Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Aarthi Narayanan,Sergey Iordanskiy,Ravi Das,Rachel Van Duyne,Steven Santos,Elizabeth Jaworski,Irene Guendel,Gavin Sampey,Elizabeth Dalby,Maria Iglesias-Ussel,Anastas Popratiloff,Ramin Hakami,Kylene Kehn-Hall,Mary Young,Caroline Subra,Caroline Gilbert,Charles Bailey,Fabio Romerio,Fatah Kashanchi

doi:10.1074/jbc.m112.438895

Abstract

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

Highlights

Exosomes are extracellular vesicles that have been implicated in intercellular communication
trans-activation response element (TAR) RNA Can Be Detected in Sera of HIV-1-infected Patients and in Culture Supernatants of Infected Cells—Earlier studies have demonstrated that HIV-1 produces its own viral miRNAs with TAR miRNA being the predominant species (46 –53)
B, total RNA isolated from culture supernatants of U1 and HLM-1 cells was analyzed by qRT-PCR with primers specific for TAR RNA and viral mRNAs, including vpu, vpr, vif, nef, tat, env, rev, and unspliced HIV-1 RNA as described in A

Summary

Background

Exosomes are extracellular vesicles that have been implicated in intercellular communication. We demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. Many viral miRNAs have been discovered in exosomes This has been demonstrated in the case of Epstein-Barr virus infections, both in cell culture systems and patient serum samples [55,56,57,58,59]. We provide evidence that exosomes derived from HIV-1-infected cells contain both 5Ј and 3Ј TAR miRNAs. Our proteomic analysis of exosomes derived from infected cells showed the presence of the viral proteins, such as Gag and Env. Within exosomes originating from HIV-1-infected cells, we detected Drosha and Dicer proteins, components of the miRNA machinery. We have detected TAR RNA and specific components of the host miRNA machinery in serum exosomes of HIV-1-infected patient samples

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION