Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

• Published in: Proceedings of the National Academy of Sciences - PNAS 2012-06, Vol.109 (24), p.9587-9592
• Main Authors: Warren, Kylie, Wei, Ting, Li, Dongsheng, Qin, Fangyun, Warrilow, David, Lin, Min-Hsuan, Sivakumaran, Haran, Apolloni, Ann, Abbott, Catherine M, Jones, Alun, Anderson, Jenny L, Harrich, David
• Format: Article
• Language: English
• Subjects: Antibodies; Biological Sciences; Cell Line; Cell lines; Cellular biology; Chromatography, Liquid; DNA; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Eukaryotes; Fractionation; gene expression regulation; HEK293 cells; HeLa cells; HIV; HIV 1; HIV Reverse Transcriptase - metabolism; HIV-1 - enzymology; Human immunodeficiency virus; Human immunodeficiency virus 1; Humans; Immunoassay; Immunoprecipitation; Peptide Elongation Factor 1 - genetics; Peptide Elongation Factor 1 - metabolism; Proteins; Reverse transcription; RNA; RNA, Small Interfering; RNA-directed DNA polymerase; Small interfering RNA; T-lymphocytes; Tandem Mass Spectrometry; Transcription, Genetic; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1204673109

Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.