25 frames/s under continuous illumination to illustrate the utility of STICS in an experimental parameter regime for which in-frame motion prevents SPT and tight confinement of fast diffusion precludes stroboscopic illumination. Overall, our application of STICS to freely diffusing cytosolic protein in small cells extends the utility of singlemolecule experiments to the regime of fast confined diffusion without requiring advanced microscopy techniques.

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Labeling of virus components for advanced, quantitative imaging analyses

et al. 2016

Self Cite

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In recent years, investigation of virus-cell interactions has moved from ensemble measurements to imaging analyses at the single-particle level. Advanced fluorescence microscopy techniques provide single-molecule sensitivity and subdiffraction spatial resolution, allowing observation of subviral details and individual replication events to obtain detailed quantitative information. To exploit the full potential of these techniques, virologists need to employ novel labeling strategies, taking into account specific constraints imposed by viruses, as well as unique requirements of microscopic methods. Here, we compare strengths and limitations of various labeling methods, exemplify virological questions that were successfully addressed, and discuss challenges and future potential of novel approaches in virus imaging.

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Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

Abstract: Analysis of the cytosolic HIV-1 Gag fraction in live cells via advanced fluctuation imaging methods reveals potential nucleation steps before membrane-assisted Gag assembly.

Cited by 87 publications

References 83 publications

Analysis of the human immunodeficiency virus-1 RNA packageome

Analysis of the human immunodeficiency virus-1 RNA packageome

Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy

Labeling of virus components for advanced, quantitative imaging analyses

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