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Author Notes:

Correspondence and requests for materials should be addressed to S.G.S. (email: sarafianoss@missouri.edu)

Maritza Puray-Chavez and Philip R. Tedbury contributed equally to this work.

Author contributions: S.G.S. conceived the research; S.G.S., M.P.-C, V.Y., and P.R.T. designed experiments; M.P.-C, V.Y., D.L., O.B.U., J.J., J.J.W. performed experiments; M.P.-C., A.D.H., O.B.U., A.N.E., P.R.T., and S.G.S. analyzed data; P.R.T., M.P., V.Y., and S.G.S. wrote the paper.

All authors reviewed the manuscript prior to submission.

The authors declare no competing financial interests.

Subjects:

Research Funding:

The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pNL4–3 from Dr. Malcolm Martin; HIV-2.ST pJSP4-27/H6 from Dr. Beatrice Hahn and Dr. George Shaw; Jurkat E6-1 from Dr. Arthur Weiss; TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc; THP-1ATCC from Drs. Li Wu and Vineet N. KewalRamani; HIV-1 p24 Gag Monoclonal (#24-2) from Dr. Michael H. Malim; Monoclonal Antibody to HIV-1 p24 (AG3.0) from Dr. Jonathan Allan; raltegravir (Cat # 11680) from Merck and Company, Inc.; HIV-1Ba-L from Dr. Suzanne Gartner, Dr. Mikulas Popovic and Dr. Robert Gallo.

We also acknowledge Dr. Michael Parniak for the gift of the RNase H inhibitor naphthyridinone {4-[(4′-aminomethyl-1,1′-biphenyl)methyl]-1-hydroxy-1,8-naphthyridin-2-one.

This work was supported in part by grants from the National Institutes of Health (AI076119, AI100890, AI120860, GM103368 to S.G.S. and AI052014 to A.N.E.), Mizzou Advantage, and Trail to a Cure.

M.P.-C and V.Y. are supported by the Fulbright Student Program.

O.U. has received support from GM056901 and the Gus T. Ridgel Fellowship.

J.J.W. is supported by a Life Sciences Fellowship from the University of Missouri.

Technical support for the confocal microscopy was provided by Alexander Jurkevich, Molecular Cytology Core, Bond Life Sciences Center, University of Missouri.

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • IMMUNODEFICIENCY-VIRUS TYPE-1
  • COMPLEMENTARY ASSAYS REVEAL
  • FUSION INHIBITOR T-20
  • TRANSCRIPTION
  • ARCHITECTURE
  • INTEGRATION
  • LYMPHOCYTES
  • PROVIRUSES
  • RESOLUTION
  • RETROVIRUS
  • Fluorescence imaging
  • Retrovirus

Multiplex single-cell visualization of nucleic acids and protein during HIV infection

Journal Title:

Nature Communications

Volume:

Volume 8

Publisher:

, Pages 1882-1882

Type of Work:

Article | Final Publisher PDF

Abstract:

Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We also report transcriptional "bursts" of nascent RNA from integrated proviral DNA, and concomitant HIV-1, HIV-2 transcription in co-infected cells. MICDDRP can be used to simultaneously detect multiple viral nucleic acid intermediates, characterize the effects of host factors or drugs on steps of the HIV life cycle, or its reactivation from the latent state, thus facilitating the development of antivirals and latency reactivating agents.

Copyright information:

© 2017 The Author(s).

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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